The caudolateral neostriatum of the avian forebrain
and its modulation by dopaminergic afferents
Inaugural-Dissertation
zur
Erlangung des Grades eines Doktors der Naturwissenschaften
in der
Fakultät für Psychologie
der
RUHR-UNIVERSITÄT BOCHUM
vorgelegt von:
Sven Kröner
März 2000
Gedruckt mit Genehmigung der Fakultät für Psychologie der Ruhr-Universität Bochum
Referent: Prof. Dr. Onur Güntürkün (Fakultät für Psychologie)Koreferent: PD Dr. Kurt Gottmann
Tag der mündlichen Prüfung: 29.05.2000
Table of contents
Introduction 1
Chapter 1 Afferent and efferent connections of the caudolateral
neostriatum in the pigeon (Columba livia): A retro- and
anterograde pathway tracing study.
23
Chapter 2 A polysensory pathway to the forebrain of the pigeon: the
ascending projections of the n. dorsolateralis posterior
thalami (DLP).
68
Chapter 3 Characterization of cell types in the chick neostriatum
caudolaterale: Electrophysiological and morphological
properties.
74
Chapter 4 The dopaminergic innervation of the avian telencephalon 102
Chapter 5 The dopaminergic innervation of the pigeon
telencephalon: Distribution of DARPP-32 and co-
occurrence with glutamate decarboxylase and tyrosine
hydroxylase.
152
Chapter 6 Effects of dopamine on the excitability of neurons in the
chick NCL.
175
General discussion 193
List of abbreviations 202
Literature 204
Acknowledgements 236
Curriculum vitae 237
Introduction 1
Introduction
One of the long-standing issues that memory research continues to confront is the dilemma of
localizing a complex function in an anatomically highly differentiated central nervous system.
Is memory a mental function that can be identified within an isolated neural structure or
specific groups of neurons? Traditionally, this question has been framed by psychological
inquiry. In the last century, clinical observations in patients with focal lesions begun to
establish a correspondence between specific symptoms and localized regions of injury, most
notably left hemisphere frontal sites with expression of speech (Broca, 1861) and left
hemisphere posterior temporal lobe sites with speech comprehension (Wernicke, 1874). These
and subsequent cases strengthened the localizationist view, and it was not unreasonable to
expect that other mental processes, like memory functions, could similarly be localized to
circumscribed areas of the cortex. The classical studies of Lashley (1950) demonstrated,
however, that the „engram“, as a single entity, does not exist, but that rather the behavioral
effects of brain lesions correlated with the size of the lesion and not their location, with
different areas making an essential contribution. Since the time of Lashley, it has become
increasingly clear that memory is divisible into different processes and takes heterogeneous
forms based on the type of memory (e.g., classical conditioning versus procedural), the
content of memory (e.g., episodic versus semantic), the temporal parameters of memory (short
term versus long term), and the level of processing (encoding or retrieval).
An important cognitive feature of „higher“1 organisms is their ability to temporarily structure
their behaviour and to actively „hold“ in mind information relevant for goal-attainment. Food-
hoarding birds for example, must remember a large number (up to 33.000) of caches in which
they hide their food (reviewed in Shettleworth, 1983). The information about the location of
the caches is certainly stored in long-term memory. In order to effectively retrieve the food,
however, the animal must actively remember both the places already visited (and update this
information with every new place visited), and must plan a route to the closest location that
contains food (c.f. Shettleworth, 1983).
The active retention of information in memory for the guidance of responses in forthcoming
situations is embedded in the concept of „working memory“ (Baddeley, 1992). Working
1 The term„higher“ is not used in the sense of „further developed“ with regard to a scala naturae, but to describethe complex abilities of the cognitive system. Therefore, the processes underlying „higher“ cognitive functionsshow some degree of abstraction and may be engaged in various behaviours. This might be reflected for examplein the ability of an organism to rapidly adapt to new situations.
Introduction 2
memory encompasses both storage and processing functions, and within cognitive psychology
it represents an extension of an earlier concept, short-term memory, a limited-capacity
temporary memory store, typified by the model proposed by Atkinson and Shiffrin (1968).
Among different species working memory probably takes different forms, and its functions
may range from the „simple“ active retention of goal-related information, to an import role in
(speech)-comprehension, thinking, and planning in primates and humans (e.g. Baddeley,
1992; Kimberg and Farah, 1993; Goldman-Rakic, 1996; Cohen et al., 1997). In order to
understand this sentence, for example, a reader must maintain the first half of the sentence in
working memory while reading the second half.
In mammals, working memory is closely related to the functions of the prefrontal cortex
(PFC) and its innervation by the mesocortical dopaminergic system (see below), and in recent
years considerable progress has been made in identifying the cellular elements that participate
in working memory in mammals (e.g. Goldman-Rakic, 1996 for review). Important aspects in
this quest relate to the specifity of the input-output relationships (i.e. the functional
heterogeneity of brain areas, and the laminar and synaptic organizations of these connections),
the distribution of receptors, and the physiological properties of the neurons involved.
In birds, a region in the posterior forebrain, called neostriatum caudolaterale (NCL), has
recently been shown to be crucially involved in working memory (Mogensen and Divac, 1982,
1993; Gagliardo et al., 1996, 1997; Güntürkün, 1997; Kalt et al., 1999), and other tasks which
in mammals target „frontal“ executive functions (see below), e.g. reversal learning (Hartmann
and Güntürkün, 1998), or response inhibition (Güntürkün, 1997; Aldavert et al., 1999). In
addition, the observation that the NCL shows a high dopamine (DA) content (Divac et al.,
1985) and a strong innervation by dopaminergic fibers (Waldmann and Güntürkün, 1993;
Divac et al. 1994), led to the notion that the NCL might be the avian equivalent of mammalian
PFC. In contrast to mammals, however, virtually nothing is known about the anatomical and
physiological properties of the circuitry that might subserve higher cognitive functions in non-
mammalian species.
The organization of the forebrain of birds and other amniotes (i.e. reptiles) is radically
different from that of mammals (see chapter 1 and 4), reflecting at least 250 million years of
divergent evolution. A comparison between birds and mammals may thus shed light on the
structural and physiological requirements of a proposed canonical circuit involved in working
memory, and more specifically, how the avian brain manages to solve these tasks in the
absence of the layered isocortex of mammals.
Introduction 3
In the studies presented here, I will describe some aspects of the anatomical and physiological
organization of the NCL and its modulation by dopaminergic afferents, to gain insights into
the neuronal circuitry that may underly working memory and other higher cognitive functions
in birds. It should be stressed, however, that within this view no structural homology between
the NCL and the PFC is assumed, but that rather a functional analogy between these brain
areas may exist with regard to some functions, but not necessarily others as well. The term
„homology“ not only implies similar functions of an organ in different species, but also
stipulates a common ancestor (Campbell and Hodos, 1970). Functional „analogies“ in contrast
may reflect parallel evolution, i.e. similar adaptive trends and corresponding selective
pressures (Rehkämper and Zilles, 1991).
For comparison with the results obtained here in birds, the following sections give a brief
overview over some of the functions of the PFC and its interaction with the mesocortical
dopaminergic system. It is sought to describe the basic neuronal circuitry that underlies these
functions (as far as it is known), and specifically its relationship to the layered structure of
mammalian neocortex.
The prefrontal cortex - connectivity and areal organization
The prefrontal cortex occupies the rostral pole of the isocortex of mammals. In various
mammalian species the PFC extends over increasingly larger areas of brain (e.g. primates >
dogs > cats > rats), and its relative growth is larger than that of the other associative cortices,
reflecting a phylogenetic development (Fuster, 1989).
The most commonly used hodological criterion to define the PFC in various species is the
projection of the thalamic nuc. mediodorsalis (MD) (Rose and Woolsey, 1948; Divac et al.,
1978; Goldman-Rakic and Porrino, 1985; Groenewegen et al., 1990). In primates this
projection is topographically organized and allows a rough anatomical division of the PFC
(figure 1). The dorsolateral PFC (Walkers areas 8, 9, 10, 45, 46; Walker, 1940) receives
afferents from the lateral part of the MD (pars parvocellularis), and the
orbitofrontal/ventromedial PFC (Walkers areas 11, 12, 13) is innervated by the medial part of
the MD (pars magnocellularis) (Goldman-Rakic and Porrino, 1985; Groenewegen, 1988;
Groenewegen et al., 1990; Barbas et al., 1991; see Fuster, 1989, for review). However, the
definition of the PFC by its afferents from MD remains a matter of debate, as the MD projects
Introduction 4
also to brain areas beyond the PFC, and on
the other hand the PFC receives substantial
innervation from other thalamic nuclei, e.g.
the anterior nuclei and the pulvinar
(Markowitsch and Pritzel, 1979; Goldman-
Rakic and Porrino, 1985; Groenewegen et
al., 1990; Barbas et al., 1991). Based on
criteria defined by Campbell and Hodos
(1970), Kolb (1984) proposed, that in
rodents the prelimbic and infralimbic
regions of the PFC might be functionally
analogous to the dorsolateral PFC in
primates (but see Preuss, 1995; Williams
and Goldman-Rakic, 1998).
In addition to thalamically relayed input,
direct subcortical and limbic afferents
reach the PFC from the brainstem
tegmentum, the pons, the hypothalamus,
and the amygdala (for reviews see Fuster, 1989; Groenewegen et al., 1990). In various
mammalian species the PFC has furthermore been shown to receive afferent fibers from the
hippocampal complex, the cingulate cortex, and from adjacent areas of the limbic cortex
(Goldman-Rakic et al., 1984; Reep, 1984; Pandya and Yeterian, 1990; Conde et al., 1995). In
the context of working memory, the connections of the PFC with the MD, the hippocampus,
the tegmentum, and the nuc. accumbens (see below) appear to be of particular importance, as
they form a unitary functional network, and lesions in theses regions result in impairments
similar to those observed after lesions of the PFC (e.g. Sakurai and Sugimoto, 1985; Floresco
et al., 1997; Seamans et al., 1998; see below).
Sensory input from other cortical areas reaches the PFC via a set of interconnected pathways
(for reviews: Jones and Powell, 1970; Pandya and Yeterian, 1990). The primary sensory area
of each modality projects first to an adjacent area in parietal, occipital, or temporal cortex
(figure 2). This is the beginning of a sequential order of cortical areas that make up a pathway
for that modality. Each area in the sequence projects not only to the next in line but also to a
discrete area of the frontal cortex, which in turn reciprocates by sending fibers back to the
Sulcus arcuatus
Sulcus principalis
Superior prefrontal convexity
Inferior prefrontal convexity
Orbitofrontalcortex
Inferior prefrontal convexity
B
A
Figure 1: Simplified scheme of the basic subdivisions of theprimate prefrontal cortex (PFC). A: lateral view. B: ventral view.Areas in the superior prefrontal convexity and the sulcusprincipalis are commonly designated as dorsolateral PFC. Theorbitofrontal cortex and areas in the inferior prefrontal convexityconstitute the ventromdedial PFC. The orbitofrontal cortex ispart of the limbic association cortex (Data from Rosenkilde,1979).
Introduction 5
projecting area. The fields that constitute the third link in each of the three pathways - namely,
parietal area 7 (somatic), temporal area 22 (auditory), and inferotemporal area 21 (visual) -
project to the prefrontal cortex and, in addition, to the cortex in the dephts of the superior
temporal sulcus, which constitutes another multimodal area. Although the termination fields
show considerable overlap, especially around the principal sulcus of primates (Area 46), a
strong topography of these afferents exists, resulting in a distinct pattern of connections for
each of the subdivisions of PFC (primates: Jones and Powell, 1970; Selemon and Goldman-
Rakic, 1988; Pandya and Yeterian, 1990; Romanski et al., 1999; rodents: Condé et al., 1995;
Reep et al., 1996).
The (reciprocal) connections of the PFC with the posterior parietal lobe2 can be considered a
prototypical example for the organization of cortico-cortical associational fibers. Both, the
sources of the afferents and their areas of termination are organized in modules. The largest
2 In the parasensory posterior cortex visual and somesthetic information are integrated for the representation ofthe spatial location of an object. The connection between area 7 in the parietal lobe and the PFC is part of anetwork responsible for voluntary movements, particularly eye-movements (Selemon and Goldman-Rakic, 1988),and the so-called „dorsal visual-stream“ conveys information about the „where“ of an object. In contrast, theventral visual stream originating in the temporal lobe, relays information necessary for object-recognition, the„what“ of an object (Wilson et al., 1993; Rao et al., 1997; see Romanski et al., 1999, for auditory processing) .
46
46
8
6
13
AA3
V1
V2AA1
VA1
VA2
VA3
V3
SA3
A1
SA1SA2
V4
AA2
86
S1
12
467b
7a
45
Figure 2: Simplified scheme of the neocortical sensory and associational afferents to the prefrontal cortex in primates. Theparasensory association areas send both hierachially organized indirect, and parallel projections to the PFC. In addition to theprojections depicted along the lateral surface of the brain, afferents from (para-)sensory areas reach the PFC via the cingulateand enthorinal cortex (Data from Jones and Powell, 1970; Pandya and Yeterian, 1990). Abbreviations: S, somatosensory; SA,somato-parasensory association areas; A, auditory; AA, auditory-parasensory association areas; V, visual; VA, visual-parasensory areas.
Introduction 6
number of cortico-cortical projection neurons can be found in layers III, V, and VI (Schwartz
and Goldman-Rakic, 1984; Selemon and Goldman-Rakic, 1988). The afferents from area 7 in
the parietal lobe terminate in a feedforward pattern mainly in layers I, IV and VI of the PFC,
while the feedback from the PFC terminates in layers I and VI, but spares layer IV (Selemon
and Goldman-Rakic, 1988). Within the gaps of the ipsilateral connections terminate callosal
fibers from the contralateral PFC or parietal cortex. These callosal connections originate from
homotopic functional modules and their termination areas show large overlap in the
contralateral hemisphere (Schwartz and Goldman-Rakic, 1984; Selemon and Goldman-Rakic,
1988; Berendse et al., 1992).
The overall pattern of cortical afferents and their coarse topography to the PFC can be
summarized such, that the less differentiated cortices, particularly structures of the limbic
system, innervate the ventromedial (orbitofrontal) PFC, while the „cognitive“, i.e. sensory
aspects of a stimulus are processed in the dorsolateral PFC (c.f. Fuster, 1989).
As already indicated, all of the above mentioned connections of the PFC are reciprocal, and
the „feedback“ from the PFC largely retains the topography of the original input (Selemon and
Goldman-Rakic, 1988; Sesack et al., 1989; Pandya and Yeterian, 1990). Of special
importance are furthermore the reciprocal connections of the PFC with areas in the premotor,
supplementary motor, and motor cortex (Brodmann´s areas 6 and 8; Brodmann, 1909), as via
these efferents the PFC initiates and controls the execution of movements. An exception from
the general rule of reciprocity are the projections of the PFC to the basal ganglia. The inputs
from the PFC to the dorsal- (caudate-putamen) and ventral striatum (nuc. accumbens) are
relayed back only indirectly via the globus pallidus, nuclei in the thalamus, and the substantia
nigra, respectively (Alexander et al., 1990; Berendse et al., 1992; Goldman-Rakic, 1995;
Parent and Hazrati, 1995). Depending on their precise location of termination, the striatal
efferents of the PFC may participate in motoric, associative and limbic aspects of behaviour
(Parent, 1990; Goldman-Rakic, 1995; Parent and Hazrati, 1995).
Microarchitecture of the prefrontal cortex
In primates, the clearest cytoarchitectonic feature of the PFC is the distinct granular layer IV,
that distinguishes the PFC from the remaining frontal cortex; and therefore the PFC has also
been dubbed „frontal granular cortex“ (Walker, 1940). In rodents, however, the granular layer
is not as clearly developed. Afferent fibers terminate in all six layers of the PFC, but the
projections of most brain regions show a distinct laminar pattern within the PFC (c.f. figure 4;
Introduction 7
and the example of area 7 above). Synaptic inputs from many other association cortical
regions converge in layer I-II, where the apical tuft of the layer III, and V-VI pyramidal
neurons extends (Sesack et al., 1989; Berendse et al., 1992; Condé et al. 1995). Inputs to the
soma or proximal/basal dendrites of pyramidal cells in layers III and V-VI, respectively, are
known to arise from neighboring reciprocally connected cells within the same cortical region
(Levitt et al. 1993; Kritzer and Goldman-Rakic, 1995; Pucak et al. 1996; Melchitzky et al.,
1998; Gonzalez-Burgos et al., 2000). This reciprocal interaction may give rise to a
reverberative ensemble of local neurons. Such local activity, confined within the PFC, may
underlie working memory processes, because the PFC must rely on sustained firing to „hold“
information in the absence of continuous presence of previously presented sensory cues (Amit
1995; Goldman-Rakic, 1996; Durstewitz et al., 1999). In analogy to the columns of primary
sensory cortex, clusters of neighbouring cells in the PFC are assumed to form functional
modules (Pucak et al. 1996; Melchitzky et al., 1998; Gonzalez-Burgos et al., 2000), which
might share the memory for a certain location (Rao et al., 1999), or display similar object
selectivity (Rainer et al., 1998b). Local interneurons that contain γ-aminobutyric-acid (GABA)
may play a pivotal role in the fine-tuning of reverberating activity within a cortical module
(Rao et al., 1999), and/or provide lateral inhibition between modules encoding different
features, e.g. opposite spatial loctions (Wilson et al., 1994; Rao et al., 1999). GABAergic
interneurons also appear to be a major target of the dopaminergic innervation (see below).
Behavioural neurophysiology of the prefrontal cortex
Lesion studies
Animals that have been subjected to extensive ablation of their PFC show severe impairments
in the planning and organization of complex behavioural sequences (Kimberg and Farah,
1993; Mogensen and Holm, 1994). The results from numerous lesion studies in mammals
indicate that the disturbance of 3 (interacting) basic functions of the PFC contribute to the
impairments observed in these „frontal animals“ (reviewed in Fuster, 1989; Dias et al., 1997).
1 Focussing of attention: The animal with prefrontal lesions has a fundamental difficulty in
supressing its attention to irrelevant stimuli and maintaining its attention on relevant
stimuli. This distractability is also responsible for hyperreaction to external stimuli, which
in turn accounts for a general motor hyperactivity (Fuster, 1989, for review).
2 Supression of competing tendencies: A loss of inhibition of false response tendencies can
be seen in tasks in which the preoperative incentive values of the discriminanda are
Introduction 8
reversed („reversal-learning“), or in tasks that demand the (successive) discrimination of
two stimuli of which one requires a given response, and the other no response at all
(Go/No-Go tasks). Especially lesions of the ventromedial PFC impair the animals ability to
supress interference from competing tendencies (Brutkowski, 1964; Sakurai and Sugimoto,
1985; Dias et al., 1997), typically leading to perseveration and a loss of response control
(Iversen and Mishkin, 1970).
3 Working memory: As outlined above, working memory describes the process of active
retention and/or manipulation of items in short-term memory, required for the guidance of
responses in forthcoming situations. Experimentally, impairments in working memory
functions can be assessed by variants of the „delayed-response“ or „delayed-alternation“
tasks, which were established in memory research by Jacobsen in the mid-thirties (e.g.
Jacobsen, 1935). Permanent or reversible lesions of the PFC (by cooling, electrical
stimulation or pharmacological blockade), result in severe impairments in the acqusition
and the performance of delay-tasks. The impairments become particulary obvious after
lesions of the dorsolateral PFC (around the sulcus principalis), or its homologue area in the
rat (Jacobsen, 1935; Sawaguchi and Goldman-Rakic, 1991; Kesner et al., 1996; Fuster,
1989, for review).
In addition to these impairments regarding attention and cognition, changes in the affective
and social behaviour are often observed in frontal animals and human patients (see below).
Cellular correlates of working memory
The polysensory character of the PFC indicated from the pattern of converging afferents, is
also reflected in the response characteristics of single neurons. Neurons in the PFC are able to
encode the behavioural significance of a stimulus across various modalities (Yajeya et al.,
1988; Watanabe, 1992). Subsets of prefrontal neurons in the area of the principal sulcus (i) are
activated phasically in the presence of a visual stimulus, (ii) are activated tonically during the
delay period over which the stimulus is kept on line, or (iii) show phasic reactivation in
relation to the initiation of a memory-guided response. Many prefrontal neurons respond in
more than one phase of the trial, i.e., during the cue, delay, and/or response periods, that
means their activity is related to the subfunctions of registration, memory, and motor control,
respectively (Fuster and Alexander, 1971; Fuster, 1973; Funahashi et al., 1989, 1993; Di
Pellegrino and Wise, 1991; Wilson et al., 1993; Funahashi and Kubota, 1994; Rao et al.,
1997, 1999; Asaad et al., 1998; Rainer et al., 1998a,b; Sawaguchi and Yamane, 1999; see
Introduction 9
Fuster, 1989, and Goldman-Rakic, 1996 for reviews). The sustained delay activity of neurons
in the mammalian PFC encodes information relevant to the current behavioural goal (Yajeya
et al., 1988; Funahshi et al., 1989; Di Pellegrino and Wise, 1993; Rao et al., 1997; Asaad et
al., 1998; Rainer et al., 1998a,b; Sawaguchi and Yamane, 1999). This delay activity has been
shown to be content specfic with individual neurons encoding e.g. the location of an object in
space (Funahashi et al., 1989; Fuster, 1973, Rao et al., 1997); the direction of a prior response
(Funahashi et al., 1993), or the identity of an object such as a face or a fruit (Quintana et al.,
1988; Wilson et al., 1993; Rao et al., 1997; Rainer et al., 1998b). In addition, the activity of
prefrontal neurons is often polarized, exhibiting excitatory responses to targets in the preferred
direction and inhibitory responses to targets at nonpreferred directions (Funahashi et al., 1989;
Rainer et al., 1998a; Sawaguchi and Yamane, 1999; Rao et al., 1999). Both a disruption of
delay activity as well as delay activity coding for the wrong stimulus or response are
correlated with subsequent behavioural errors (Fuster, 1973; Quintana et al., 1988; Funahashi
et al., 1989). In contrast to neurons in the temporal and parietal cortices that also show delay-
activity during a working memory task (Miller et al., 1996; Constantinidis and Steinmetz,
1996), networks in the PFC are able to sustain delay activity even in the pressence of
interfering inputs (Miller et al., 1996). It has been hypothesized that this unique feature of the
PFC is due to a „protective“ action of DA (Durstewitz et al., 1999).
Data from functional imaging studies in humans have spawned the discussion about the
functional segregation of the PFC. While all of these studies have demonstrated the
participation of the PFC in different task-specific working memory networks (e.g. Cohen et
al., 1997; Belger et al., 1998; D´Esposito et al., 1999; Haxby et al., 2000), it remains a matter
of debate, whether differential activation of dorsal and ventral areas within the PFC, reflects
content-specific or process-specific domains. One view proposes that the dorsal-ventral
subdivisions of the PFC might be concerned with spatial and object working memory,
respectively. Alternatively it has been suggested that the dorsal PFC is related to the
manipulation of objects in memory, while the ventral PFC merely subserves the active
retention of this information (Belger et al., 1998; D´Esposito et al., 1999; but see Haxby et al.,
2000).
Pathophysiology of the prefrontal cortex in humans
The clinical symptoms following lesions of the PFC in man, most likely also relate to the
basic functions of the PFC reviewed for animals above. In humans the deficits following
Introduction 10
frontal lesions are categorized with respect to symptoms pertaining to perception and motility,
cognition, and affect and emotion (Fuster, 1989).
Perception and Motility. The perceptive abilities of frontal patients are often impaired in as
much as they show increased distractability, or, quite contrary, sensory neglect as a specific
form of attention deficit. Other attentional or „sensory“ deficits regard to spatial orientation
and gaze control (in Fuster, 1989). The motoric deficits of frontal patients depend on the focus
of the lesion. Dorsolateral lesions greatly reduce spontaneous motor activity, leading to
hypokinesis which ranges from a certain „aspontaneity“ to severe mutism (Stuss and Benson,
1986). In contrast, lesions of the orbitofrontal PFC often result in hyperkinesis, and excessive
and aimless motility (in Fuster, 1989).
Cognitive Symptoms. The common cognitive motif following lesions of the PFC are deficits in
the temporal integration of stimuli and actions (Fuster, 1989). Lesions of the dorsolateral PFC
result in disturbances of short-term memory, and the prospective planning of sequences
(Milner and Petrides, 1984; Robbins, 1996). In patients, the ability to sequentially organize a
task is often tested with the „Tower of London“ (Robbins, 1996), while short-term memory
impairments can also be probed with variants of delay-tasks (Milner and Petrides, 1984;
Fuster, 1989). Lesions in the orbitofrontal PFC, however, result in the inability to suppress
interfering tendencies and result in perserverative behaviour (Fuster, 1989; c.f. also Dias et al.,
1997), which can be assessed with the „Wisconsin-Card-Sorting-Test“ (Robbins, 1996).
Affect and Emotion. Symptoms of disturbed affect and emotion can coarsly be divided into
two complexes. The so-called „apathetic syndrome“ is characterized by a low awareness, lack
of initiative and hypokinesis. Most specific, however, is the generalized blunting of affects
and emotional responses, therefore it has also been termed „pseudodepression“ (Stuss and
Benson, 1986). The counterpart of this apathy is the „euphoric syndrome“ which results from
orbitofrontal lesions, and which along with euphoria is characterized by distractibility,
hypermotility, and a disinhibited hunger and sexual drive (Fuster, 1989).
In addition to deficits resulting from lesions of the PFC, multiple lines of evidence suggest
that certain PFC regions may be a locus of dysfunction or structural pathology in such mental
illnesses as schizophrenia (reviewed in Yang et al., 1999), or obsessive-compulsive disorder
(reviewed in Fuster, 1989). The PFC also appears to be markedly affected in Alzheimer’s
disease (Hof et al., 1990; Terry et al., 1991). It has been reported for example that one major
correlate of cognitive deficiency in alzheimers disease is the synaptic loss in the PFC,
Introduction 11
contributing about 70% of the strength of the correlation with psychometric scores (Terry et
al., 1991).
The mesocortical dopamine system
The dopaminergic innervation of the
forebrain of mammals and birds is
constituted by a small number
(!30.000 - 40.000 neurons on each
side of the rat brain) of highly
collateralized neurons residing in the
ventral mesencephalon (e.g.
Björklund and Lindvall 1984; Fallon
and Loughlin, 1995; Williams and
Goldman-Rakic, 1998; c.f. chapter 4
for birds). These mesencephalic cell
groups are designated as A8, A9, and
A10, according to the nomenclature
of Dahlström and Fuxe (1964), and
they generally correspond to the DA
cells of the substantia nigra (SN, A9),
ventral tegmental area (VTA, A10),
and the retrorubral area (A8)3 (Porrino
and Goldman-Rakic 1982; Berger et
al., 1991; Williams and Goldman-
Rakic, 1998).
Functionally, different mesencephalic dopaminergic projections exist which have been
designated as mesostriatal-, mesolimbic-, or mesocortical DA system, respectively, based on
their main targets of innervation (figure 3; Björklund and Lindvall, 1984; Cooper et al., 1996).
The mesolimbic DA system projects to various limbic areas, including the amygdaloid
complex, hippocampus, septum, nucleus accumbens, and entorhinal cortex . The mesostriatal
DA system provides strong innervation to the striatal aspects of the basal ganglia (i.e. caudate-
3 Due to the similarity of their projection, DA cells in the retrorubral field are often grouped with the substantianigra, pars compacta (Björklund and Lindvall, 1984; Reiner et al., 1994)
Figure 3: Organization of the mesencephalic dopamine system inmammals (brain of a rat). A: The mesocortical- and mesolimbic DAsystem. B: The mesostriatal DA system. In primates the mesocorticaldopaminergic innervation is much expanded and extends over largeareas of the posterior cortices. See text for details.
A
locuscoeruleus
lat. nucleusparabrachialis
entorhinalcortex
amygdala
piriformcortex
ant. cingulatecortex
prefrontalcortex
lateralseptum
hippocampus
habenula
A10
A8A9
Bcaudate-putamen
bed nuc.-stria terminalis
tuberculumolfactorium
nuc. accumbens
perirhinalcortex
A10
A9A8
Introduction 12
putamen), and to a lesser extent to the globus pallidus and tuberculum olfactorium (Björklund
and Lindvall, 1984; Cooper et al., 1996).
The organization and extent of the cortical innervation by the mesocortical DA system varies
with the species studied (reviewed in Berger et al., 1991). In primates, tyrosine hydroxylase-
or dopamine-immunoreactive fibers, originating in a continuum of cells in A8-A10, are found
in the motor, premotor, supplementary motor area, parietal, temporal, and posterior cingulate
cortices (sensorimotor), in addition to prefrontal, anterior cingulate, insular, piriform,
perirhinal, and entorhinal cortices (association). This innervation shows a strong rostro-caudal
gradient (also seen in rodents), the innervation being densest in the frontal cortex, particularly
in areas 4 and 6 (Björklund and Lindvall, 1984; Berger et al., 1988, 1991; Goldman-Rakic et
al., 1992; Williams and Goldman-Rakic, 1993). The rodent mesocortical DA innervation is
mainly confined to the limbic cortices, including the prefrontal, anterior cingulate, insular,
piriform, perirhinal, and entorhinal cortices (Björklund and Lindvall 1984; Berger et al. 1991).
Input to the rodent PFC derives from largely separate populations of dopaminergic neurons
located in the SN and VTA, respectively (Swanson 1982; Björklund and Lindvall, 1984;
Fallon and Laughlin, 1995).
The laminar organization of the mesocortical DA innervation generally shows a bilaminar
pattern, but again with distinct species-differences between rodents and primates (c.f. figure
3). In the rodent PFC, afferents from the medial VTA provide dense DA input to the deep
layers V-VI. The considerably weaker DA innervation to the superficial layer I-III originates
in the medial SN and the lateral VTA (van Eden et al., 1987; Berger et al., 1991).
Furthermore, on the ground of developmental, morphological, and pharmacological
differences (reviewed in Berger et al., 1991), these distinct inputs have been divided into two
classes, with class 1 DA neurons innervating the deep cortical layers. There is also evidence
that class 1 and 2 neurons show functional diverences, as class 1 neurons are more susceptible
to the effects of stress (Deutch et al., 1991). It has been hypothesized that the two modes of
dopaminergic innervation in the avian forebrain might relate to these two classes of DA
neurons in rodents (Wynne and Güntürkün, 1995; c.f. chapter 4).
In the primate granular cortices, the superficial layers I-III are generally innervated even
denser than layers V-VI, whereas in the motor and anterior cingulate (agranular) cortices all
layers receive a similarly dense dopaminergic input (Berger et al., 1988, 1991; Berger and
Gaspar, 1994; Goldman-Rakic et al., 1992; Lewis et al., 1992; Williams and Goldman-Rakic,
1993; Krimer et al., 1997). In the PFC of rats and primates dopaminergic fibers make about 40
Introduction 13
- 90 % specialized synaptic contacts (!39 % in the sulcus principalis of primates, Smiley and
Goldman-Rakic, 1993; !56 % in the suprarhinal, and !93 % in the anteromedial PFC of rats,
Séguéla et al., 1988; but see Smiley and Goldman-Rakic, 1993 for technical considerations),
which are mostly of the symmetric type. The remaining axonal varicosities constitute non-
synaptic release sites and probably contribute to the actions of DA via volume transmission
(Smiley and Goldman-Rakic, 1993). The most common synaptic target of the DA terminals in
the PFC of rodents and primates appear to be the dendritic spines and shafts of pyramidal
neurons (van Eden et al., 1987; Goldman-Rakic et al., 1989; Smiley and Goldman-Rakic,
1993). Many of the postsynaptic spines innervated by DA terminals also receive unlabeled
asymmetric (putative excitatory) terminals. Through this „triadic” synaptic arrangement DA
might both, pre- and postsynaptically, modulate the excitatory afferents to pyramidal cells
(van Eden et al. 1987; Séguéla et al. 1988; Goldman-Rakic et al. 1989; Smiley and Goldman-
Rakic 1993; c.f. chapter 4 for the situation in birds). However, dopaminergic fibers in the PFC
have also been observed to terminate on smooth GABAergic, stellate cells (Goldman-Rakic et
al., 1989; Benes, et al., 1993; Smiley and Goldman-Rakic, 1993). In addition, the localization
of DA receptors in interneurons and the physiological actions of DA in the PFC strongly
implicate a dopaminergic modulation of GABAergic interneurons (see below).
Dopamine receptors in the PFC
Dopamine receptors belong to the superfamily of seven transmembrane spanning G protein-
coupled receptors. In recent years, molecular cloning studies have led to a revision of the
traditional classification of dopamine receptors into D1 and D2 subtypes simply based on their
effects on the adenylyl-cyclase system (Kebabian and Calne, 1979). So far at least 5 different
DA receptors (D1 - D5) have been identified. The pharmacological properties in response to
„classical“ D1 and D2 receptor ligands, respectively, distinguish a D1-like family (D1 and
D5), and a D2-like family (D2, D3, D4) of DA receptors (Seeman and van Tol, 1993; Cooper
et al., 1996). In accordance with the classical view, receptors of the D1 family are coupled
positively to the adenylyl-cyclase, while D2 receptors inhibit adenylyl-cyclase activity
(Kebabian and Calne, 1979; Cooper et al., 1996; Robinson and Caron, 1997).
Insight into the distinct pharmacological profiles and the differential distribution of the DA
receptor subtypes are of high clinical relevance for the development of new drug treatments.
For example, the therapeutic effects of neuroleptics are mediated via receptors of the D2
family. The blockade of D2 receptors by „typical“ neuroleptics such as haloperidol, however,
Introduction 14
often results in side-effects on the extrapyramidal motoric system, leading to tardive
dyskinesia or parkinson-like symptoms. This is related to the fact that D2 receptors are most
abundant in the neostriatum. „Atypical“ antipsychotics such as clozapine, on the other hand,
have only a low affinity - only about 10 % of the classical neuroleptics - for the D2 receptor,
but a much higher affinity for D3 or D4 receptors, which are highly abundant in the nuc.
accumbens and the PFC. The latter structures and their dopaminergic innervation appear to be
critically involved in both the positive and negative symptoms of schizophrenia (Seeman and
van Tol, 1993).
The best studied effector protein modulated following the activation of DA receptors in
neurons is DARPP-32 (c.f. chapter 5). DARPP-32 is a Dopamine- and cAMP-Regulated
PhosphoProtein of molecular weigth 32,000 that serves as a „third messenger" in the
intracellular cascade that follows D1-receptor stimulation (Hemmings et al., 1987a, 1987b,
1995; Fienberg et al., 1998). Via activation of the adenylyl cyclase, D1-receptor action
stimulates cAMP synthesis, which through protein kinase A (PKA) leads to phosphorylation
of DARPP-32. Phosphorylated DARPP-32 in turn inhibits protein phosphatase-1. In striatal
neurons protein phosphatase-1 controls the state of phosphorylation of a wide array of
phosphproteins, including neurotransmitter receptors (e.g. for α-amino-3-hydroxy-5-
methylisoxazole-4-propionic acid [AMPA] and N-methyl-D-aspartate [NMDA]), ion channels
(e.g. Ca++ channels and voltage dependent Na+ channels), ion pumps (e.g. the Na+-K+-
ATPase), and transcription factors (Hemmings et al., 1995; Nishi et al., 1997; Fienberg et al.,
1998). Conversely, DA, acting on D2-like receptors, through both inhibition of PKA and
activation of calcium/calmodulin-dependent protein phosphatase (protein phosphatase
2B/calcineurin), may cause the dephosphorylation of DARPP-32 (Nishi et al., 1997), enabling
a possible interplay of D1 and D2-like receptors.
The laminar distribution of DA receptors in the frontal cortex largely parallels the bilaminar
pattern of the innervation by dopaminergic fibers. In rodent frontal cortex there is
considerable expression of mRNA for the D1 receptors in deep layers V-VI. A comparatively
lower expression of D2 receptor mRNA is distributed in superficial layers I-III and in deep
layer V (Gaspar et al. 1995; see Yang et al., 1999 for review). In general, in the frontal
cortices of rats and primates, the density of D1 receptors has been estimated to be about five-
to tenfold higher than that of D2 receptors (Lidow et al., 1991; Joyce et al., 1993). In rodent
frontal cortex there is also evidence for a considerable number of D4 receptors, but not for D3
and D5 receptors (Joyce et al., 1993; Ariano et al. 1997, see Yang et al., 1999 for review). In
Introduction 15
primate PFC expression of mRNA for all 5 DA receptor subtypes has been found (Lidow et
al., 1998). The largest numbers of receptor proteins were found for the D1, D4 and D5
subtypes in pyramidal neurons of superficial layers II-III and deep layers V-VI, with layer V
neurons showing a stronger expression of D4 and D5 receptors (Lidow et al. 1991, 1998).
Immunoreactivity and mRNA expression for D1, D2 and D4 receptors has also been found in
GABAergic interneurons of rat and primate frontal cortex (Mrzljak et al., 1996; Ariano et al.,
1997; LeMoin and Gaspar, 1998; Muly et al., 1998).
contralateral PFCipsilateral PFC
entorhinal cortexhippocampus
amygdalamidline thalamus
intracortical associational
hippocampusintracortical associational
MDhippocampus
midline thalamus
intracortical associational
entorhinal cortex
contralateral PFC
hippocampus
amygdala
intracortical associational
contralateral PFC
hippocampusmidline thalamus
ventromedial thalamus
intracortical associational
rodents primates
SN (A 9)
VTA (A 10)
1
2
4
5/6
3
Layers AfferentsDopamine
column 1 column 2
dendrite
spine
sensoryafferent
D1D2?
Glu
D1/D2?
DAGlu
DAvaricosity
Figure 4: A simplified scheme of the functional architecture of the prefrontal cortex and the innervation from themesocortical dopamine system. On the left, the laminar distribution of known afferents to the PFC, and the pattern ofdopaminergic fibers in rodents and primates is shown. On the right, the neuronal elements of a proposed canonical circuitare shown. Neurons in the PFC (shown are pyramidal cells - green - in layers III and V/VI and interneurons - blue) appearto be arranged in columns, or „stripes“, which are believed to belong to distinct functional modules. The axon collaterals ofnon-pyramidal interneurons (yellow) can either provide „fine tuning“ between the pyramidal neurons of a functional module(not shown), or may exert strong lateral inhibition onto neurons of a distinct functional column/module. Reverberatingactivity between neurons of a certain lamina and/or a functional module could provide a means for the „holding on-line“ ofa certain information. Insert: Dopamine released from varicosities of dopaminergic fibers may act directly at receptorswithin a synapse of the spine, or may diffuse over longer distance to activate extrasynaptic receptors, e.g. at other sites of thedendrite, or on afferents presynaptically to the neuron (forming a synaptic triad). See text for details.
Introduction 16
Physiology of the mesocortical dopaminergic system
Midbrain dopaminergic neurons exhibit a burst of action potentials immediately following
unexpected salient events, which include sudden novel stimuli (Schultz and Romo, 1990;
Ljungberg et al., 1992), and primary rewards (Ljungberg et al., 1992; Schultz et al., 1995).
During the learning-phase of a task, reward-related activation of DA neurons first occurs in
response to the (unexpected) primary reward (the UCS). If the UCS becomes predictable,
activation of DA neurons „shifts“ to the occurence of the conditioned stimulus (CS)
(Ljungberg et al., 1992), and finally to the occurence of a cue that signals the CS (Schultz et
al., 1993; Schultz, 1998 for review). Schultz and coworkers (Schultz et al., 1995; Schultz,
1998; c.f. also Montague et al., 1996) have argued that the activity of dopaminergic neurons
provides an essential signal for reinforcment learning. This model is based on the observation
that dopamine neurons report rewards relative to their prediction rather than signaling primary
rewards unconditionally. The dopamine response is positive (activation) when primary
rewards occur without being predicted. The response is nil when rewards occur as predicted.
The response is negative (depression) when predicted rewards are omitted. (Ljungberg et al.,
1992; Schultz et al., 1993; Schultz, 1998 for review). The dopamine reward signal is
supplemented by reward-processing neurons in the striatum, amygdala, and frontal cortex
(Apicella et al., 1991, 1992; Schultz et al., 1992; Schultz, 1998), and these regions provide
direct and indirect feedback to the mesencephalic DA neurons via a cortico-striato-tegmental
loop (Alexander et al., 1990; Goldman-Rakic, 1995). However, Redgrave and coworkers
(Redgrave et al., 1999) have recently proposed another functional interpretation of the activity
of DA midbrain neurons. In their view DA release is responsible for reallocating attentional
and behavioural resources, in a sense of „switching“ attention to new or salient stimuli.
In accordance with both models, dopaminergic midbrain neurons are activated at the onset of
working memory tasks (Schultz et al., 1993) and DA levels in the PFC increase during delay-
task performance (Watanabe et al., 1997). In contrast, the behavioural impairments following
lesion of the mesencephalic dopamine neurons or the blockade of dopaminergic transmission
in the PFC are very similar to those observed after lesions of the PFC itself (Brozoski et al.,
1979; Simon et al., 1980; Sokolowski and Salamone, 1994; Sokolowski et al., 1994).
Destruction of dopaminergic neurons in area A 10 (Simon et al., 1980) or of dopaminergic
fibers within the PFC by 6-hydroxydopamine (6-OHDA) (Brozoski et al., 1979) result in
deficits in delay-tasks, and an impairment in response control (Sokolowski and Salamone,
1994). The effect of DA on performance in delay-tasks is mediated by the D1 receptor, and an
Introduction 17
optimal level of stimulation by DA appears to be critical for proper functioning (Sawaguchi et
al., 1990b; Sawaguchi and Goldman-Rakic, 1991, 1994; Williams and Goldman-Rakic, 1995;
Murphy et al., 1996; Sawaguchi, 1997; Zahrt et al., 1997; Arnsten and Goldman-Rakic, 1998;
Seamans et al., 1998).
The cellular bases of the effects of DA in the cortex, however, remain enigmatic. In vivo
extracellular single-unit recording studies have shown that iontophoretically applied DA either
increased or decreased spontaneous neuronal firing in the neocortex (Bradshaw et al. 1985;
Sesack and Bunney 1989; Yang and Mogenson, 1990). Dopamine applied iontophoretically or
released by VTA stimulation, suppressed spontaneous, as well as presumed glutamate-
mediated mediodorsal thalamic-evoked firing in the rat PFC in-vivo (Ferron et al. 1984;
Sesack and Bunney 1989; Godbout et al. 1991; Pirot et al. 1992). In contrast, neuronal firing
induced by iontophoretic application of acetylcholine or NMDA is enhanced by very low
doses of iontophoretically applied DA (Yang and Mogenson 1990; Cepeda et al. 1992).
These findings are in good agreement with the long proposed neuromodulatory action of DA.
By definition, a neuromodulator, in contrast to the classical neurotransmitters, is not inhibitory
or excitatory per se, but when applied in conjunction with other substances it has the ability to
alter their actions. The modulatory nature of DA also appears to extend to a voltage- (or
„activity-“) dependence of its effects, in that DA has inhibitory effects at hyperpolarized
membrane potentials, but enhancing effects at depolarized potentials (see below).
Another important methodological difference that hampers comparison across different in-
vivo studies may result from the use of anesthetics that block N-methyl-D-aspartate (NMDA)
conductances and thus one of the major excitatory effects of DA. Both in the striatum and
PFC, DA appears to differentially modulate AMPA and NMDA receptors. Application of DA
reduces AMPA currents, but enhances NMDA conductances (Cepeda et al., 1992, 1998;
Durstewitz et al., 2000; but see Law-Tho et al., 1994). This in turn is partly due to the
different voltage dependence of these currents. Activation of NMDA receptors requires prior
depolarization of the membrane, whereas AMPA receptors show no voltage-dependent
activation. Similarly in vitro, the effects of DA on striatal neurons, acting via the D1 receptor,
are inhibitory when the membrane potential is hyperpolarized (which resembles the so called
„down“-state of striatal neurons observed in-vivo), but are excitatory when the cell is in the
depolarized „up“-state (Hernández et al., 1997). There is evidence that in the cortex DA has
comparable enhancing effects particularly on highly activated neurons (Sawaguchi et al.,
1988, 1990a; Durstewitz et al., 2000). Although DA inhibits spontaneous activity within the
Introduction 18
PFC of anesthetized animals (see above), it enhances task-related single-unit activity more
than background activity in the behaving animal (Sawaguchi et al., 1988, 1990a).
In the cortex, this „content-specific“ enhancement of neuronal activity results from a complex
interplay of ionic conductances that govern the action-potential threshold of pyramidal
neurons and regulate the transmission of synaptic input through the apical dendrite (where
most of the cell´s input arrives).
In the cortex DA, via its action at the D1 receptor, appears to shift the threshold of the
persistent Na+ current (INaP) towards more hyperpolarized potentials (Yang and Seamans
1996; Durstewitz et al., 2000; but see Geijo-Barrientos and Pastore, 1995). Activation of INaP
or its prolonged activation will reduce the threshold for generating an action potential, and
render the cell more susceptible for excitatory inputs (Schwindt, 1992; Yang and Seamans,
1996). Dopamine might also act on a slowly inactivating K+ current (IKS) that usually delays
spike generation and lengthens inter-spike intervals, which in turn suppresses repetitive firing
(Schwindt et al. 1988; Kitai and Surmeier, 1993; Yang and Seamans, 1996). A reduction of
IKS shortens inter-spike-intervals and reduces accomodation of firing (Yang and Seamans,
1996; Nisenbaum et al., 1998). The concurrent actions of DA on both INaP and IKS might
reduce spike treshold and contribute to an increased firing rate observed in vivo (Sawaguchi et
al., 1988; 1990a,b) and in vitro (Cepeda et al., 1992; Yang and Seamans, 1996).
In addition, DA has been shown to excert a differential effect on high-voltage-activated
(HVA) Ca++ currents. Dopamine augments the activation of L-type Ca++ channels, which
functionally serve to enhance the amplitude and duration of excitatory synaptic inputs
(Seamans et al., 1997), thus further increasing the neurons excitability (Hernández-López et
al. 1997; Cépeda et al. 1998). Finally, in both cortical and striatal neurons, DA reduces the
amplitude of isolated N-type Ca++ currents, which has been interpreted as a „filtering“-
mechanisms that permits only strong synaptic inputs (e.g. behaviourally salient stimuli) to
reach the soma (Surmeier et al., 1995;Yang and Seamans, 1996). The properties of these
conductances modulated by DA are described in more detail in the discussion of chapter 6.
In the mammalian PFC, DA also appears to modulate the activity of GABAergic interneurons
and spontaneous GABA release. Dopamine directly depolarizes interneurons in the PFC and
enhances spontaneous and evoked IPSPs recorded in pyramidal neurons (Penit-Soria et al.,
1987; Godbout et al., 1991; Rétaux et al., 1991; Pirot et al., 1992; Zhou and Hablitz, 1999;
Durstewitz et al., 2000). Accordingly, part of the suppressive effects of DA on spontaneous
Introduction 19
firing of pyramidal PFC neurons observed in vivo might be due to the action of DA on
GABAergic interneurons.
Aims of the study
As outlined above, the experiments in this thesis try to unravel the anatomical and
physiological properties of a proposed canonical circuit involved in working memory and
other executive functions in a non-mammalian species. The NCL of pigeons (Columba livia)
and chicks (Gallus gallus domesticus) serves as a model structure in this quest as behavioural
studies have shown its involvement in working memory (e.g. Mogensen and Divac, 1982,
1993; Kalt et al., 1999), reversal learning (Hartmann and Güntürkün, 1998), and response
inhibition (Güntürkün, 1997; Aldavert et al., 1999). Furthermore, the strong dopaminergic
innervation (e.g. Waldmann and Güntürkün, 1993) and recent evidence from a behavioural
study investigating the effects of pharmacological blockade of dopaminergic transmission
(Güntürkün and Durstwitz, in press), support the involvement of DA in the executive
functions of the NCL.
The studies undertaken here to uncover the cellular bases of these behaviours adress two main
topics.
1. The functional organization of the NCL.
2. The influence of the dopaminergic system on identified cell types within the NCL.
The functional organization of the NCL
The term working memory refers to the control of reactions depending on past experiences
and the ongoing stream of sensory information that is currently being experienced (c.f.
Baddeley, 1992; Goldman-Rakic, 1995). As exemplified for the PFC above, this „memory for
action“ requires (i) the retrieval of learned responses from long-term memory (possibly from
memory stores in the hippocampus or posterior cortices), (ii) the simultaneous
integration/evaluation of sensory information about the environment (provided by the
posterior cortices and the thalamus) for the selection of the appropiate response, and (iii) the
initiation of the response. Thus, a clear prerequisite for a structure in non-mammalian species
that might subserve working memory is the ability to integrate all available sensory
Introduction 20
information and to exert influence over motor and limbic structures. While the available
behavioural data (e.g. Mogensen and Divac, 1982, 1993; Hartmann and Güntürkün, 1998)
strongly indicate that such sensorimotor integration takes place in the NCL, the anatomical or
electrophysiological basis for these computations is unclear. The experiments described in
chapter 1 have thus examined the areal termination pattern of afferents from other
telencephalic structures to the NCL, by means of anterograde and retrograde pathway tracing
techniques. In chapter 2, these findings are complemented by the anatomical description of
thalamic input from a multimodal nucleus in the dorsal thalamus. In addition, the organization
of efferents from the NCL to (para-)sensory structures, as well as limbic and motoric areas in
the avian forebrain, through which the NCL might modulate its own input and initiate motoric
responses, respectively, are characterized in chapter 1. Taken together, these anatomical
results reveal the nature of polymodal processing within NCL and possible functional
subdivisions of the NCL.
The projection from the mediodorsal thalamus to the PFC serves as a hallmark for the
definition of the PFC (Rose and Woolsey, 1948; Divac et al., 1978; Goldman-Rakic and
Porrino, 1985; Groenewegen et al., 1990), and lesions of the MD result in deficits in delay-
tasks comparable to those following lesions of the PFC (Sakurai and Sugimoto, 1985). It has
been suggested that in birds the projection from the multimodal nuc. dorsolateralis posterior
(DLP) to the NCL might form a functional equivalent to the MD-PFC connection (Güntürkün,
1997). In chapter 2 the nature of short-latency visual evoked potentials in the NCL is
examined by extracellular electrophysiological recordings in-vivo, and the effects of lesions to
the DLP on visual responses in the NCL are studied. Together with the anatomical data also
presented in chapter 2 (see above), the results demonstrate direct thalamic modulation of NCL
activity.
To understand the neuronal circuitry that underlies higher cognitive functions in vertebrates, a
detailed knowledge about the celltypes and their possible function within the proposed
canonical circuit is required. It is axiomatic in neuroscience that the informational output of
most neurons is defined by their temporal patterns of action potentials (e.g. McCormick et al.,
1985). This notion is implicit in all contemporary models of the function of the brain, making
it essential to understand how each neuron transforms its input into output. Intracellular or
patch-clamp electrophysiological recordings give the opportunity to determine the intrinsic
electrophysiological properties of neurons by measuring the membrane´s response to
hyperpolarizing and depolarizing current pulses. The intrinsic membrane properties of
Introduction 21
neurons in the mammalian and avian forebrain are not homogeneous, but instead produce
several categories of transform characteristics that can be used to categorize cells into certain
types (e.g. McCormick et al., 1985; Larkman and Mason, 1990; Kubota and Taniguchi, 1998).
In chapter 3 the principal cell types of the chick NCL are characterized both
electrophysiologically and morphologically in vitro, using whole-cell patch-clamp recordings
in combination with intracellular staining. The distinct firing patterns and axonal connections
of the principal neurons can be interpreted with respect to the integrative role they play within
the circuitry of the NCL, thus unraveling the „building blocks“ of mental computation within
NCL.
The influence of the dopaminergic system on identified cell types within the NCL.
The dopaminergic innervation in the avian telencephalon takes two different, and possibly
discrete, forms. The first, which is also predominant in the mammalian cortex (e.g. Williams
and Goldman-Rakic, 1993), is characterized by dopaminergic fibers that travel in close
vicinity along the somata and dendrites of target-neurons while forming „en-passant“' a large
number of bouton-like axonal swellings. The second one, called the „basket“'-type, is a
peculiar arrangement of dopaminergic fibers in the avian telencephalon. In this case, single
fibers densely coil around the somata and intial dendrites of postsynaptic targets, enwrapping
them in basket-like structures (Wynne and Güntürkün, 1995; c.f. chapter 4). It can be
speculated that this particularly strong dopaminergic innervation also differs functionally from
the more diffuse „en-passant“ innervation. The relative frequency of these modes of
innervation differs among various brain areas (Wynne and Güntürkün, 1995; chapter 4), but
the NCL is characterized by a strong innervation from both types of dopaminergic fiber
systems, thus in principle enabling a direct comparison of the two modes of DA innervation.
Chapter 4 provides a review about the dopaminergic innervation of the avian forebrain and
discusses anatomical and behavioural findings with regard to the functional organization of
the avian brain, and some general implications of these findings for sensorimotor integration
and learning. Chapter 5 presents original data also reviewed in chapter 4. This study
attempted to identify the cell type that receives dense basket-type DA innervation by
colocalizing the dopaminergic fibers4 with postsynaptic markers of neuroactive substances
4 In fact, immunoreactivity against tyrosine-hydroxylase (TH), which is the rate limiting enzyme in the synthesisof all catecholamines, is used for the demonstration of presumed dopaminergic fibers, as there is a goodcorrespondence between TH immunoreactivity and a direct marker of DA (c.f. chapter 4), comparable to thesituation in the prefrontal cortex (Williams and Goldman-Rakic, 1993).
Introduction 22
(i.e. glutamate-decarboxylase, as a marker of GABAergic neurons) or receptor proteins (i.e.
DARPP-32 as an indicator for D1 receptors). Specifically it was investigated whether
GABAergic interneurons are a main target of dopaminergic fibers, and if so, whether the
effects of DA might be mediated via the DA D1 receptor as is the case in the PFC (Krimer et
al., 1997; Muly et al., 1998).
Finally, the effects of DA on membrane excitability are tested directly in chick NCL neurons
in-vitro. In chapter 6, patch-clamp recordings from neurons of all cell types are made (c.f.
chapter 3) and changes in the firing patterns in response to DA application are analyzed. The
observed changes in action-potential firing provide insight into the mechanisms and substrate-
specifity (i.e. cell types and receptors involved) by which DA might enable sustained activity
during a delay task. Furthermore, double-labeling of intracellularly filled neurons with
dopaminergic fibers is used to identify classes of neuron that receive basket-type DA
innervation. The responses of neurons receiving basket-innervation to application of DA are
compared to the responses of neurons that receive only en-passant innervation of
dopaminergic fibres. Taken together, this will elucidate whether the basket-type innervation
selctively innervates a distint class of neurons, and/or whether the effects of DA excerted
through this strong somatic innervation differs qualitatively or quantitatively from the
dendritic en-passant innervation.
Taken together, the results show the structural and physiological bases of polymodal sensory
integration and motor control in the avian telencephalon, which appear to underlie higher
cognitive functions. Furthermore, the functional organization of the mesotelencephalic
dopaminergic system and dopaminoceptive elements in the caudal forebrain are studied,
which appear to be critically involved in cognitive processing and motor control, both in birds
and mammals.
Chapter 1: Connections of the avian neostriatum caudolaterale 2323
Afferent and efferent connections of the caudolateral neostriatum in the
pigeon (Columba livia): A retro- and anterograde pathway tracing study.
Sven Kröner and Onur Güntürkün
ABSTRACT
The avian caudolateral neostriatum (NCL) was first identified on the basis of its dense dopaminergic
innervation. This fact and data from lesion studies have led to the notion that NCL might be the avian equivalent
of prefrontal cortex (PFC). A key feature of the PFC is the ability to integrate information from all modalities
needed for the generation of motor plans. By using antero- and retrograde pathway tracing techniques we
investigated the organization of sensory afferents to the NCL and the connections with limbic and
somatomotor centers in the basal ganglia and archistriatum. Data from all tracing experiments were compared
with the distribution of tyrosine-hydroxylase (TH) immunoreactive fibers, serving as a marker of dopaminergic
innervation. The results show that NCL is reciprocally connected with the secondary sensory areas of all
modalities and with at least two parasensory areas. Retrograde tracing also demonstrated further afferents from
the deep layers of the Wulst and from the frontolateral neostriatum as well as the sources of thalamic input.
Efferents of NCL project onto parts of the avian basal ganglia considered to serve somatomotor or limbic
functions. Projections to the archistriatum are mainly directed to the somatomotor part of the intermediate
archistriatum. In addition, cells in caudal NCL were found to be connected with the ventral and posterior
archistriatum, which are considered avian equivalents of mammalian amygdala. All afferents and projection
neurons were confined to the plexus of densest TH innervation. Our results show that the NCL is positioned to
amalgamate information from all modalities and to exert control over limbic and somatomotor areas. This
organization might comprise the neural basis for such complex behaviours as working memory or spatial
orientation.
Journal of Comparative Neurology 407:228-260 (1999)
INTRODUCTION
The neostriatum caudolaterale (NCL) of the pigeon has been compared with the mammalian
prefrontal cortex (PFC), due to its dense dopaminergic innervation (Divac et al., 1985, 1994;
Divac and Mogensen, 1985; Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995),
and the behavioural deficits in working memory (Mogensen and Divac, 1982, 1993, Gagliardo
et al., 1996, 1997, Güntürkün, 1997), reversal learning (Hartmann and Güntürkün, 1998), and
Chapter 1: Connections of the avian neostriatum caudolaterale 2424
go/no-go tasks (Güntürkün, 1997) that follow its ablation. Working memory tasks selectively
target the control of reactions depending on past experiences and the ongoing stream of sensory
information that is currently being experienced, which in mammals is a key feature of the PFC
(Goldman-Rakic, 1987; Fuster, 1989). A clear prerequisite for a structure in non-mammalian
species that can subserve such functions and may thus be regarded as an analogue of the PFC is
the ability to integrate all available sensory information and to exert influence over motor and
limbic structures.
A number of previous studies have demonstrated afferent input to the lateral neostriatum from
visual (Ritchie, 1979; Shimizu et al., 1995;), auditory (Wild et al., 1993) and somatosensory
(Shimizu et al., 1995) areas, as well as a projection from the multimodal thalamic nucleus
dorsolateralis posterior (DLP); (Waldmann and Güntürkün, 1993; Leutgeb et al., 1996). Based
on cytoarchitectonic and hodological data, Rehkämper and Zilles (1991) have proposed that the
complete posterior neostriatum, including its caudolateral aspect, might represent an area of
multimodal integration. Furthermore, in a recent retrograde labeling study focused on the NCL
(Leutgeb et al, 1996) it has been shown that the NCL is reached by afferents from all major
secondary sensory areas within the pigeon brain. Until now, however, a detailed study of the
termination pattern of these afferents and a possible functional segregation within the pigeon´s
NCL is still lacking. Retrograde tracing data from a very recent study in chicks (Metzger et al.,
1998) revealed distinct and non-overlapping termination zones for the trigeminal, tectofugal,
and auditory systems within the NCL. Since retrograde tracing in pigeons (Leutgeb et al., 1996)
suggested a substantially different organization of the NCL with largely overlapping sensory
compartments, this report describes the afferent and efferent connections of the NCL as studied
by retro- and anterograde pathway tracing and compares it with the distribution of tyrosine
hydroxylase-immunoreactive fibers.
MATERIALS AND METHODS
Neuroanatomical pathway tracing experiments
Fifty-seven adult pigeons (Columba livia) from local stock provided the data presented here. Treatment of
animals conformed to NIH guidelines and specifications of the German Animal Welfare Act. Accordingly,
prior to surgery, the animals were deeply anaesthetized with 0.33 - 0.4 ml Equithesin per 100 g body weight.
Animals received pressure-injections of either the sensitive antero- and retrograde tracer Cholera Toxin,
subunit b (CTb, 1% in A.dest.; List Labs, Campbell, CA) or biotynilated dextran amines as anterograde tracer
(BDA, 10,000 molecular weight form, 10% in sodium phosphate buffer, pH 7.3; Molecular Probes, Leiden,
The Netherlands). Tracer was delivered through glass micropipettes (tip diameter 15-20 µm) attached to a
Chapter 1: Connections of the avian neostriatum caudolaterale 2525
nanoliter-injector (World Precision Instruments, Sarasota, FL). Since previous studies (Leutgeb et al., 1996;
Metzger et al., 1998) and our own data have indicated that the majority of connections of the NCL are
restricted to the ipsilateral hemisphere, most animals received bilateral injections to minimize the number of
pigeons used. Stereotaxic coordinates for the injections were determined by using the atlas of Karten and
Hodos (1967).
TABLE 1: Experimental Parameters1
Tracer
BDA CTbInjection site Number of
animalsNumber of
cases L R L R
NCL 7 11 − − 6 5Nd 3 4 − − 2 2field L 3 6 1 − 2 3Ep 6 10 − 1 5 4NFT 3 5 − − 2 3rostral HA 3 5 1 − 2 2caudal HA 4 6 1 1 2 2NIMm 4 6 1 − 3 2NIMl 6 10 − − 5 5Ai and Av 6 10 − 3 2 5Ap 3 5 − 1 2 2LPO 5 8 − − 4 4PA 4 6 − 1 3 2
1 R, Right side; L, Left side; for other abbreviations, see list
The afferent sources of the NCL were determined by injections of CTb (30 - 54 nl) into medial and lateral
parts of the NCL along its rostro-caudal extent, as well as into the directly adjacent neostriatum dorsale (Nd,
Bonke et al., 1979; Wild et al., 1993; Wild, 1994). We included Nd in our analysis because the pattern of
catecholaminergic innervation suggests that Nd is continuous with NCL (figure 1) and might constitute its
auditory subcomponent. To study the areal pattern of afferent input to the NCL, injections of CTb (9 - 80 nl)
and BDA (40 - 100 nl) were made into most of the telencephalic regions which by means of retrograde
transport had been shown to constitute afferent sources of the NCL. Efferent connections of the NCL to motor
and limbic structures were studied by placing injections into the somatomotor and limbic parts of the
archistriatum (Zeier and Karten, 1971) as well as into parts of the avian basal ganglia that are comparable to
parts of mammalian striatum.
Immunohistochemical procedures
Survival times were 3 days for CTb and 4-6 days for BDA. Fifteen minutes prior to perfusion the animals were
injected with 1,000 IU heparin and deeply anaesthetized with 0.4 - 0.5 ml Equithesin per 100 g body weight.
Pigeons were then perfused transcardially with 200 ml 0.9% saline (40°C) followed by 1,000 ml of 4%
paraformaldehyde in 0.12 M phosphate buffer (PB; 4°C, pH 7.4). After perfusion, brains were dissected and
postfixed in the same fixative to which 30% sucrose was added, and then transferred to 30% sucrose in
Chapter 1: Connections of the avian neostriatum caudolaterale 2626
phosphate buffer containing 0.9% NaCl (PBS; pH 7.4) for approximately 18 hours at 4°C. Brains were cut in
frontal slices of 40 µm on a freezing microtome and collected in PBS containing 0.01% NaN3 as a
preservative. Representative sections were then processed for the avidin-biotin-conjugate technique (ABC).
Immunohistochemical labeling for Cholera Toxin b: Endogeneous peroxidases were blocked by
preincubating slices in a solution of 0.5% H2O2. Slices were washed and for immunohistochemistry of CTb
free-floating sections were incubated overnight at 4°C in anti-CTb from goat (Jackson, West Grove, PA;
1:20,000) in PBS containing 0.3% Triton X-100 (Sigma, Deisenhofen, Germany). The following steps were
carried out at room temperature, separated by three washes in PBS of 10 minutes each. After washing, slices
were first incubated for 1 hour in biotinylated donkey anti-goat (Jackson,1:500 in 0.3% Triton X-100 PBS) for
1 hour and then in the avidin-biotin complex (ABC Elite, Vector Labs, Burlingame, CA; 1:100 in PBS with
0.3% Triton X-100) for 1 hour. Washes in PBS were followed by two additional washes in 0.12 M acetate
buffer (pH 6). Staining was achieved by the 3,3'-diaminobenzidine (DAB) technique with heavy-metal
amplification (modified from Adams, 1981) by adding H8N2NiO8S2 (2.5 g/ 100 ml), NH4Cl and CoCl2 (both
40 mg/ 100 ml). After 15 minutes of preincubation the reaction was catalyzed with a solution of 0.5% H2O2.
The reaction was stopped by rinsing the tissue in 0.12 M acetate buffer and PBS. Slices were then mounted,
dehydrated and coverslipped.
Labeling for BDA: Visualization of BDA was identical to that of CTb, except that primary and secondary
antibodies could be omitted and slices were directly incubated in ABC, followed by the staining procedure
described above. Selected series labeled for BDA or CTb were counterstained with cresyl violet.
Immunohistochemical labeling for tyrosine hydroxylase: To compare the distribution of afferent fibers and
retrogradely labeled neurons within NCL with the distribution of putative dopaminergic fibers, several sections
were processed for tyrosine hydroxylase (TH). Therefore, additional sections stained for CTb or BDA were
either double-labeled with TH, or occasionally, sections adjacent to these were single-labeled for TH. The
same basic procedure as for immunohistochemistry of CTb was applied for labeling of TH: After blocking of
endogeneous peroxidases and thorough washing, free floating sections were incubated overnight at 4°C in
monoclonal mouse anti-TH (Boehringer, Mannheim, Germany) diluted 1:200 in PBS containing 0.3 %Triton
X. Slices were then incubated at room temperature in biotinylated rabbit anti-mouse (Chemicon, Temecula,
CA; 1:200 in 0.3% Triton PBS) for 1 hour and finally in ABC for 1 hour. Staining was achieved using the DAB-
technique as described for CTb. In those cases that were double-labeled, enhancement of the DAB staining by
nickel ammonium sulfate was ommitted, resulting in a light brown signal for TH which could be clearly
distinguished from the black reaction product of the tracers.
RESULTS
Retrograde tracing of afferent sources of the NCL
Our injections of CTb into medial and lateral parts of NCL generally replicated the results of an
earlier study (Leutgeb et al., 1996). All injections were confined to NCL as defined by TH and
DA-like immunoreactivity (Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995;
Chapter 1: Connections of the avian neostriatum caudolaterale 2727
present study), and extended from the caudal tip of the telencephalon at approximately A 4.25 to
rostral A 7.00.
Telencephalic afferents to NCL
Areas that were found to project to the NCL included the medial aspect of the hyperstriatum
accessorium (HA) throughout its visual (Karten et al., 1973; Shimizu et al., 1995) and
somatosensory part (Delius and Benetto, 1972, Wild, 1987b, Funke, 1989; but see Deng and
Wang, 1992, 1993), the ectostriatal belt (Ep) surrounding the ectostriatum dorsally and laterally
(Karten and Hodos, 1970) and the adjoining lateral neostriatum, the anterior neostriatum
overlying the nucleus basalis (neostriatum fronto-trigeminale, NFT, of Wild et al., 1985), and
field L1 and L3 of the auditory field L complex. A large number of cells were labeled in the
medial part of the intermediate neostriatum (NIM of Veenman et al., 1995b) and the overlying
intermediate and medial parts of the hyperstriatum ventrale (HV); part of these neurons
probably correspond to the previously described parasensory area in the intermediate
NCL
ApH
AvAi
AiddAidv
NCm
NCL NCL
Ap
NCm
ApH
Av
NCL
Aidd
Ai
TPO
BNST
ApH
PPPA
Hv
Tn
NCL
ApH
Aidd
PA
Hv
AvAi
Tn
A 5.75A 4.25 A 5.00
A 6.75 A 7.25
Figure 1: Camera lucida drawing of tyrosine hydroxylase (TH) immunoreactive fibers in the caudal telencephalon of the pigeon.Staining for TH produces a pattern very similar to that seen with an antibody against dopamine which has been previously usedto define the neostriatum caudolaterale (NCL); (Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995; Metzger et al.,1996). For abbreviations see list.
Chapter 1: Connections of the avian neostriatum caudolaterale 2828
neostriatum, situated between the rostral pole of field L and the caudomedial border of the
visual ectostriatum, that receives multimodal input via the DLP (Gamlin and Cohen, 1986;
Wild, 1987a, 1994; Funke, 1989; Korzeniewska and Güntürkün, 1990). These cells were
predominantly labeled if injections were made into more rostral and medial (including Nd)
parts of the NCL.
After injections into caudal NCL, a distinct cell group occupying the remaining medialmost
aspect of the NIM between approximately A 8.50 and A 12.50 was labeled. Cells within this
area also extended across the lamina hyperstriatica (LH) into the intermediate and medial parts
of the HV. These two cell groups roughly followed the cytoarchitectonic borders of the regions
Ne 9 or Ne 8 and Ne 3, respectively, as described by Rehkämper et al., (1985). For the reason
of simplicity we will refer to them as the medial part of the NIM (NIMm) and the lateral part of
the NIM (NIMl), respectively.
In the caudomedial neostriatum (NCm) scattered cells were found dorsal to the lamina
medullaris dorsalis (LMD). Another band of cells stretched along the border between the
hyperstriatum dorsale (HD) and the dorsal division of the ventral hyperstriatum (HVdv). These
cells could be found in most cases, irrespective of the injection site within NCL. In cases that
were centered more medially or caudally within NCL this band of cells also extended into a
small triangular shaped zone within the vallecula. Finally, in several cases a number of cells
were observed in the frontolateral part of the neostriatum (NFL). Injections of CTb into this
area did indeed confirm a reciprocal connection with the ventrolateral part of the NCL (data not
shown). The large majority of fibers from this part of the anterior neostriatum, however,
terminated within the area corticoidea dorsolateralis (CDL) and the area temporo-parieto-
occcipitalis (TPO) overlying the NCL. Therefore we can not fully exclude that part of the
labeling within NFL was attributable to tracer spread into CDL.
In addition to labeling in sensory areas, retrograde transport revealed a number of labelled
neurons in the dorsal and ventral parts of the intermediate archistriatum. Cells in the ventral
archistriatum were the only telencephalic connection found to project bilaterally onto the NCL.
In contrast to Leutgeb et al., (1996) we never observed any labeling within the nucleus taeniae
(Tn). Furthermore, a few cells within the ventral striatum just dorsal to the fasciculus
prosencephali lateralis (FPL) were also found to project to the NCL.
Chapter 1: Connections of the avian neostriatum caudolaterale 2929
HV
PA
NCL
NCm
Aidd
Ai
AvTn
Field L
APH
Hp
NCL
TPc
A 6.75
A 3.00
HV
Aidd
Ai
Av
APH
HpCDL
NCL
CDL
Nd
A 5.75
A 4.25AVT
rostral NCLcaudal NCL Nd
NCm
TnDLP
DIP
SRt
Ov
HpAPH
Figure 2: Schematic representation of retro- and anterograde labeling following injections of Cholera Toxin b (CTb) into variouslocations within NCL. Retrogradely labeled cells are represented by filled symbols (dots, triangles or squares for the differentinjection sites) and anterogradely labeled fibers and terminals are represented by short lines. Anterograde labeling is shown onlyfor selected regions. The left and middle column illustrate the topographic ordering of NCL afferents and efferents afterinjections into the caudalmost NCL (left column) or the far rostral NCL (middle column), respectively. The right column showsan example of an injection into Nd. Labeling after Nd injections was very similar to that observed after tracer deposits intomedial NCL. In this and all following figures medial is to the left and dorsal is at the top.
Chapter 1: Connections of the avian neostriatum caudolaterale 3030
Figure 2 (continued)
HA
NFT
NFL
HVvv
HD
A 12.50
A 14.00
A 10.50
A 8.25
HA
Bas
NFT
NFL
LPO
Va
HV
HD
HA
NILE
Ep
NIMm
NIMl
HV
Va
HD
LPOPAPP
E
Ep
HA
TPO
NIMlNIMm
HV
PAPP
FPL
AC
rostral NCLcaudal NCL Nd
Chapter 1: Connections of the avian neostriatum caudolaterale 3131
In addition to labeling in sensory areas, retrograde transport revealed a number of labelled
neurons in the dorsal and ventral parts of the intermediate archistriatum. Cells in the ventral
archistriatum were the only telencephalic connection found to project bilaterally onto the NCL.
In contrast to Leutgeb et al., (1996) we never observed any labeling within the nucleus taeniae
(Tn). Furthermore, a few cells within the ventral striatum just dorsal to the fasciculus
prosencephali lateralis (FPL) were also found to project to the NCL.
In general, injections into NCL resulted in a similar pattern of retrograde labeling, but regional
differences exist. With respect to a mediolateral topography our results resemble those of
Leutgeb and coworkers (1996) and are thus not described in detail here. However, we found a
strong rostro-caudal topography in the organization of afferent and efferent connections of the
NCL. The nature of this topography was further elucidated in anterograde tracing experiments.
Figure 2 illustrates the results after two injections into the far caudal and rostral aspects of
NCL.
Subtelencephalic sources of afferent input to NCL were observed in the thalamus, midbrain and
tegmentum. In the lower brainstem the avian equivalent of the substantia nigra, the nucleus
tegmenti pedunculopontinus, pars compacta (TPc), the locus coeruleus (LoC) and the area
ventralis tegmentalis (AVT) were labeled. These cell groups have been shown to be
immunoreactive for TH and dopamine and contain the sources of dopaminergic afferents to the
telencephalon (Waldmann and Güntürkün, 1993; Metzger et al., 1996; present study). The
projection from the LoC and some cells in the formatio reticularis was bilaterally organized. In
the thalamus retrogradely labeled cells were mainly found in the nucleus subrotundus (SRt) and
the DLP. Yet, we also found a projection from the n. dorsointermedius posterior thalami (DIP)
and a band of cells that seemed to intersperse between DLP and DIP and also extended ventral
to the latter two. Therefore we termed these cells collectively the ventrointermediate area of the
posterior nuclei (VIP). Figure 3 shows examples of retrogradely labeled neurons from sensory
forebrain areas (A - F) and the sources of thalamic input to NCL following injections of CTb
into NCL.
Anterograde labeling of efferents of the NCL
The pattern of anterograde labeling resulting from injections into NCL was studied within the
basal ganglia and the archistriatum. Fibers showing terminal-like varicosities were found
throughout most parts of the archistriatum. For the description of archistriatal subdivisions we
adopted the terminology of Wynne and Güntürkün (1995).
Chapter 1: Connections of the avian neostriatum caudolaterale 3232
Figure 3: Photomicrographs of retrogradely labeled cells resulting from injections of CTb into the NCL similar to those shown infigure 2. Afferents arise from secondary sensory „belt“ regions adjacent to primary thalamorecipient areas. See text for details.Retrogradely labeled cells in the telencephalon include the ectostriatal belt (A), field L 1 (B), HA and HD (C), the borderbetween HD and HVdv (D), the presumed parasensory area NIMm (E) and the NFT (F). Labeling in the thalamus was mostabundant in the DLP and SRt (G). H: Cells in the ventrointermediate area of the posterior nuclei (VIP) labeled after an injectioninto far lateral NCL. Scale bars are 100 µm (B, H) and 250 µm (A, C - G). See list for abbreviations.
Chapter 1: Connections of the avian neostriatum caudolaterale 3333
Injections into the very caudal aspect of NCL predominantly labeled fibers in parts of the
central (Ai) and ventral parts (Av) of the intermediate archistriatum and in some cases also in
the posterior archistriatum (Ap). Injections into more rostral NCL resulted in terminal labeling
that was confined to the dorsal and central parts of the intermediate archistriatum. After
injections into the caudal NCL we observed terminal-like labeling in the medial lobus
parolfactorius (LPO) and the ventral striatum, i.e. the nucleus accumbens (AC) and the bed
nucleus of the stria terminalis (BNST), which was not attributable to tracer spread into the
piriform cortex (CPi) or the caudal archistriatum. Injections into rostral NCL on the other hand
resulted in dense terminal labeling in the lateral aspects of LPO and large parts of the
paleostriatum augmentatum (PA). Labeling in the PA was most abundant in its caudal extent.
Both termination patterns within the archistriatum and the basal ganglia thus suggest that the
anterior NCL may be connected to areas concerned with somatomotor functions, i.e. the dorsal
and lateral striatum (Veenman et al., 1995a,b), as well as the dorsal and central archistriatum
(Zeier and Karten, 1971), while the caudal aspect of NCL might project to limbic parts of the
striatum (Veenman et al., 1995b) and the presumed avian homologue of the amygdala, e.g. the
ventral and caudal archistriatum (Zeier and Karten, 1971, Dubbeldam et al., 1997). Figure 4
shows examples of anterograde labeling in the basal ganglia and the archistriatum following
injections of CTb into NCL.
Labeling after Nd injections
Labeling after CTb injections into Nd largely paralled the results after medial NCL injections.
A large number of retrogradely labeled cell bodies and fibers could be observed throughout the
NIM, which seemed to cluster in two distinct cell groups as also observed after injections into
the NCL. Strong labeling was also present in the caudomedial neostriatum and areas L1 and L3
of the field L complex. Furthermore, cells were found in the medial HV and along the border of
HV and HD, the caudal and rostral aspects of HA, the valleculla (Va), and the NFL. Finally,
somata and terminal-like labeling could be observed in the ventromedial archistriatum and
lateral parts of the NCL. Terminal-like labeling in the basal ganglia after Nd injections was
restricted to the medial and central parts of the caudal PA. In the thalamus labeling was most
prominent in the SRt and DLP. A number of cells was also found within the shell of the nucleus
ovoidalis (Ov) and the nucleus semilunaris parovoidalis. In the midbrain AVT, TPc and LoC
contained labeled cell bodies. The results following an injection into Nd are summarized
schematically in figure 2 (right column).
Chapter 1: Connections of the avian neostriatum caudolaterale 3434
Figure 4: Photomicrographs of anterograde labeling of NCL efferents to the basal ganglia and archistriatum. A: Labeled fibers inthe presumed „limbic“ parts of the avian striatum (i.e., nucleus accumbens and medial LPO) after an injection of CTb into farcaudal NCL. B and C: Labeling in the presumed „somatomotor“ parts of the basal ganglia, the lateral LPO (B) and central PA(C). D and E: Labeled fibers and somata in somatomotor and limbic parts of the archistriatum. Injections into the most caudalNCL label numerous fibers in the central part of the intermediate archistriatum, but also extend into the ventral archistriatumwhich forms part of the avian amygdala (D). In contrast, injections into the rostral NCL lead to labeling in the dorsal and centralparts of the intermediate archistriatum which are considered somatomotoric in function. Cells from most parts of thearchistriatum project back to the NCL. The projection from Av is the only bilaterally organized connection of the NCL. Scalebars are 250 µm (A), 50 µm (B), 100 µm (C), and 500 µm (D, E).
Chapter 1: Connections of the avian neostriatum caudolaterale 3535
Anterograde tracing experiments
Injections into the ectostriatal belt
After injections into the dorsal and lateral ectostriatal belt and the adjoining neostriatum
intermedium (NI) underneath the external pallium (Veenman et al., 1995b) a massive projection
of labeled fibers was observed to move caudally through the NI and to innervate the anterior
and lateral NCL. This projection seemed to be topographically organized and extended in the
case of caudal injections posteriorly to about A 5.00. In addition to terminals, a number of
retrogradely labeled cell bodies also were found, indicating a reciprocal connection between
the NCL and the ectostriatal belt region. As shown in figure 5 a massive reciprocal connection
of the Ep exists with the deeper hyperstriatal layers, especially with the lateral part of HV.
Cells within HV and the NI or NFL, respectively, were distributed up to far rostrally within the
hemisphere. Relatively few cells and fibers were found within the outer rind of the pallium, i.e.
the lateral NI (NIL), TPO and CDL, the larger portion of them remaining underneath the border
that separates the NI and anterior NCL from the overlying external pallium. Furthermore, in the
dorsal and ventral aspects of the rostral archistriatum small clusters of cells were labeled, as
well as a number of somata in the paleostriatum primitivum (PP). Within the ectostriatal core
labeled cells were found in a narrow area below the injection site. In the thalamus large
numbers of neurons were labeled in nucleus rotundus which probably are due to tracer spread
into the ectostriatal core.
Injections into field L
In accordance with previous reports (Bonke, et al., 1979; Wild et al., 1993) tracer deposits into
the auditory field L complex which were mainly centered on the secondary sensory field L1
lead to massive terminal-like labeling in Nd. Yet, injections of CTb revealed a diffuse
projection that also extended further laterally in caudal NCL (figure 6). In the medioventral
aspect of NCL also a small number of retrogradely labeled cells could be observed. Field L
injections also resulted in retrograde labeling of neurons throughout the HA, NIM and HV, as
well as in ventral and intermediate archistriatum. Diencephalic neurons were labeled within
shell and core regions of n. ovoidalis and the n. semilunaris parovoidalis.
Chapter 1: Connections of the avian neostriatum caudolaterale 3636
LPO
PA
PP
NILNIM
HA
HD
HV
E
A 9.75
NCL
APH
PA
Tn Av
Hp
AiddAi
CDL
NCm
RtA 7.50
Hp
TPO
CDL
PA
PP
Av
Ai
Aidd
NCm
A 6.50
NCL
APH
PA
TnAv
Hp
Aidd
Ai
CDL
Rt
NCm
A 6.00
TnAv
Aidd
Ai
Aidv
SRt Rt
NCm
NCL
CDLHp
APHA 7.00
NCL
APH
Ap
TO
NCm
Nd
A 6.50
A 5.00
Rt
Ov
NCL
NCm
CDL
Hp
APH
Nd
SRt
Av
Aidd
AiAidv
Am
A 5.50
HV
HV
1 mm
A
Figure 5: Results of a 15 nl CTb injection into the ectostriatal belt region, which receives afferents from the ectostriatum, theprimary visual forebrain area of the tectofugal pathway. A: Schematic representation of labeling witin the caudal telencephalon.B: Photomicrograph showing the innervation of the anterior NCL and retrogradely labeled cell bodies at about A 7.00. Scale baris 200 µm.
Chapter 1: Connections of the avian neostriatum caudolaterale 3737
Injections into the hyperstriatum accessorium
After injections into the Wulst that were centered on the medial aspect of the dorsal
hyperstriatum accessorium, fibers were found to travel through the lamina frontalis superior
(LFS) and further laterally along the border that separates the anterior NCL from the overlying
LPO and CDL. Fibers showing terminal-like varicosities remained in close apposition to the
ventricle as they moved further caudally through NCL. Neurons that project back to the injected
site generally followed the distribution of terminating fibers but extended somewhat ventrally to
these. Injections along the complete rostro-caudal extent of the Wulst revealed a topographic
pattern of termination within NCL with more caudal injections leading to stronger labeling in
the posterior NCL, while afferents from most rostral HA were confined to more anterior
aspects of the NCL. This termination pattern may thus reflect the previously described
functional segregation of the Wulst into a caudal visual and a rostral somatosensory area. Yet,
the projections from HA onto NCL were not restricted to separate termination fields but
showed considerable overlap (compare figures 7 and 8).
Injections into all parts of the HA evinced retrogradely labeled cells in the HD, frontolateral
neostriatum, field L1, external pallium (CDL and TPO), a caudomedial portion of the area
parahippocampalis (APH), and the ventral and central archistriatum. Injections into rostral HA
additionally labeled cells in the NIM. In several cases in which tracer spread occured into the
thalamorecipient granular layer of the intercalated nucleus of the HA (IHA), retrogradely
labeled cells were observed in the thalamic relay stations of the thalamofugal visual and
somatosensory system, respectively: deposits of tracer into the caudal Wulst evinced cells in
the dorsal aspect of the lateral geniculate nuclei (GLd; Karten et al., 1973; Güntürkün et al.,
1993). Injections into the rostral HA on the other hand predominatly labeled cells in the
somatosensory nucleus dorsalis intermedius ventralis anterior (DIVA; Wild, 1987b; Funke,
1989; Medina et al., 1997).
Injections into the neostriatum frontotrigeminale
After injections into the NFT dorsal to the nucleus basalis (Bas), fibers gathered in the tractus
fronto-archistriatalis and passed caudally until they terminated within the lateral part of the
anterior archistriatum and the rostral and ventral part of NCL. As can be seen in figure 9 the
terminal field of this projection occupied a large part of the anterior and lateral NCL and
extended caudally up to about A 5.00. Within this dense terminal field a comparatively small
number of neurons were found to project back to the injection site. In those cases that showed
Chapter 1: Connections of the avian neostriatum caudolaterale 3838
A 6.50Tn
Av
Aidd
Ai
Aidv
SRtRt
NCm
NCL
CDLHp
APH
LH
Ov
Av
Aidd
Ai
Aidv
DLP AmDIP
Ov
RtSRt
NCL
NCm
CDLHp
APH
NdNCL
APH
PA
Tn Av
Hp
Aidd
Ai
CDL
Rt
HV
NCm
LH
L1
NCL
APH
Ap
TO
NCm
Nd
TO
NCL
APH
Nd
NCL
APH
TO
Nd
A 5.00 A 4.50 A 4.001 mm
L2
L3
A 6.00A 5.50
HV
A
Figure 6: Results of a 9.2 nl CTb injection centered onto the secondary sensory field L1 of the auditory field L complex withslight tracer spread into the HV. A: Schematic representation of labeling within Nd and the adjoining parts of NCL. B:Retrogradely labeled neurons in the nucleus ovoidalis, the main thalamic relay of auditory input to the telencephalon, resultingprobably from tracer spread into the primary field L2. C: Photomicrograph of labeled fibers in Nd and NCL. Scale bars are 500µm (B) and 250 µm (C).
Chapter 1: Connections of the avian neostriatum caudolaterale 3939
no or minimal tracer spread into Bas, retrogradely labeled fibers and cells within Bas were
restricted to the area directly ventral to the injection site. Furthermore, cells were labeled in the
HV dorsal to the injection site, the anterior archistriatum, along the tractus fronto-archistriatalis,
and in medial HA.
Injections into the neostriatum intermedium, pars medialis
Injections into the intermediate neostriatum attempted to evaluate whether two separate
projections from this area onto NCL exist as indicated by the results from our retrograde tracing
experiments reported above. Tracer deposits were thus aimed at the terminal field of the DLP
just medially and caudally to the ectostriatum, termed NIMl here, and the medialmost aspect of
NI, the NIMm, respectively.
Injections into NIMm consistently produced dense terminal labeling and a considerable number
of retrogradely labeled cell bodies in the most caudal aspect of NCL. Fiber bundles left the
injection site laterally and travelled caudally within the LH. They reached the caudal
neostriatum at the rostral end of Nd where they made massive terminations. The remainder of
labeled fibers coursed laterally and caudally within the periventricular roof of NCL. On their
way towards the caudal pole of the hemisphere they gradually fanned out towards the midline
until they occupied a large proportion of the hemisphere (figure 10).
Small injections that were entirely confined to NIMl resulted in anterograde labeling that was
largely restricted to Nd and the dorsal roof of the medial NCL. Labeling in these cases was
virtually identical to that seen after field L injections (compare figure 6). However, labeling
within the NCL varied considerably with the amount of tracer used. Larger NIMl injections that
also extended into one of the adjacent areas HV, NIMm or Ep, led to a much higher number of
labeled fibers and somata within NCL, while at the same time the terminal field also expanded
further lateral and medial (figure 11). However, the distribution of these fibers did not
resemble the terminal fields resulting from injections into NIMm or Ep. In addition, all of these
cases showed a similar labeling in the thalamus that was distinct from the pattern observed after
NIMm injections (below).
Deposits of CTb into the medialmost NI yielded numerous neurons within the medial HV,
dorsal to the injection site, HA, HD, in Ai and Av. Retrogradely labeled cells from Av were
also found on the contralateral side. Massive anterograde labeling was seen in medial LPO.
Injections into lateral NIM resulted in a similar pattern of labeling which included the
Chapter 1: Connections of the avian neostriatum caudolaterale 4040
somatosensory and visual Wulst, medial HV dorsal to the injection site, as well as Av and Ai.
In addition, cells were labeled in field L1 and L3, as well as in large aspects of the
caudomedial neostriatum. In these cases labeled fibers were seen in the PA ventral to the
injection site, but not within LPO. Labeling in the thalamus further strengthened the notion of
TnAv
AiddAi
Aidv
SRt
Hv
Rt
NCm
NCL
CDLHp
APH
DLL
Av
Aidd
Ai
Aidv
DLP Am
OvRt
SRt
NCL
NCm
CDLHp
APH
Nd
A 5.50A 6.00
NCL
APH
TO
NdNCL
APH
TO
Nd
1 mm A 4.50 A 4.00
NIL
LPO
PAPP
NI
NIM
HA
Hd
HVdv
HV
A 11.00
E
NCL
APH
PA
Tn Av
Hp
Aidd
Ai
CDL
Rt
Hv
NCm
DLL
L1
NCL
APH
Ap
TO
NCm
Nd
A 6.50
A 5.00
A
Figure 7: Labeling observed after a 40 nl injection of CTb into the caudal HA. A: Schematic representation of labeled fibers andcell bodies in the dorsal NCL. Injections of BDA revealed a similar amount of anterogradely labeled fibers but only very fewretrogradely labeled cells. B: Photomicrograph showing retrogradely labeled cells in the nuclei of the dorsolateral geniculatecomplex, as could be observed in some cases in which the injection site involved the thalamorecipient intercalated nucleus of theHA. Scale bar is 200 µm.
Chapter 1: Connections of the avian neostriatum caudolaterale 4141
NCL
APH
TSM
L 1
PA
TnAv
Hp
Aidd
Ai
CDL
NCm
1 mm
TPO
L 1
CDL
PA
PP
Av
Ai
Aidd
AL
Hp
NCm
A 14.00
NCL
APH
TSM
L 1
PA
TnAv
Hp
Aidd
AiDIVA
DLL
CDL
SPCDLM
VIA Rt
HV
NCm
Av
Aidd
Ai
Aidv
TSM
AL
AmT
Rt
NCL
NCm
DIVA
TnAv
Aidd
Ai
Aidv
DLL
SPC
DLM
TSM
AL
VIASRt
Hv
Rt
NCm
NCL
A 5.50A 6.00
NCL
APH
ApTSM
TO
NCm
A 5.00
A 7.00A 6.50
A 7.50
NFL
HIS
HD
HA
HVHV
A
Figure 8: Results following a 13.8 nl injection of CTb into the rostral HA. A: Schematic representation of labeled fibers and cellbodies in the NCL. The projection from rostral HA is largely restricted to the anterior NCL. B: Photomicrograph showingretrogradely labeled cells in the somatosensory thalamic n. dorsalis intermedius ventralis anterior resulting from tracer spreadinto the intercalated nucleus of the HA. Scale bar is 200 µm.
Chapter 1: Connections of the avian neostriatum caudolaterale 4242
two distinct subareas within NIM: injections into the NIMl confirmed the projection from DLP
(Kitt and Brauth, 1982; Gamlin and Cohen, 1986; Wild, 1994) and in addition labeled cells in
SRt, the shell of Ov and DIP. In contrast, after tracer deposits into the medialmost NI with slight
spread into the overlying HV, but not into PA or LPO, a large number of retrogradely cells
were found in the medial part of the dorsal thalamus, namely the n. dorsomedialis posterior
thalami (DMP) and DIP, and only very few within DLP.
Injections into the archistriatum
One of the most intriguing results of our retrograde labeling experiments was the extremely high
number of cells seen in the NCL that project to the archistriatum and the close match between
the distribution of these cells and the distribution of TH-immunoreactive fibers (figure 12).
After injections of CTb into the anterior two-thirds of the archistriatum that involved the central
and ventral parts of the intermediate archistriatum, cells along the whole rostro-caudal extent of
the NCL were labeled (figure 13). These cells clustered underneath the dorsal roof of the NCL
and gradually fanned out towards the medial neostriatum. This distribution thus follows closely
the original description of the NCL based on the distribution of dopaminergic and
catecholaminergic fibers (Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995;
Metzger et al., 1996; present study) and it thus seems that all parts of the NCL, including Nd,
project upon the archistriatum.
According to the injection site within the archistriatum this projection showed an anterior to
posterior gradient with more rostral archistriatal injections labeling relatively more cells in
anterior NCL. Yet, in general, the highest absolute number of cells was seen in the caudalmost
aspect of NCL where labeled cells extend far medially and ventrally. There might exist a
further parcellation with respect to dorsal and ventral subdivisions of the archistriatum as
suggested from the anterograde tracing experiments, yet with our injections which always
involved various subdivisions of the intermediate archistriatum we could not confirm this. A
different pattern was seen after injections into the posterior archistriatum which is the source of
the tractus occipitomesencephalicus, pars hypothalami (HOM) and which together with parts of
the ventromedial archistriatum has been suggested to constitute the avian homologue of the
amygdala (Zeier and Karten, 1971, Dubbeldam et al., 1997; Davies et al., 1997). In these cases,
a small numer of retrogradely labeled cells could be observed in the periventricular rim of the
NCL and Nd (figure 14). Their number increased towards the caudal tip of the hemisphere
supporting the notion that the most caudal NCL projects to the limbic/amygdaloid part of the
Chapter 1: Connections of the avian neostriatum caudolaterale 4343
1 mm
LPO
NFL
HA
HVvv
Bas
NFT
A 6.50
NCL
APH
PA
Tn Av
Hp
Aidd
Ai
CDL
Rt
HV
NCm
A 7.00Hp
NCL
APH
PA
Tn Av
Aidd
Ai
CDL
NCm
HV
A 7.50
TPO
CDL
PA
PP
Av
Ai
Aidd
NCm
A 6.00
TnAv
Aidd
Ai
Aidv
SRt
HV
Rt
NCmNCL
CDLHp
APH
A 5.50
Hp
Av
Aidd
AiAidv
DLP Am
RtSRt
NCL
NCm
CDL
APH
Nd
A 5.00
NCL
APH
Ap
TO
NCm
Nd
A 12.50
A
Figure 9: Results of a 13.8 nl CTb injection into the neostriatum frontotrigeminale overlying the n. basalis. B: Photomicrographshowing the dense anterograde labeling in the rostral and lateral aspects of NCL. C: Detail from B showing the border betweenNCL and the adjacent medial neostriatum. Scale bars are 500 µm (B) and 100µm (C).
Chapter 1: Connections of the avian neostriatum caudolaterale 4444
A 9.25
LPO
PAPP
NIL
NI
NIM
HAHd HVdv
HV
LH
TnAv
Aidd
AiAidv
SRt
Hv
Rt
NCm
NCL
DMA
Hp
LH
Av
Aidd
Ai
Aidv
DLPAmDIP
Ov
RtSRt
NCL
NCm
CDL
DMP
Hp
APH
Nd
NCL
TO
APHNd
APH
TO
NCL
NdNCL
APH
Ap
TO
NCm
Nd
A 5.001 mm
A 6.00
A 5.50
A 4.00A 4.50
DIP
DLP
DMP
AC
E
A
Figure 10: Pattern of labeling observed after a 11.5 nl injection into the medialmost NIM. As shown in A fibers travel from theinjection site in a compact bundle caudally within the lamina hyperstriatica until they reach the medialmost NCL from wherethey gradually fan out to diffusely innervate the caudalmost part of NCL. B, C: Photomicrographs showing the abundantdistribution of anterogradely labeled fibers and retrogradely labeled cells in the caudalmost NCL, following injections ofbiotinylated dextran amine (B) and CTb (C) into the medialmost NIM. D: Retrogradely labeled cells in the posterior thalamus,specifically in DMP. In B and C dorsal is to the right. Scale bars are 100 µm (B, C) and 300 µm (D).
Chapter 1: Connections of the avian neostriatum caudolaterale 4545
archistriatum. A very large number of cells, however, was labeled in the ventralmost
neostriatum surrounding the archistriatum dorsally and caudally.
After injections into the central and ventral archistriatum a large number of retrogradely labeled
cells was seen in the ventral hyperstriatum and the intermediate neostriatum. Other areas that
project to the central archistriatum include the caudomedial neostriatum, medial HA, rostral HD
and the vallecula, the field L complex as well as cells within NFT and NFL. Strong terminal-
like labeling was seen in LPO and medial PA as well as in HV. Descending fibers leave the
intermediate archistriatum through the tractus occipitomesencephalicus (OM), from which they
eventually split off to provide massive input to the deep layers of the optic tectum. Terminal-
like labeling was furthermore seen in the dorsal thalamus, predominantly in the posterior
thalamic nuclei DLP, DIP and SRt, all three of which also contained labeled cell bodies, as
well as in the nucleus spiriformis medialis (SPm) and the nucleus intercollicularis (ICo). At
midbrain level, terminal-like labeling and retrogradely labelled neurons could be observed in
TPc, AVT, and LoC. The OM further descended onto various nuclei in the brainstem (see Zeier
and Karten, 1971, Dubbeldam et al., 1997; Davies et al., 1997).
Injections into the posterior archistriatum labeled somata in the mediodorsal HV and along the
LFS. The Ap seemed to be reciprocally connected with cells in CDL and APH, as well as the n.
accumbens. Within the archistriatal complex, cells in the „limbic“ subcomponents, i.e. the
nucleus taeniae and Av, were labeled. The Ap was also found to project heavily onto the BNST
and tuberculum olfactorium. Descending fibers of the HOM terminated mainly within the lateral
hypothalamus (lHy). In the diencephalon cell bodies were observed in SRt and along the whole
track of the HOM. At the level of the midbrain cells could be found within TPc, AVT, and LoC.
Injections into the paleostriatum augmentatum
Injections into medial and central parts of the paleostriatum augmentatum between A 7.75 and A
9.00 labeled neurons along the complete rostrocaudal extent of NCL and Nd. As illustrated in
figure 15 these cells could be found throughout the whole depth of the NCL although their
number seemed to be highest within or just medially of the tractus archistriatalis dorsalis, at the
medial border of the TH-rich caudal neostriatum. A large number of retrogradely labeled cells
were seen in the dorsal part of the archistriatum. A smaller bilateral projection further-
Following page - Figure 11: Results of a comparatively large (23 nl) CTb injection into lateral NIM which also extended slightlyinto HV. See text for details. B: Photomicrograph of retrogradely labeled cells in the thalamus. C: Labeled fibers and cells in thedorsal NCL at about A 6.00. D: Detail showing the border of the densest innervation. Scale bars are 200 µm (B), 500 µm (C)and 50 µm (D).
Chapter 1: Connections of the avian neostriatum caudolaterale 4646
DIP
Ov
Rt
NCm
Hp
APH
Nd
Av
Aidd
AiAidv
Am
SRt
DLP
A 5.50
NCL
TO
NCL
APH
Nd
A 4.00
TnAv
AiddAidv
RtSRt
DLP
Av
Aidd
AiAidv
Am
NCm
Hv
CDLHp
APH
A 6.00
NCL
APH
Nd
TO
DLP
NCL
A 4.50
TPO
Hp
PA
PP
FPL
HA
Hv
E
NCL
APH
PA
Tn
Hp
AiddAi
CDL
Rt
Hv
NCm
Av
A 6.50APH
Ap
TO
NCm
Nd
DLPDIP
A 5.00
NCL
A
Figure 11
Chapter 1: Connections of the avian neostriatum caudolaterale 4747
more seemed to originate from portions of the anterior archistriatum. In addition, our injections
into PA labeled cells in HV, lateral HD, and the caudal pallium (e.g. NIL, TPO and CDL). A
few cells were also seen along the needletrack in NI, the caudomedial HV and the NCm. In the
thalamus cells were labeled within the lateral „somatic“ area of the dorsal thalamic zone,
namely DIP, VIP and to a lesser extent in DLP, SRt, n. suprarotundus (SpRt), and nuclei of the
ansa lenticularis. Sometimes marked neurons were also observed within the ventrointermediate
area of the thalamus (VIA) and n. rotundus (Rt) as well as n. triangularis (T). While labeling
within VIA was most likely due to tracer spread into the dorsal pallidum, i.e. PP (Medina and
Reiner, 1997), labeling in Rt and T derived from tracer spread into the ectostriatum (Benowitz
and Karten, 1976). At the level of the midbrain AVT and TPc also contained a number of
labeled cells.
Figure 12: Photomicrographs illustrating the close correspondence between NCL projection neurons to the archistriatum and thecatecholaminergic innervation of the caudal telencephalon. Cells in A were retrogradely labeld after a CTb injection into theintermediate archistriatum similar to that shown in figure 13. B shows an adjacent slice from the same animal processed for THimmunoreactivity. Scale bar is 1 mm.
Chapter 1: Connections of the avian neostriatum caudolaterale 4848
Figure 13: Results from a large (81 nl) injection of CTb into the archistriatum which involved parts of the central and ventralintermediate archistriatum. A: Diagrammatic representation of retrogradely labeled projection cells within NCL. B:Photomicrograph showing cells in the far caudal NCL. C: Retrogradely labeled cell in the NCL following a BDA injection in theintermediate archistriatum colocalized with TH immunoreactive fibers. Scale bars are 1 mm (B) and 50 µm (C).
A 6.25
Ai
A 3.75A 4.25
A 4.75
A 5.25
A 7.25
A 6.00A 5.75
A 6.75
NCL
Nd
OMCDL
APH
Aidd
Av Aidv
Tn
AVT
DIP
DLP
HV
Am
SRt
PA
NCm
APH
OM
OMOM
NCL
NCL
NCL
Nd
OM
CDL
APH
Aidd
Av
AidvTnDIP
DLP
HV
Am
PA
NCm
APH
OM Av
Aidd
Aidd
NCL
DLP
Nd
Nd
NCm
SRt
DIP
AiAi
APHCDL
SGC
SACSGP
SGC
SACSGP
SGC
SACSGPPT
SPL
SCI
DMP
ICo
DMP
OM
NCL
APH
APH
APH
CDL CDL
NCmNCL
Aidd
AiTn
L1L1L1
NCm
Aidd
Ai
Av Av
PA
HV
HV
NCLNCL
CDL
Av
Ov
Tn
Am
NCm
HV
A
Chapter 1: Connections of the avian neostriatum caudolaterale 4949
Injections into the medial lobus parolfactorius
Labeling within NCL after deposits of CTb into medial LPO between A 9.50 and A 11.50 was
very similar to that observed after PA injections. Again, retrogradely labeled neurons were
distributed diffusely along the medioventral and rostrocaudal extension of NCL, yet cells were
predominantly located along the medial border (figure 16). This was also true for two cases in
which the injection accidentally was centered onto lateral LPO and PA. Regarding a possible
rostrocaudal topography of NCL projections to the basal ganglia, we found a relative larger
number of retrogradely labeled neurons projecting to the LPO in caudal NCL while neurons
projecting to the PA tended to be located more rostrally and medially. However, as noted
APH
NCL
Nd
CDL
A 5.25
Hp
CPi
SPC AiAm Aidd
Aidv
NCL
APH
AVT
Hp CDL
A 4.25
HOM
NCL
Hp
SRt
SPC
Nd CDL
CPi
APH
A 5.75
AiddAidvAm
Ai
Av
NCL
APH
LHy
Ap
NdHp
A 4.75
CDL
Figure 14: Schematic representation of labeling in the caudal telencephalon after a deposit of 15 nl CTb into the posteriorarchistriatum. All injections into the archistriatum were placed using a lateral approach. Cells within NCL project only relativesparsely onto Ap. Their number increases towards the caudal pole of the hemisphere. A strong projection arises from cellsimmediately dorsal of the archistriatum. This area seems to intersperse between NCL and archistriatum, in contrast to the lattertwo it also labels considerably weaker for TH (compare also figure 1).
Chapter 1: Connections of the avian neostriatum caudolaterale 5050
above, these projections largely overlapped. Labeling in other areas of the telencephalon
included the NIM and HV dorsal to the injection site, medial HA and HD, and the area
prehippocampalis (APrH). Many cells were also seen in TPO, CDL and up to far rostral levels
within NFL, but only few in NIL. In the archistriatum large parts of the Av and Ai were labeled.
The projection from the Av seemed to be bilaterally organized. We found that in the thalamus
CTb injections which were centered on medial LPO yielded numerous neurons within n.
dorsomedialis anterior thalami (DMA) and DMP, and to a lesser extent also in the n.
subhabenularis (SHL) and DIP. In the mesencephalon cells were found in AVT, TPc and LoC.
DISCUSSION
The main results of this study can be summarized as follows: The NCL has reciprocal
connections with the secondary sensory areas of all modalities, as well with at least two
parasensory areas in the NIM and the deep layers of HV. The afferents from these areas have
diffuse and largely overlapping termination fields within NCL (see figure 17). These
connections are restricted to the plexus of the densest innervation by catecholaminergic and
presumably dopaminergic fibers as demonstrated here by TH immunoreactivity. Furthermore,
all parts of the so defined NCL project onto the basal ganglia and the archistriatum. The
importance of these findings for sensorimotor integration in the avian telencephalon and their
possible relevance for the evolution of cortex-equivalent structures will be discussed
separately in detail below.
Sensory integration within NCL
All five secondary sensory areas were found to project to NCL. The organization of these
pathways conforms with a general pattern of sensory processing in the avian telencephalon
(summarized in Veenman et al., 1995b): the primary receptive fields of subtelencephalic input
Following pages:Figure 15: Results following an injection of 28 nl CTb into the „somatic“ central PA. A: Schematic representation of retrogradelylabeled cells in the caudal telencephalon. The most abundant labeling can be seen in the dorsal part of the archistriatum and therostral NCL. See text for details. B: Photomicrograph illustrating the location of projection neurons at the medial border of theNCL and within Aidd. C: Photomicrograph of labeling in the posterior dorsal thalamus. In addition to labeled neurons within DIP,cells could often be observed in the VIP, DLP and SRt. Scale bars are 500 µm (B) and 200 µm (C).
Figure 16: Projections to the limbic medial striatum. Results of a 22 nl injection of CTb into the medial LPO with no obvioustracer spread into AC or lateral LPO. Labeling in the caudal telencephalon was complementary to that observed after injectionsin the somatic parts of the basal ganglia. Most abundant labeling was seen in the ventral part of the intermediate archistriatumand the caudal NCL. B: Photomicrograph showing the location of NCL projection neurons (arrows) at the medial border of thetractus archistriatalis dorsalis as well as in Ai and Av in a slice double labeled for TH. The DA shows only weak staining forTH. C: Retrogradely labeled cells in the posterior neostriatum cluster at the medial extent of the NCL. D: Photomicrograph ofretrogradely labeled thalamic neurons within DMA.
Chapter 1: Connections of the avian neostriatum caudolaterale 5151
TPO
NIM
Hp
PAPP
FPL
HA
HV
A 7.00
NCL
APH
PA
Tn Av
Hp
AiddAi
CDL
NCm
A 7.50
TPO
CDL
PA
PP
AvAi
Aidd
Hp
NCm
NCL
APH
PA
TnAv
Hp
Aidd
Ai
CDL
Rt
Hv
NCm
TnAv
Aidd
AiAidv
SRt
Hv
Rt
NCm
NCL
CDL
DMA
Hp
APH
Av
Aidd
AiDLPAmDIP
OvRt
SRt
NCL
NCm
CDL
DMP
Hp
APH
Nd
NCL
APH
TO
NdNCL
APH
TO
Nd
A 8.25
A 6.50 A 6.00A 5.50
A 4.50 A 4.00
DLPDIP
AVT
1 mm
NCL
APH
Ap
TO
NCm
Nd
A 5.00
A
Figure 15
Chapter 1: Connections of the avian neostriatum caudolaterale 5252
NCL
APH
L 1
PA
Tn Av
Hp
AiddAi
CDL
NCm
FPL
LPO
PA
PP
NIL
NI
NIM
HA
Hd HVdv
HVL 1 TPO
CDL
PA
PP
Av
AiAidd
OM
Hp
NCm
NCL
APH
L 1
PA
Tn Av
Hp
Aidd
Ai
CDL
SPCDLM
VIA
HV
NCm
FPL
DMA
TnAv
Aidd
Ai
Aidv
HV
Rt
NCm
NCL
CDL
DMA
Hp
APH
SRt
SPC
DLPDIP
Ov
Rt
SPC
NCL
NCm
CDL
DMP
Hp
APH
Av
Aidv AiddAiAm
SRt
A 11.25 A 7.50
NCL
APH
OM
TO
Nd
NCL
TO
OM
APH
Nd
NCL
APH
Ap
TOOM
NCm
Nd
A 7.00
A 6.50 A 6.00A 5.50
A 4.00A 4.50A 5.001 mm
AVT
DIP
DMP
DLP
HVHV
A
Figure 16
Chapter 1: Connections of the avian neostriatum caudolaterale 5353
relay the information to adjacent secondary sensory structures, which in turn project to areas of
the external pallium. In the case of the tectofugal visual pathway the ectostriatum is the primary
sensory structure (Kondo, 1933, Benowitz and Karten, 1976). From there intratelencephalic
projections lead to the ectostriatal belt (Karten and Hodos, 1970; Watanabe et al., 1985). The
present study demonstrates in accordance with Leutgeb et al. (1996) that the NCL is the tertiary
telencephalic component of the tectofugal complex. This pattern also holds for the other sensory
systems. Caudal IHA and the caudolateral component of HD are the primary sensory areas of
the thalamofugal visual pathway (Karten et al., 1973), from where projections lead to HA
(Shimizu et al., 1995), which by itself projects to NCL (Shimizu et al., 1995, Leutgeb et al.,
1996). More rostrally, somatosensory thalamic fibers terminate in HD/HIS and IHA (Delius
and Bennetto, 1972, Funke, 1989, Wild, 1997) which in turn project to rostral HA (Wild,
1987b). Field L2 is the primary telencephalic area of the auditory system (Wild et al., 1993),
which projects to L1 and L3, from where efferents lead to Nd/NCL (Bonke et al., 1979, Wild et
al., 1993, Leutgeb et al., 1996). N. basalis is the primary telencephalic area of the trigeminal
system (Schall et al., 1986), which projects to NFT (Wild et al., 1985; Wild and Farabaugh,
1996), from where efferents lead to NCL (Schall et al., 1986, Wild et al., 1985; Wild and
Farabaugh, 1996). Thus, the NCL is the tertiary telencephalic component of all sensory
modalities examined in the present study. Due to this multimodal input and the broad overlap of
terminations, the NCL is a true associative forebrain structure. Additionally, parts of the caudal
neostriatum may also have access to olfactory information: after injections into the posterior
archistriatum we found a large number of cells in the ventralmost neostriatum overlying the
archistriatum. This region has been suggested to be reciprocally connected with the avian
piriform cortex and olfactory bulb, which, as their mammalian counterparts process olfactory
information (Reiner and Karten, 1985; Haberly and Bower, 1989; Bingman et al., 1994). In
concert with the CPi and limbic archistriatum this area might thus be concerned with
viscerolimbic functions. However, from our combined immunocytochemical and pathway
tracing data it seems that this part of the NC is not a genuine subdivision of the NCL, although it
might be ventrally continuous with it. Olfactory information can reach the NCL nevertheless via
its connections with the HD. In addition to afferents from the visual dorsolateral thalamus
(Karten et al., 1973; Güntürkün and Karten, 1991; Güntürkün et al., 1993) and non-specific
input from the mediodorsal thalamus (Bagnoli and Burkhalter, 1983), the HD is also
reciprocally connected with the hippocampal formation and the CPi (Bingman et al., 1994;
Casini et al., 1986; Shimizu et al., 1995).
Chapter 1: Connections of the avian neostriatum caudolaterale 5454
The pattern of afferents within NCL might provide clues to the functional architecture of the
pigeon´s nervous system. The only two pathways which show no or little overlap within NCL
are those from the auditory and the trigeminal systems. Indeed, conditioning paradigms have
shown that pigeons have severe constraints to associate auditory signals with food reward in an
appetitive learning paradigm in which pecks on a key serve as an operant (LoLordo and
Furrow, 1976, Delius and Emmerton, 1978). The same animals are rapidly able to associate in
a classical conditioning paradigm the very same auditory signals with a mild shock applied to
the body (Delius and Emmerton, 1978). Delius and Emmerton (1978) speculated that a
granivorous animal like a pigeon is simply not in need of associating acoustic cues with food
objects since grains are mostly silent. However, grains can be quite noisy during pecking due to
the direct auditory feedback generated by the impact of the beak on the substrate. Indeed, Delius
(1985) could show that pigeons are easily able to learn an auditory tone discrimination in an
appetitive paradigm if the acoustic signals are generated by the pecks. Yet, delaying the
acoustic feedback by more than 0.5 s abolishes learning. Since the n. basalis not only receives
trigeminal projections but also some auditory afferents from the lemniscal nuclei (Delius et al.,
1979, Schall et al., 1986, Arends and Zeigler, 1986; Wild and Farabaugh, 1996) it is likely that
the specialised auditory-trigeminal associations occuring during pecking are directly processed
within the basalis system. Indeed, lesions of the n. basalis eliminate the ability to discriminate
acoustic signals generated by the animals own pecks (Schall and Delius, 1991). Thus, it is
conceivable that the separation of auditory and trigeminal afferents within NCL is related to the
severe constraints of pigeons in associating acoustic and trigeminal stimuli. Both the tectofugal
and the thalamofugal systems project to NCL, but their common area of termination seems
limited. This fact might be related to differences in visual field representations between the
thalamo- and the tectofugal pathway. The retina of pigeons has two areas of enhanced vision
(Galifret, 1968, Binggeli and Paule, 1969). One is the central fovea which looks into the
monocular lateral field. The other is the so called “red field“ in the dorsotemporal retina with
which the animal views stimuli in the inferior frontal visual field (Güntürkün, 1998). The
representation of the red field within the GLd of the thalamofugal pathway is extremly sparse,
while the central fovea is heavily represented (Remy and Güntürkün, 1991). Thus, the
thalamofugal pathway mainly processes stimuli from the lateral rather than the frontal visual
field. Accordingly, thalamofugal lesions produce minor deficits when tested with frontal
stimuli, but severe deficits when tested with lateral stimuli (Güntürkün and Hahmann, 1998).
Contrarily, there is evidence that tectofugal rotundus lesions impair frontal acuity, while
Chapter 1: Connections of the avian neostriatum caudolaterale 5555
Figure 17: Schematic representation of terminal fields within NCL showing the large overlap of afferents from secondarysensory areas. Note that the afferents from the presumed parasensory areas within NIM, which occupy large parts of thecaudalmost NCL are not included in this figure. See text for details.
1 mm
A 6.50
A 6.00
A 5.50A 5.00
A 7.50
visual afferents from Ectostriatal belt
trigeminal afferents from frontal neostriatum
somatosensory afferents from rostral HA visual afferents from caudal HA
auditory afferents from Field L
"border" of the NCL as defined by dopaminergic innervation
A 7.00
Chapter 1: Connections of the avian neostriatum caudolaterale 5656
leaving lateral acuity intact (Güntürkün and Hahmann, 1998). This in turn may be due to the fact
that ventral tectal cells, which represent the lower frontal visual field, project heavily onto
rotundus, while the contribution of dorsal tectal cells to the tectofugal pathway is limited
(Hellmann and Güntürkün, 1997). Thus, thalamo- and tectofugal visual pathways in pigeons
seem to differentially represent lateral and frontal vision, respectively. Their largely
complementary representation within NCL might thus be related to their differential
representation of the visual field. Despite the small overlap of the tecto- and the thalamofugal
visual fields within NCL, these domains extensively overlap with the trigeminal and the
auditory projections, respectively. The large common territory of the tectofugal visual and the
trigeminal system might be related to the specialization of the tectofugal pathway to the lower
and frontal visual field (Hellmann and Güntürkün, 1997, Güntürkün and Hahmann, 1998) which
within the egocentric space overlaps with trigeminal inputs from the beak. Thus, a common
sensory focus of both systems could create a need for common coding which might be
accomplished by extensive areas of terminal overlap in an associative structure like NCL. The
large overlap between thalamofugal visual and auditory domains might be similarly interpreted.
As outlined above, the thalamofugal pathway in pigeons is oriented laterally (Remy and
Güntürkün, 1991, Güntürkün and Hahmann, 1998). Like most other birds, pigeons fixate distant
objects laterally with their central fovea (Blough, 1971, Martinoya et al., 1981, Bischof, 1988),
since their lateral monocular acuity is about twice as high as their frontal monocular one
(Hahmann and Güntürkün, 1993, Güntürkün and Hahmann, 1994). Thus, the thalamofugal visual
system is more related to distant visual objects and might therefore need common processing
with audition, which also is a distance sense.
Connections of the NCL with the NIM and ventral hyperstriatum
Our retrograde and anterograde tracing experiments have suggested the existence of two
different, albeit continuous, sources of afferents from NIM to NCL. While the NIMl projection
innervates the Nd region and the laterally adjacent parts of the NCL, afferents from NIMm
terminate abundantly throughout the caudalmost NCL. Both areas receive differential input from
the dorsal thalamus (Kitt and Brauth, 1982; Gamlin and Cohen, 1986; Wild, 1987a, 1994;
Metzger et al., 1996; present study) and differ in their cytoarchitectonic characteristics
(Rehkämper et al., 1985). The NIMl has been shown to receive visual and somatosensory
information from telencephalic areas (Funke, 1989; Wild, 1987b, 1994; Shimizu et. al., 1995;
present study). Since we found retrogradely labeled cells in L1 and L3 after injections into
Chapter 1: Connections of the avian neostriatum caudolaterale 5757
NIMl, this structure probably also integrates auditory information. In addition, polysensory
input reaches the NIMl via DLP (Kitt and Brauth, 1982; Gamlin and Cohen, 1986;
Korzeniewska and Güntürkün, 1990; present study).
The NIMm, as defined here, resembles in its location and connectivity the mediorostral
neostriatum/hyperstriatum ventrale (MNH) of the chick which has been extensively studied in
the context of imprinting (e.g. Metzger et al., 1996; Bredenkötter and Braun, 1997; Gruss and
Braun, 1996; for review: Scheich, 1987). Injections into NCL which labeled cells in NIMm
also always produced cells in the overlying HV, suggesting a similar translaminar organization
of this area in the pigeon as is characteristic for the MNH. Data on the connections of either the
MNH or NIMm with telencephalic regions are very limited (Metzger et al., 1996). Our results
indicate that the NIMm, as the NIMl, might also receive input from the visual and
somatosensory areas of the Wulst. Thalamic input to NIMm originates mainly in the DMP and to
a much lesser extent in the DMA, a situation which is reversed for the MNH of chicks (Kitt and
Brauth, 1982; Wild, 1987a; Metzger, et al., 1996; present study). The sensory properties of
these afferents are not entirely clear, but the nuclei of the dorsomedial thalamus receive input
from the hypothalamus and tractus solitarius (Berk and Butler, 1981, Berk and Hawkin, 1985,
Arends et al., 1988; Wild et al., 1990) and in turn project to the viscerolimbic striatum and CPi
(Metzger et al., 1996; Bingman et al., 1994; Kitt and Brauth, 1982; Wild, 1987a; Veenman et
al., 1995a, 1997). Furthermore, the NIMm and to a lesser extent also the NCL of pigeons
display strong immunoreactivity for the calcium-binding protein parvalbumin (S. Kröner,
unpublished observations), as has previously been shown for the MNH of chicks (Braun et al.,
1991a). Parvalbumin is associated with fast spiking GABAergic neurons (DeFelipe et al.,
1989; Kawaguchi and Kubota, 1993), and in songbirds it is characteristic of nuclei displaying
high metabolic activity, notably all nuclei of the vocal motor system (Braun et al., 1985,
1991b). As outlined above, both areas of the NIM appear capable of multisensory processing.
This draws attention to our observation that their afferents terminate predominantly in those
aspects of the NCL which receive relatively little input from secondary sensory regions
(compare figure 17). Although it is possible that other telencephalic areas which send
projections to NCL (e.g. HA or NFT) are also parasensory in nature (Deng and Wang, 1992,
1993; Schall et al., 1986; Wild and Farabaugh, 1996) the nuclei within NIM clearly are a step
progressed in an assumed hierarchy of stimulus processing, as they, like the NCL, receive their
afferents from secondary sensory structures. It thus seems that parasensory inputs from NIM to
the NCL complement the pattern of multimodal integration.
Chapter 1: Connections of the avian neostriatum caudolaterale 5858
It should be noted, that our injections into NCL also labeled cells in the HV which based on
their location resemble the intermediate and medial part of the hyperstriatum ventrale (IMHV),
another area which in the chick has been extensively studied in the context of imprinting and
memory formation (Bradley et al., 1985; Davies et al., 1988; review by Horn, 1998). Although
we did not target these cells in the anterograde tracing experiments, results from the study of
Shimizu and coworkers (1995) suggest that afferents from the HV also terminate abundantly
throughout the caudalmost neostriatum (Shimizu et al., 1995).
Projections to the archistriatum
We found a continous band of cells in the NCL which project in a topographically ordered
manner onto most parts of the intermediate archistriatum. In addition, the posterior archistriatum
receives a weak projection from cells in the caudal- and dorsalmost aspects of the NCL. The
connections with the intermediate archistriatum are reciprocally organized (Davies et al., 1997;
Leutgeb et al., 1996; Metzger et al., 1998; present study) and bilaterally projecting cells in the
Av provide a means of interhemispheric comparison (Wild and Farabaugh, 1996; Metzger et
al., 1998; present study). Functionally, the avian archistriatum has generally been divided into
two main subdivisions (Zeier and Karten, 1971; Davies et al., 1997; Dubbeldam et al., 1997).
These are a somatic sensorimotor part which in the pigeon comprises parts of the anterior and
intermediate archistriatum (Aa, Ai, Aidd, Aidv), and a viscerolimbic division that includes the
posterior and medial archistriatum, as well as the ventral parts of the intermediate archistriatum
(Ap, Am, Av). The sensorimotor archistriatum receives widespread afferent projections from
higher order sensory areas of the telencephalon (e.g., Ritchie, 1979; Wild et al., 1985, 1993;
Shimizu et al., 1995) and is involved in high-level motor control (Zeier, 1971; Knudsen et al.,
1995) and memory function (Knudsen and Knudsen, 1996). Furthermore, via the OM the
archistriatum projects to premotor areas of the brainstem (e.g. reticular formation and lateral
pontine nuclei) in all birds (Zeier and Karten, 1971; Wild et al., 1985, 1993; Davies et al.,
1997; Dubbeldam et al., 1997; present study) and to some specific groups of brainstem
motoneurons involved in respiration and vocalization in songbirds (Nottebohm et al., 1976,
Wild, 1993). The limbic portion of the archistriatum on the other hand is considered
homologous to the mammalian amygdala (Zeier and Karten, 1971; Davies et al., 1997;
Dubbeldam et al., 1997). It seems to be crucially involved in such viscerolimbic functions as
agonistic behaviour and homeostasis. (e.g. Cohen, 1975; Ramirez and Delius, 1979; Lowndes
and Davies, 1995). The archistriatum´s functional segregation is reflected in the
Chapter 1: Connections of the avian neostriatum caudolaterale 5959
extratelencephalic projections via the OM or HOM, respectively (Zeier and Karten, 1971;
Davies et al., 1997; Dubbeldam et al., 1997), and in its connections with limbic or somatic
striatum (Veenman et al., 1995b; present study). While the main output from the NCL might be
directed to the sensorimotor part of the archistriatum (Leutgeb et al., 1996), especially the
caudalmost NCL also appears to be capable of modulating the amygdaloid division of the
archistriatum (present results). The very large number of neurons from NCL that project to the
archistriatum, and the close correspondence between the position of these output neurons and
the densest catecholaminergic innervation, underline the importance of this projection for an
understanding of the functions of the NCL. In addition, we believe that the projection from the
caudal neostriatum to the archistriatum might serve as a criterion for the delineation of NCL.
Given the fact that injections into the archistriatum labeled a continuos band of cells that
extended from the ventrolateral NCL up to Nd, we think it reasonable to consider Nd the
´auditory subcomponent` of NCL. This view is also supported by the facts that the Nd shares
afferents with the adjacent NCL from areas other than the field L complex, shows the same
dense dopaminergic innervation and shows a similar organization of efferent connections other
than those to the archistriatum.
Projections to the basal ganglia
The connectivity, neurotransmitter content, and cytoarchitecture of the avian basal ganglia are
highly similar to that in mammals (Karten and Dubbeldam, 1973; Reiner et al., 1984; Veenman
and Reiner, 1994; Veenman et al., 1995b). In mammals corticostriatal inputs from association,
sensorimotor and limbic cortices project in a segregated manner onto three distinct striatal
regions referred to as associative, sensorimotor and limbic striatal territories (Parent, 1990;
Parent and Hazrati, 1995). These corticostriatal inputs provide the basal ganglia with
exteroceptive sensory information as to the location of objects in space, interoceptive sensory
information on relative body position in space, and neural feedback and feedforward
information on ongoing and impending body movements (McGeorge and Faull, 1989; Alexander
et al., 1990; Romo et al., 1992; Schultz et al., 1992; Aldridge and Berridge, 1998). Based on its
palliostriatal connections a similar functional segregation has recently been suggested for the
avian striatum (Veenman et al., 1995b): ventral striatal structures such as the AC, the BNST,
and olfactory tubercle constitute the „limbic“ parts of the avian striatum. These „limbic“ parts
also include the medial LPO and lateralmost PA. The remaining dorsal striatal parts (lateral
LPO, medial PA) on the other hand are considered sensorimotor in nature. We show here that
Chapter 1: Connections of the avian neostriatum caudolaterale 6060
all parts of the NCL project to the ventral and dorsal aspects of the striatum. Our combined
retro- and anterograde tracing experiments suggest a rostrocaudal topography of these
projections, with caudal parts of NCL projecting predominantly onto limbic medial LPO and
more rostral parts of NCL projecting to lateral LPO and mainly medial PA (figure 18). The
projection onto the limbic striatum had already been indicated by the study of Veenman and
coworkers (1995b); but as their injections also involved the CPi and Ap (see their figure 18
and discussion), which also project heavily onto the limbic parts of the avian basal ganglia
(Bingman et al., 1994; Veenman et al., 1995b; present study), until now the existence of this
connection from the NCL was not entirely clear. In addition to this direct pathway, the NCL is
also capable of influencing the ventral basal ganglia via its connection with the limbic
archistriatum. The ventral striatum of mammals receives input from limbic cortical regions such
as hippocampus, cingulate cortex, amygdala and piriform cortex (McGeorge and Faull, 1989;
Alexander et al., 1990), and similar projections have been shown for the avian homologues of
these structures (Bingman et al., 1994; Veenman et al., 1995b). In mammals, a further prominent
input to the ventral striatum originates in the frontal cortex (Berendse et al., 1992, Sesack et al.,
1989; Alexander et al., 1990), and particularly the prefronto-accumbal network plays a crucial
role in motivated behaviour (Apicella et al., 1991; Schultz et al., 1992; Floresco et al., 1997).
The demonstration of a projection from caudal NCL onto the AC thus further strengthens the
notion that the NCL might be functionally equivalent to the PFC (Veenman et al., 1995b; present
study). As in mammals, this projection might provide the sensory information required, e.g. for
the evaluation of appetitive and aversive stimuli. In addition to the projection upon limbic
striatum we found a prominent projection onto large parts of PA and lateral LPO which
comprise the somatomotoric divisions of the avian striatum (Veenman et al., 1995b), providing
the NCL with a further direct pathway for movement initiation. It is unclear why the projection
from NCL to the somatic PA was not described by Veenman and coworkers (1995b), but it
seems possible that this reflects differences in the sensitivity of the different tracers used, or,
more likely, that their injections into PA were not centered on those regions in which we found
the most abundant anterograde labeling, namely the caudal aspects of the central and lateral PA
(compare their figures 5 and 6 with figures 2 and 15 of the present study).
The palliostriatal projections form asymmetric and probably excitatory synapses with spiny
striatal neurons (Veenman and Reiner, 1996), a situation comparable to that in mammals (Dube,
et al., 1988). Based on these and similar findings large parts of the external pallium, including
the NCL and the archistriatum, have been compared with a subpopulation of corticostriatal
Chapter 1: Connections of the avian neostriatum caudolaterale 6161
projection neurons from layer III and V in the rat (Zeier and Karten, 1971; Veenman et al.,
1995b; Akintunde and Buxton, 1992; Cowan and Wilson, 1994). In sum, the NCL is positioned
to amalgamate sensory inputs from all modalities and to relay them to the sensorimotor division
of the basal ganglia, providing the latter with information that has been further processed and
integrated with input from other modalities.
Comparison with vocal control pathways in songbirds:
In songbirds much work has focused on the pathways that control the acquisition and production
of learned song. Within oscines, the forebrain vocal system comprises two major pathways that
converge on the same premotor nucleus of the archistriatum. The two pathways diverge from a
nucleus in the caudolateral neostriatum, the „high vocal center“ (HVC, Nottebohm et al., 1976,
1982). The main descending vocal motor pathway leads from the HVC to the robust nucleus of
the archistriatum (RA), which in turn projects onto mesencephalic and medullary nuclei
involved in vocalization (Nottebohm et al., 1976, 1982; Bottjer et al., 1989; Vates et al., 1997).
rostral NCL
Ai
Av
caudal NCL
PA
PA
lateral LPO/ PA
medial LPO/ventral striatum
Figure 18: Diagrammatic illustration of the organization of presumed „limbic“ (grey) and „somatic“ (black) pathways from theNCL to the archistriatum and the basal ganglia. All parts of the NCL send projections to both limbic and somatomotor areas, butwith a complementary rostral to caudal topography (see text for details and compare figures 15 and 16). The relativecontribution of the caudal and rostral NCL to these pathways is indicated by the width of the arrows. In addition to directconnections indirect pathways exist, as limbic and somatomotor parts of the archistriatum also project to corresponding areaswithin the basal ganglia.
Chapter 1: Connections of the avian neostriatum caudolaterale 6262
HVC also projects to RA by a second pathway that involves nuclei in the anterior forebrain.
This circuit sequentially connects HVC, area X of the LPO; the dorsolateral thalamus, the
magnocellular nucleus of the anterior neostriatum (MAN) and RA (Bottjer et al., 1989; Vates et
al., 1997). Auditory input is thought to reach both major pathways of the vocal system via HVC
and the adjacent „shelf“ region, which receive auditory information from the field L complex
and associated regions of the caudal forebrain (Kelley and Nottebohm, 1979; Fortune and
Margoliash, 1995; Vates et al., 1996). In nonsongbirds the projection from field L 1 and L3 to
the Nd/NCL is similar to this ascending auditory pathway in oscine songbirds (Wild et al.,
1993; present study), while the projection from NIMl to the Nd/NCL has been compared with
another polysensory projection upon HVC which arises from the nucleus interfacialis
(Nottebohm et al., 1982; Wild, 1994; Fortune and Margoliash, 1995). The present
demonstration of a prominent input from NIMm to Nd/NCL suggests a correspondence of this
pathway with yet another ascending projection onto HVC: in oscine songbirds MAN shares a
similar location in the intermediate neostriatum with the NIM. The MAN can also be divided
into a lateral (lMAN) and a medial (mMAN) part which receive differential input from the
medial portion of the dorsolateral thalamus (DLM) and the DMP, respectively (Bottjer et al.,
1989; Johnson, et al., 1995; Foster et al., 1997; Vates et al., 1997), thus resembling the thalamic
projections to NIMl and NIMm (Kitt and Brauth, 1982; Wild, 1987a; present study). The
mMAN then gives rise to a projection onto HVC and the adjacent „shelf“ region (Nottebohm et
al., 1982; Fortune and Margoliash, 1995; Vates et al., 1996, 1997; Foster et al., 1997). A
similar recursive pathway through the anterior forebrain also exists in budgerigars: the central
nucleus of the lateral neostriatum (NLc) integrates information from other forebrain nuclei and
relays it to the premotor neurons of the archistriatum (Brauth et al., 1994; Striedter, 1994;
Durand et al., 1997). It has previously been suggested that the HVC complex, as might be true
for the NLc of parrots, is concerned not only with translating auditory signals into vocal output
but with the integration of multimodal inputs, and that the song system arose from an elaboration
of pathways generally present in all birds (e.g. Wild, 1994; Margoliash et al., 1994). The fact
that the shelf region adjacent to HVC shares the afferent inputs of HVC (Fortune and
Margoliash, 1995; Vates et al., 1996; Foster et al., 1997) further strengthens the notion that
HVC represents a specialized nucleus for the control of learned song, while neural populations
in the adjacent neostriatum might be concerned with other aspects of multisensory integration
and motor control. However, judging from the presently available data important differences
seem to exist: the HVC of Passeriformes, or NLc of parrots, respectively, do not receive
Chapter 1: Connections of the avian neostriatum caudolaterale 6363
similarly abundant sensory input as described for NCL (e.g. Hall et al., 1993; Brauth et al.,
1994; Striedter, 1994; Fortune and Margoliash, 1995; Leutgeb et al., 1996; present study).
Furthermore, in pigeons the NCL and the archistriatum have direct reciprocal connections with
all sensory and parasensory areas investigated here, and in addition both project onto the
striatum (Veenman et al., 1995b; present study).
Comparison with mammalian prefrontal cortex
The PFC of mammals is actually not a single functional region but, rather, a group of
anatomically and functionally heterogenous areas (e.g. Goldman-Rakic, 1987). It is commonly
thought to involve the dorsolateral PFC, as well as the orbitofrontal and anterior cingulate
cortices (Walker, 1940; Akert, 1964; discussed in Preuss, 1995; Condé et al., 1995). These
areas participate to different extents in the complex cognitive, limbic and premotor functions
that the PFC subserves, depending on their respective afferent and efferent connections (Sesack
et al., 1989; Pandya and Yeterian, 1990). Thus, in primates, deficits resulting from damage to
the lateral frontal region involve mainly attentional, temporal and integrative functions and are
different from changes relating to emotion following orbitofrontal, and motivational changes
following medial frontal damage (summarized in Fuster, 1989). Sensory input reaches the PFC
via a set of interconnected pathways (for reviews: Jones and Powell, 1970; Pandya and
Yeterian, 1990). The primary sensory area of each modality projects first to an adjacent area in
parietal, occipital, or temporal cortex. This is the beginning of a sequential order of cortical
areas that make up a pathway for that modality. Each area in the sequence projects not only to
the next in line but also to a discrete area of the frontal cortex, which in turn reciprocates by
sending fibers back to the projecting area. The fields that constitute the third link in each of the
three pathways - namely, parietal area 7 (somatic), temporal area 22 (auditory), and
inferotemporal area 21 (visual) - project to the prefrontal cortex and, in addition, to the cortex
in the dephts of the superior temporal sulcus, which constitutes another multimodal area.
Although the termination fields show considerable overlap, especially around the principal
sulcus of primates, a strong topography of these afferents exists, resulting in a distinct pattern of
connections for each of the subdivisions of PFC (primates: Jones and Powell, 1970; Pandya
and Yeterian, 1990; rodents: Condé et al., 1995; Reep et al., 1996).
Chapter 1: Connections of the avian neostriatum caudolaterale 6464
Taken together, the organization of sensory input to the NCL strongly resembles the pattern
described above for mammalian PFC (Leutgeb et al., 1996; present study). In addition, it is
reciprocally connected with the avian homologue of the amygdala (present study), a connection
which is also characteristic for mammalian PFC (Condé et al., 1995; Reep et al., 1996; Sesack,
et al., 1989; Barbas and DeOlmos, 1990), and projects to most parts of the somatic and limbic
striatum, as well as the sensorimotor archistriatum (c.f. figure 19; Veenman et al., 1995b;
Leutgeb et al., 1996). Furthermore, the NCL and PFC share a dense dopaminergic input (Divac
and Mogensen, 1985, Waldmann and Güntürkün, 1993, Divac et al., 1985, 1994, Wynne and
Güntürkün, 1995), and a functional importance for processes which serve executive functions
(working memory: Mogensen and Divac, 1982, 1993, Gagliardo et al., 1996, 1997, Güntürkün,
1997; behavioral flexibility: Hartmann and Güntürkün, 1998; behavioral inhibition: Güntürkün,
1997). With regard to memory functions it has been proposed that the PFC performs a unitary
function, that of holding information gathered by the other association cortices „on-line“ in short
term memory (Goldman-Rakic, 1987; Kimberg and Farah, 1993). Judging from the anatomical
LPO
TeO
LHy
thalamus
brainstem
Acc/BNST
OM HOM
IHA
Ai
OMDLP/DIP
vis. HA
som. HA
BasNFT
PA
PP
L2
L1/L3NCLNIMIMHVEp
E
Ap
Figure 19: Diagrammatic summary of the circuitry in which the NCL participates as it emerges from this study. The NCL isreciprocally connected with the secondary sensory of all modalities as well as at least two parasensory areas. It projects to theavian equivalents of the amygdala (Ap, Av) and premotor cortex (Ai), as well as limbic and somatic parts of the avian striatumwhich taken together by means of their long descending projections exert control over limbic and motoric centers in thediencephalon, midbrain and brainstem.
Chapter 1: Connections of the avian neostriatum caudolaterale 6565
data and the behavioural studies cited above the NCL seems well capable to occupy the
position of such a central excecutive in the avian brain.
However, at least one important difference hampers comparisons between NCL and PFC: As a
common hallmark for the delination of the PFC across mammalian species serve the afferents
from the mediodorsal (MD) nucleus of the thalamus (Rose and Woolsey, 1948; Akert, 1964;
Preuss, 1995). Thalamic afferents to the NCL arise mainly from DLP and surrounding
structures. DLP, however, is very likely not the avian version of mammalian MD. Based on its
afferents and electrophysiological properties, DLP was suggested to be equivalent to the
posterior complex of nuclei and especially its suprageniculate component (Korzeniewska,
1987, Korzeniewska and Güntürkün, 1990). Like the posterior nuclei, DLP is dominated by
afferents from the vestibular nuclei (mammals: Mickle and Ades, 1954; birds: Wild, 1988,
Korzeniewska and Güntürkün, 1990), dorsal column nuclei (mammals: Feldman and Kruger,
1980, Berkley et al., 1986; birds: Funke, 1989, Wild, 1989, Korzeniewska and Güntürkün,
1990), superior colliculus (mammals: Hicks et al., 1986, Katoh and Benedek, 1995; birds:
Gamlin and Cohen, 1986, Korzeniewska and Güntürkün, 1990), and reticular formation
(mammals: Hicks et al., 1986; birds: Korzeniewska and Güntürkün, 1990). Amygdalar or
hypothalamic afferents to mammalian posterior thalamic nuclei (Jones, 1985) or avian DLP
(Korzeniewska and Güntürkün, 1990) have not been reported. Contrary to this pattern,
mammalian MD is reported to receive, among others, afferents from the amygdaloid complex,
diagonal band of Broca, and different hypothalamic and preoptic structures (Siegel et al., 1977,
Sapawi and Divac, 1978, Velayos and Reinoso-Suarez, 1982, Irle et al., 1984, Russchen et al.,
1987, Cornwall and Phillipson, 1988, Groenewegen, 1988). Based on this evidence, any
general connectional similarity between MD and DLP could be denied. This view results from
a comparison between DLP and the whole MD. If, however, DLP is compared with only lateral
MD (rats: paralamellar MD portion, Cornwall and Phillipson, 1988, Groenewegen, 1988; cats:
intermediate and lateral MD component, Velayos and Reinoso-Suarez, 1982; monkeys:
parvocellular and multiform lateral MD portion, Russchen et al., 1987) the picture changes. The
lateral MD portion does not receive afferents from the amygdala but is reached by inputs from
vestibular nuclei, superior colliculus, and reticular formation. It thus resemble the pigeon's DLP
in many aspects. It is the lateral portion of MD in cats and monkeys which heavily projects onto
the dorsolateral component of prefrontal cortex (Markowitsch and Pritzel, 1979, Giguere and
Goldman-Rakic, 1988, Ray and Price, 1993), which is known to be of prime importance in
delay tasks. The paralamellar MD in rats is innervated by deep layers of superior colliculus
Chapter 1: Connections of the avian neostriatum caudolaterale 6666
and projects to medial precentral cortex (Groenewegen, 1988), an area which has been
compared with the frontal eye field in the monkey (Reep, 1984), a caudal portion of prefrontal
cortex (Fuster, 1989; Pandya and Yeterian, 1990). But despite these similarities between lateral
MD of mammals and avian DLP, important differences also exist. For example, the pigeon's
DLP receives a prominent innervation by the cuneatus-gracilis complex and consequently the
majority of neurons in this structure respond to somatosensory stimuli either as uni-, bi- or
multimodal units (Korzeniewska and Güntürkün, 1990, Wild, 1994), which is clearly different
from the anatomical and physiological pattern found in lateral MD. Thus, at the present state of
knowledge, the initial suggestion of Korzeniewska and Güntürkün (1990) that the DLP is
equivalent to the mammalian posterior complex of nuclei and not to MD still seems to be the
most valid concept. Nevertheless the pigeon’s DLP might serve functions similar to MD, since
DLP-lesions were shown to disrupt working memory tasks (Güntürkün, 1997).
Veenman and coworkers (1997) have suggested that DLP might be homologous to the
intralaminar nuclei in mammals. In our view this seems not very likely since the mammalian
intralaminar nuclei receive extensive afferents from medial hypothalamus, preoptic area, lateral
septum, bed nucleus of the stria terminalis, ventral subiculum, perirhinal cortex, and amygdala
(Price, 1995). Additionally they are characterized by a dense cholinergic input (Tohyama and
Takatsuji, 1998), and their lesion effects are well characterized (Orem et al., 1973). A
comparable pattern does not exist for DLP (Korzeniewska and Güntürkün, 1990, Güntürkün,
1997, Veenman et al., 1997). The suggestion of Miceli and Repérant (1985) that the avian SRt,
which also projects onto NCL, is an intralaminar structure thus seems to be more likely, given
the hodological data provided by these authors.
Recently, Metzger et al. (1996, 1998) suggested that the MNH of chicks might constitute the
avian PFC. This hypothesis is based on the observation that MNH is characterized by a medium
density of dopaminergic afferents (Metzger et al., 1996), a high density of dopaminoceptive
neurons (Schnabel et al., 1997), and afferents from the DMA (Metzger et al., 1996). If avian
DMA should prove homologous to mammalian MD (Veenman et al., 1997), the hypothesis of
Metzger et al. (1996, 1998) would obviously gain weight. Yet, as discussed above, data on the
connections of DMA and MNH are limited. However, the DMA is known to project not only
onto MNH, but also to the accumbens (Veenman et al., 1997), medial LPO (Székely et al.,
1994), and piriform cortex (Bingman et al., 1994), projections which have not been shown for
mammalian MD (Groenewegen, 1988, Price, 1995). Given these contradictions in the DMA -
MD comparison and the complete lack of functional data on DMA, clearly more information is
Chapter 1: Connections of the avian neostriatum caudolaterale 6767
needed to clarify the comparison between avian MNH and mammalian PFC. These discussions
on the organization of avian associative structures and mammalian PFC demonstrate that simple
equivalencies between avian and mammalian prefrontal systems will not hold, even if two
entities resemble each other with regard to their anatomical organization and function.
However, difficulties in establishing homologous areas are not specific to comparisons
between vertebrate classes but also apply to intraclass comparisons, as reflected in the recent
discussion about the criteria that delineate prefrontal cortex in rats (Condé et al., 1995; Preuss,
1995).
Chapter 2: Projections of the DLP to the forebrain of the pigeon 68
A polysensory pathway to the forebrain of the pigeon: the ascending
projections of the n. dorsolateralis posterior thalami (DLP)
Onur Güntürkün and Sven Kröner
ABSTRACT
Visual evoked potentials (VEPs) in the associative neostriatum caudolaterale (NCL) have shorter latencies than
those recorded in other visual forebrain areas. Therefore visual input into NCL probably stems from a
subtelencephalic relay. Tracing experiments revealed a projection of the n. dorsolateralis posterior thalami
(DLP) into those portions of NCL in which visual, auditory, and somatosensory afferents from
intratelencephalic parasensory areas terminate. Since VEPs in NCL are abolished after DLP-lesions, this
structure has to be the critical relay. However, DLP also projects to other associative forebrain areas and parts
of the basal ganglia. Previous experiments had furthermore revealed that DLP-neurons integrate visual,
auditory, and somatosensory inputs. Thus, the DLP-projection onto various associative forebrain areas
represents a true polysensory thalamo-telencephalic system.
European Journal of Morphology 37:124-128 (1999)
INTRODUCTION
This study aims to clarify two questions regarding the organization of the avian neostriatum
caudolaterale (NCL), an area thought to be equivalent to the prefrontal cortex (PFC). First, are
visual evoked potentials (VEPs) that can be recorded in and around NCL due to a projection of
the n. dorsolateralis posterior thalami (DLP) onto the caudal forebrain? Second, what are the
detailed patterns of projections established by ascending DLP-fibers?
The thalamo- and the tectofugal pathways which terminate in the wulst and the ectostriatum,
respectively, constitute the main ascending visual systems in birds. Latencies of VEPs are about
22 ms for the wulst and more than 50 ms for the ectostriatum (Parker and Delius, 1972). Since
Güntürkün (1984) could record VEPs with latencies of 15 ms in the caudal neostriatum, and
since these VEPs were unaffercted by wulst lesions, it was concluded that an independent ‘third
primary visual system’ had to exist which terminated in the caudal forebrain. However, the
thalamic relay of this pathway remained unknown.
Due to its dense dopaminergic innervation (Divac and Mogensen, 1985, Waldmann and
Güntürkün), and its multimodal organization (Metzger et al., 1998, Kröner and Güntürkün,
Chapter 2: Projections of the DLP to the forebrain of the pigeon 69
1999), the NCL was suggested to be comparable to the mammalian PFC. Indeed, behavioral
studies could demonstrate NCL-lesions to cause deficits in working memory and behavioral
flexibility tasks (Gagliardo et al., 1996, Hartmann and Güntürkün, 1998), similar to the
situation in PFC. The main thalamic afferents of NCL are the DLP and cells in the adjacent
ventrointermediate area of the posterior nuclei (VIP) (Kröner and Güntürkün, 1999). The DLP
has been shown to constitute a polysensory entity in which visual, somatosensory, and auditory
afferents converge on single neurons (Korzeniewska and Güntürkün, 1990) and which plays a
role in working memory tasks (Güntürkün, 1997). Since the DLP is known to project to NCL
(Waldmann and Güntürkün, 1993), the aim of the present study was to clarify if DLP is the
missing thalamic link of the ‘third primary visual system’ and whether all parts of NCL receive
input from DLP.
METHODS
Due to the multitude of approaches methods are described only briefly and we rather refer to studies, in which
these are described in more detail.
Anterograde tracing: A 2.5% solution of Phaseolus vulgaris leucoagglutinin (PHA-L) diluted in 0.1 M
phosphate buffer (PB, pH 7.2) was injected in two animals over 30 min into the DLP using pulsed positive DC
current of 4.5 µA. After 5 days animals were anesthetized and perfused first with 0.9% NaCl (40°C) followed
by 4% paraformaldehyde (PFA) in sodium acetate buffer (pH 6.5) and finally by 4% PFA in sodium borate
buffer (pH 9.5). Brains were postfixed in 4% PFA in acetate buffer (pH 6.5) for 1 hour and stored in 30%
sucrose in PB at 4°C. Then, 25 µm frozen frontal brain sections were cut and processed for ABC immuno-
cytochemistry. For details of the staining procedure see Gerfen and Sawchenko (1983).
Retrograde tracing: In 12 pigeons, small amounts of Fast Blue (FB) or rhodamine isothiocyanate (RITC), each
dissolved at 2% in 1 % DMSO in distilled water were injected stereotaxically into NCL or the terminal field of
DLP within the neostriatum intermedium medialis (NIM). An additional 16 animals received pressure
injections of cholera toxin b (CTb) into the same areas within NCL or NIM. After 2-3 days animals were
perfused with 0.9% NaCl followed by 4% PFA in PB (pH 7.2, 4°C). Brains were postfixed between 1 - 24 h
(4°C) in the fixative with 30% sucrose added, and cut frontally in frozen sections of 40 µm. Fluorescent tracers
were visualized using a fluorescence microscope. For details see Korzeniewska and Güntürkün (1990).
Immunohistochemistry for CTb followed the procedure described by Shimizu et al. (1995). In brief, slices
were incubated overnight (4°C) in a primary antibody against CTb (1:20.000, containing 0.3% Triton X),
followed by a goat biotinylated antibody (1:500 in 0.3% Triton PB; both Jackson) and finally the avidin-biotin
complex (1:100; Vector). Staining was achieved by the 3,3'-diaminobenzidine-(DAB) technique.
Electrophysiology: Eight adult pigeons were anesthetized with equithesin and four 0.12 mm thick insulated
stainless steel wires were implanted under stereotaxic guidance into the DLP-termination zone of the NCL of
each hemisphere. Electrodes tips were staggered in 1 mm steps. An uninsulated loop of wire placed under the
Chapter 2: Projections of the DLP to the forebrain of the pigeon 70
scalp served as reference. Free ends of electrodes terminated in a miniature connector glued onto the skull.
Recordings were performed in awake animals. A red light diode (665 nm, half-bandwidth: 30 nm, intensity: 0.8
mcd, stimulus duration: 5 ms, interstimulus interval: 2.3 s) was placed 5 mm from the eye contralateral to
recording. Potentials from electrodepairs were bandpass filtered between 7 and 120 Hz with a notch filter at 50
Hz, and were averaged over 32 trials. At the end of recordings small radiofrequency lesions were made at
electrode tips to histologically verify their locations. In three pigeons a lesion electrode was placed in a
second surgery into the DLP ipsilateral to the recording site and thermal coagulations (20 mA, 8 s) were made.
NCL-recordings were made a few days before and one week after lesions. For further details see Güntürkün
(1984).
RESULTS
Visual evoked potentials (VEPs) with first inflection latencies 16 to 22 ms were recorded from
NCL. The area examined reached from A 4.5 to A 6.75 and from L 2.00 to L 8.00. Structure and
latencies of VEPs were in close accordance with Güntürkün (1984). To test whether the initial
VEP components derived from the DLP-projection onto NCL, the DLP was thermally lesioned
(fig. 1a). Postoperative recordings revealed a complete absence of VEPs (fig. 1a).
PHA-L injections were centered on DLP but also slightly encroached upon surrounding VIP, n.
dorsointermedius posterior thalami (DIP) and n. dorsomedialis anterior thalami (DMA) (Fig.
1b). Labeled axons were observed to proceed rostrally to the fasciculus prosencephali lateralis
(FPL). On their ascending route, fibers made dense terminal endings on neurons of n.
dorsointermedius ventralis anterior (DIVA) and n. reticularis superior dorsalis (Rsd). Many
labeled axons could also be seen in the „somatic“ parts of the basal ganglia, namely the lateral
lobus parolfactorius (LPO) and the medial paleostriatum augmentatum (PA). Some fibers were
seen dorsal to the lamina hyperstriatica (LH) in the ventral hyperstriatum (HV), the largest
terminal field however extended underneath the LH just medially and dorsally of the
ectostriatum in the NIM. Some fibers coursed frontally and terminated on neurons of the lamina
frontalis superior (LFS). Others proceeded caudally to NCL, where fibers were seen to be
loosely distributed and making terminal endings (fig. 1c,f). These axons mainly concentrated in
the caudodorsal aspect of NCL (Waldmann and Güntürkün, 1993).
Following page - Figure 1: A: Diagrammatical summary of the electrophysiological experiments. The left part of the diagrammshows the location of the recording sites within NCL and the extent of the DLP lesion. The right part shows examples of VEPsbefore and after DLP lesion. B: Injection site of PHA-L in the dorsal thalamus. C: Anterogradely labeled fibers in the NCLfollowing the PHA-L injection shown in B. D: Terminal field of DLP afferents in NIM. E: Retrogradely labeled cells in DLPafter a CTb injection into the neostriatal area shown in D. F: Diagrammatic representation of the DLP terminal fields in NIMand NCL. Scale bars are 500 µm (B,D), 50 µm (C), and 250 µm (D).Abbreviations used in the figures: ectostriatum (E), n. dorsointermedius posterior thalami (DIP), n. dorsolateralis posteriorthalami (DLP), lobus parolfactorius (LPO), neostriatum caudolaterale (NCL), neostriatum intermedium medialis (NIM),paleostriatum augmentatum (PA), ventrointermediate area of the posterior nuclei (VIP).
Chapter 2: Projections of the DLP to the forebrain of the pigeon 71
20µV
25 ms stimulus artifact
postoperative
preoperative
E
NIM
LPOPA
HV
NCL
DLPVIP
DIP
A 5.00
NCL
A 6.50 A 10.50
1 mm
DLP
NCL
DLP
DLPE
HV
NIM
LPO
B C
D E
A
F
Chapter 2: Projections of the DLP to the forebrain of the pigeon 72
The majority of fibers terminated in an area reaching from A 4.75 to A 7.25 and L 4.5 to L 8.5
within NCL (Fig. 1d,f). Only very few fibers extended into far lateral and ventral parts of NCL.
Injections of retrograde tracers into NCL caudal to A 5.00 revealed few labeled cells in DLP.
Injections placed further rostrally labeled neurons in VIP and throughout the complete
rostrocaudal extent of DLP. Injections of retrograde tracers into the dense terminal field of the
DLP within NIM resulted in a very large number of cells within DLP (fig 1e). Injections into
LFS revealed a small number of labeled cells in dorsal DLP.
DISCUSSION
This study clearly reveals that the DLP projects to a number of forebrain somatic and
association areas. It could additionally be shown that the thalamic component of the ‘third
visual system’ proposed by Güntürkün (1984) is the DLP, projecting to large parts of NCL.
The prime argument to propose an independent third visual system to the avian forebrain had
been the latencies of VEPs which were shorter in caudal forebrain then in wulst and
ectostriatum (Güntürkün, 1984). DLP integrates besides auditory, and somatosensory afferents
also a massive visual input from the tectum. Since DLP projects onto NCL, the VEPs of the
caudal forebrain are likely due to ascending DLP-projections. This assumption is clearly
supported by the absence of VEPs in NCL after DLP-lesions. In addition to afferents from DLP,
NCL integrates further visual input from secondary visual forebrain structures like caudal
hyperstriatum accessorium (HA) and ectostriatal belt (Eb) (Metzger et al., 1998, Kröner and
Güntürkün, 1999). It is possible that these afferents contribute to the late VEP components.
However, DLP-lesions had completely diminished VEPs, including their late components. This
might be due to the cellular sensory properties of the structures involved. Korzeniewska and
Güntürkün (1990) demonstrated that DLP-neurons had broad receptive fields and responded to
simple stimulus properties. Given the possible complexity of reponse properties in secondary
visual association areas of the forebrain, the simple diode-flash induced VEPs are likely to
activate DLP-, but not HA- and Eb-neurons. The dorsal part of NCL which receives the densest
DLP innervation has also been shown to receive auditory input via field L, afferents from visual
and somatosensory parts of HA, as well as a projection from cells in the terminal field of the
DLP within NIM (Kröner and Güntürkün, 1999). It thus seems that the dorsal NCL is reached
by intratelencephalic input from those modalities that are also transported by DLP afferents,
and, in addition, those parts of NCL receive an indirect input from DLP via NIM. In sum, the
DLP supplies the NCL with both a fast and a slow copy of incoming sensory information from
Chapter 2: Projections of the DLP to the forebrain of the pigeon 73
all main modalities, which within NCL can be further compared with other input from the same
modalities that originates in higher sensory areas of the telencephalon.
Acknowledgements
Thanks to our old master Juan Delius for his continuing support, his never ending enthusiasm, and his bright ideas. Supported by
the Deutsche Forschungsgemeinschaft through its Sonderforschungsbereich 509 NEUROVISION.
Chapter 3: Cell types in the chick neostriatum caudolaterale 74
Characterization of cell types in the chick neostriatum caudolaterale:
Electrophysiological and morphological properties.
Sven Kröner, Kurt Gottmann, Hanns Hatt, and Onur Güntürkün
ABSTRACT
The neostriatum caudolaterale (NCL), in the chick also referred to as dorsocaudal neostriatal complex (dNc),
is a polymodal associative area in the forebrain of birds that is involved in sensorimotor integration and
memory processes. In chick it is part of an „imprinting network“, and might serve to integrate the various
sensory and emotional components of natural imprinting objects. We have used whole-cell patch-clamp
recordings in chick brain slices to characterize the principal cell types of the NCL. Electrophysiological
properties distinguished four classes of neuron. The morphological characteristics of these classes were
examined by intracellular injection of Lucifer Yellow. Type I neurons characteristically fired a brief burst of
action potentials. A number of these cells showed distinct low-threshold transient depolarizations.
Morphologically, type I neurons had lar somata and thick dendrites with many spines. Type II neurons were
characterized by their repetitive firing pattern from which conspicuous frequency-adaptation was observed.
Cells in this class could be subdivided by the presence or absence of a time-dependent inward rectification.
Type II neurons also had large somata and thick dendrites with many spines. Type III neurons showed a high-
frequency firing with little accommodation and a prominent time-dependent inward rectification. They had thin,
sparsely spiny dendrites and extensive local axonal arborizations. Type IV neurons had a longer action-potential
duration, a larger input resistance, and a longer membrane time constant than the other classes. Type IV neurons
had small somata and short dendrites with few spines. The axons of cells in all spiny cell classes (types I, II, IV)
showed similar projection patterns to targets outside the NCL. The classes of neuron described here may play
distinct roles in sensorimotor integration and learning.
Submitted
INTRODUCTION
The neostriatum caudolaterale (NCL), in the chick also referred to as dorsocaudal neostriatal
(dNc) complex (Metzger et al., 1998), is a multimodal association area in the forebrain of birds
(Leutgeb et al., 1996; Metzger et al., 1998; Kröner and Güntürkün, 1 999). In the chick it has
mainly been studied as part of a network responsible for early learning, specifically imprinting
(Metzger et al., 1996, 1998; Bock et al., 1997; Bock and Braun, 1999a,b; Braun et al., 1999),
whereas in the pigeon most work has focused on tasks which in mammals invoke „frontal“
Chapter 3: Cell types in the chick neostriatum caudolaterale 75
executive functions, e.g. working memory (Mogensen and Divac, 1982, 1993, Gagliardo et al.,
1996, 1997, Güntürkün, 1997; Kalt et al., 1999), reversal learning (Hartmann and Güntürkün,
1998), response inhibition (Güntürkün, 1997, Aldavert-Vera et al., 1999), and spatial
orientation (Gagliardo and Divac, 1993). Anatomically, the NCL seems to be particularly well
suited for the association between external stimuli and the animals behaviour, as it integrates
information from all modalities and exerts influence over motor and limbic structures. In chicks
(Metzger et al., 1998) and pigeons (Leutgeb et al., 1996; Kröner and Güntürkün, 1999) the NCL
has been shown to be reciprocally connected with all major secondary sensory areas and at
least three further multimodal brain areas (i.e. the intermediate and medial part of the
hyperstriatum ventrale, IMHV, the mediorostral neostriatum/hyperstriatum ventrale, MNH, and
the archistriatum). Thalamic input reaches the NCL from several nuclei in the dorsal thalamus,
including the multimodal thalamic nuclei dorsolateralis posterior (DLP) and subrotundus (SRt)
(Korzeniewska and Güntürkün, 1990; Leutgeb et al., 1996; Metzger et al., 1998; Güntürkün and
Kröner, 1999; Kröner and Güntürkün, 1999). The NCL´s descending projections are directed
mainly to limbic and somatomotor regions within the basal ganglia and the archistriatum
(Kröner and Güntürkün, 1999). Thus, the NCL represents a nexus of sensorimotor integration in
the avian forebrain. In the chick, the NCL has therefore been postulated to be a polymodal
associative part of the „imprinting pathway“, in which the various sensory and emotional
components of natural imprinting objects are integrated during both, the learning process and
memory recall (Braun et al., 1999). While imprinting traditionally is conceptualized as a
relatively specialized form of learning in young animals that occurs during a sensitive phase
and which has permanent behavioural effects (Lorenz 1935), it shares certain features with
other forms of associative learning (Bolhuis, et al., 1990), such as stimulus generalization
(Bolhuis and Horn, 1992), stimulus comparison (Honey and Bateson, 1996), and the association
of stimuli in time (Bateson and Chantrey, 1972). If we are to explain the mechanisms that
underlie the sensorimotor integration and associative learning processes, we need to understand
in detail how sensory coordinates are mapped onto motor ones according to the learning history
of the animal and its current behavioural goals. Similar to the modules of mammalian cortical
structures (Douglas and Martin, 1992), the avian forebrain might be constructed from a
relatively small number of principal or canonical circuits, that are repeated in large quantities
to achieve parallel computing power. Furthermore, it has been shown in numerous preparations
that there is generally good correspondence between a neurons intrinsic firing pattern and its
morphology (e.g. McCormick et al., 1985; Chagnac-Amitai et al., 1990; Larkman and Mason,
1990; Kang and Kayano, 1994; Kasper et al., 1994a). Thus, to further our understanding of the
functions of the NCL on a cellular level, it is important to characterize its intrinsic neuronal
Chapter 3: Cell types in the chick neostriatum caudolaterale 76
organization and the cell types that might be involved in the processing of different aspects of
information. In this report, we have used in vitro whole-cell patch-clamp recording in
combination with intracellular staining to characterize the principal cell types in the chick NCL
electrophysiologically and morphologically.
MATERIALS AND METHODS
Preparation of slices and electrophysiological recording
Fertilized eggs were obtained from a commercial supplier (Sörries Trockels, Möhnesee, Germany) and chicks
(Gallus domesticus) were hatched and kept in small groups on a 12:12 hour dark/light cycle. Treatment of
animals conformed to NIH guidelines. A total of 29 chicks (2 - 11days post-hatch, median 6 d) were
decapitated and the brains were transferred to iced extracellular solution. Coronal slices (350 µm thick) of the
caudal telencephalon were cut on a vibratome. Slices were incubated in extracellular solution consisting of (in
mM): 119 NaCl, 2.5 KCl, 1 NaH2PO4, 26.2 NaHCO3 ,10 D-glucose, 4.5 CaCl2, and 1.3 MgCl2, saturated with
95% O2 /5% CO2, pH 7.3. Slices were allowed to recover for at least 1 hour, before being transferred to the
recording chamber. All recordings were made at room temperature (21 - 23°C) in a submerged slice chamber
perfused with extracellular solution.
Whole-cell patch-clamp recordings were obtained in the caudolateral subventricular region of the forebrain,
using the blind-patch technique (Blanton et al., 1989). In current-clamp experiments recording electrodes (6 –
8 MΩ resistance) were filled with an intracellular solution consisting of (in mM): 135 K-gluconate, 20 KCl, 2
MgCl2 , 10 N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 0.1 ethylene glycolbis (β-
aminoethyl ether)- N,N,N´,N´,-tertraacetic acid (EGTA), 4 Na2-ATP, and 0.5 Na2 -GTP, and adjusted to pH 7.3
with KOH. In some recordings 3 - 3.5 mg/ml of the dipotassium salt Lucifer Yellow (LY, Sigma, Deisenhofen,
Germany) were added to the intracellular solution and neurons were filled by diffusion during 30 - 90 minutes
of recording. In a further set of experiments K-gluconate and KCl in the intracellular solution were substituted
with 135 mM CsCl and 20 mM tetraethylamonium (TEA) to block potassium channels. Recordings were made
using a HEKA (Lambrecht, Pfalz, Germany) EPC-7 patch-clamp amplifier, filtered at 3 kHz, and sampled at 20
kHz. The voltage drop across the pipette (which was usually about 8 - 10 mV) could not be bridge-balanced.
Sampling was done with a Digidata 1200 interface using pClamp 6.0 software (Axon Instruments, Foster City,
CA), and data were stored on computer for off-line analysis with the CLAMPFIT module of pClamp. Input
resistance was determined from the voltage deflection induced by a hyperpolarizing current pulse in the linear
range of the current-voltage relation. Membrane time constants were calculated by fitting a single exponential
function to the voltage response to injections of hyperpolarizing currents in the linear range of the voltage
response. The percentage sag that occurred in some neurons in response to hyperpolarizing and depolarizing
current pulses was calculated as 100 x (Vmax -Vend)/Vmax, where Vmax was the peak voltage deflection and Vend
the voltage at the end of the current pulse. Resting membrane potentials were assessed in current-clamp mode
3 - 5 minutes after establishing the whole-cell configuration. Spike duration was measured at the half-maximal
amplitude from threshold, and the amplitude and time-to-peak value for the afterhyperpolarization (AHP) which
followed the action potential were measured from the equipotential-point on the repolarizing phase.
Chapter 3: Cell types in the chick neostriatum caudolaterale 77
Data analysis. Data are presented as means ± standard error (SE). Statistical analyses were done using the
SPSS 8.0 software package. The differences among cell classes for the various parameters were compared by
analysis of variance (ANOVA) and the statistical significance of the differences was examined with a Scheffé
test for multiple comparisons. Subsequent comparisons between and within cell classes were done using
Student´s t test for unpaired samples.
Histological procedures
Following recording, slices were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in 0.12 M phosphate
buffer (4°C, pH 7.4) for about 15 hours. They were then transferred to a solution of 30% sucrose in phosphate
buffer containing 0.9% NaCl (PBS; pH 7.4) and 0.01% NaN3 as a preservative. Slices were resectioned at 70
µm on a freezing microtome and collected in PBS. For immunohistochemistry of LY, endogeneous
peroxidases were blocked by preincubating slices in 0.5% H2O2. Slices were then washed in PBS and floating
sections were incubated overnight at 4°C in biotynilated anti-LY from rabbit (Molecular Probes, Leiden, The
Netherlands; 1:200 working dilution) in PBS containing 0.3% Triton X-100 (Sigma). After washing, slices
were incubated for 2 hours in the avidin-biotin complex (ABC Elite, Vector Labs, Burlingame, CA; 1:100 in
PBS with 0.3% Triton X-100). Washes in PBS were followed by additional washes in 0.12 M acetate buffer
(pH 6). Staining was achieved by the 3,3'-diaminobenzidine (DAB) technique with heavy-metal amplification
(modified from Adams, 1981) by adding H8N2NiO8S2 (2.5 g/ 100 ml), NH4Cl and CoCl2 (both 40 mg/ 100
ml). After 20 minutes of preincubation the reaction was catalyzed using 0.3% H2O2. The reaction was stopped
by rinsing the tissue in acetate buffer and PBS. Slices were then mounted, dehydrated and coverslipped.
Quantative measures of cell morphology were done using a Leica DMR (Leica, Wetzlar, Germany) and the
analySIS software package (Soft-Imaging Software, Münster, Germany). Camera lucida reconstructions of
filled cells were drawn using a Leitz BioMed with a drawing tube at x 12.5 or x 50 magnification.
RESULTS
Stable whole-cell recordings were obtained from 90 cells in the NCL. Neurons were selected
only if they exhibited a resting membrane potential (RMP) more negative than -50 mV and an
overshooting action potential. In this voltage range, there was a general lack of spontaneous
firing in all NCL cells, however, in voltage clamp recordings spontaneous postsynaptic currents
could be observed in most cells, indicating synaptic activity (data not shown). Neurons were
classified into 4 distinct types according to differences in their intrinsic firing properties and in
the voltage responses to hyperpolarizing current pulses. For 36 of the neurons thus
characterized basic morphological features were analyzed after filling with LY. With a single
exception in which a second cell was weakly stained, we found no evidence of dye-coupling
between cells in the NCL. Dye-coupling results from neuronal signalling via gap junctions and
is a phenomenon commonly observed in adult (Connors et al., 1983; Benardo, 1997), but
Chapter 3: Cell types in the chick neostriatum caudolaterale 78
particularly in developing mammalian (Connors et al., 1983; Kasper et al., 1994b) and avian
(Dutar et al., 1998) neurons, where it is believed to be involved in the functional specification
of brain areas (Peinado et al., 1993).
Type I neurons:
Type I cells displayed a characteristic firing pattern that consisted of a single action potential
or a brief burst of spikes. Type I cells represented 34 of the 90 cells characterized.
Firing properties: Cells in this class displayed the highest threshold for firing an action
potential, especially in relation to their low resting membrane potential (table 1). Type I cells
were marked with their tendency to fire a single action potential or 2 - 8 clustered spikes at
high frequency at the onset of depolarizing pulses (fig. 1A). The number of spikes in a burst and
the duration of the interspike-intervals strongly depended on the current amplitude injected (fig.
1A, C). No repetitive bursting was observed during long (1.1 sec.) depolarizing current pulses.
However, in the course of other experiments (data not shown) spontaneous occuring all-or-none
bursts were found when fast GABAergic transmission was abolished by adding picrotoxin (100
µM) to the extracellular solution (see below).
Type I cells varied with respect to their behaviour in the subthreshold voltage range: In
response to subthreshold depolarizing current pulses 13 type I cells showed prominent low-
threshold potentials that were not seen in the remaining neurons (c.f. fig. 1A1 and 1A2). This
subset of neurons also differed from the remaining type I cells in that their action potentials had
significantly larger amplitudes (60.9 ± 3.3 mV vs. 51.6 ± 2.9 mV) and shorter durations at half
maximal amplitude (1.72 ± 0.1 ms vs. 2.0 ± 0.08 ms), the latter being mostly due to shorter
rise-times of the action potentials (0.58 ± 0.05 ms vs. 0.74 ± 0.05 ms; Student´s t test for
unpaired samples; df = 32; all values for t > ± 2.1; p < 0.05). Further, 6 of the 13 cells that
showed these transient „hump“-like depolarizing responses also displayed a complex pattern of
afterpotentials that followed the first spikes. These consisted of an initial, fast
afterhyperpolarization (fAHP) that was followed by an intercalated depolarizing afterpotential
(DAP) and a late medium-duration AHP (mAHP; see fig. 1A1). The remainig type I cells
showed only a monophasic AHP (c.f. fig. 1A2).
Membrane rectification: Type I cells had low apparent input resistances and intermediate
membrane time constants (table 1). In response to hyperpolarizing current pulses that drove the
membrane potential more negative than about -80 mV, all type I neurons showed a pronounced
fast-activated inward rectification resulting in an upward bend in the current-voltage relation
(fig. 1D). Most type I cells also showed membrane outward rectification at the end of the
Chapter 3: Cell types in the chick neostriatum caudolaterale 79
-75 mV
A1
-74 mV
A2
B
C
Spi
ke fr
eque
ncy
(Hz)
Time during stimulus (ms)
400 pA450 pA500 pA550 pA600 pA650 pA700 pA
0 200 400 600 800 1000 1200
10
20
30
40
50
60
70 D
Pot
entia
l (m
V)
Current (pA)
-120
-100
-140
-80
-60
-40
-400 -200-300 -100 100 200
0.5 nA 0.5 nA
Figure 1: Intrinsic firing properties and membrane rectification of type I neurons. A: Examples of voltage responses tohyperpolarizing and depolarizing current pulses (-400 - 700 pA in A1 and -300 - 800 pA in A2) in two neurons that showed rapidadaptation of action potential firing. Type I cells typically responded with „bursts“ of action potentials to current pulses ofincreasing intensity. Both cells show membrane inward rectification to relatively large hyperpolarizing currents and outwardrectification to depolarizing currents in the spike-threshold range. The cell in A1 displays a prominent subthreshold „hump“-likepotential and a depolarizing afterpotential following single spikes. The cell in A2 shows only monophasic afterhyperpolarizationsof comparatively long duration. Note that there is also apparently less membrane rectification in this cell than in the cell shown inA1. B: Camera lucida reconstruction of a type I neuron. The soma and dendrites are drawn in black, the axon collaterals areshown in grey. Scale bar is 50 µM. C: Relationship between the instantaneous spike frequency (as the inverse of inter-spike-interval) and time for different current amplitudes for the cell shown in A1. D: Current-voltage relationship of the cell shown inA. Deviations of the potential from the extrapolated ohmic responses close to resting potential reflect inward and outwardrectification, respectively.
Chapter 3: Cell types in the chick neostriatum caudolaterale 80
current pulse, that was responsible for a downward bend in the I-V plot at depolarized
potentials (fig. 1A).
Morphology: Of the 34 cells designated
as type I cells 15 were successfully
recovered and reconstructed. Single or
burst firing neurons had large round or
fusiform somata (diameter 15 - 22 µm;
table 2) and multipolar arranged
dendrites. Dendrites were usually long
(dendritic field diameters 225 - 320 µm;
table 2) and displayed medium to high
spine densities (figs. 1B, 2, and 10). The
main axon branched near the soma and
gave rise to thin collaterals that ramified
mainly in a loose local plexus (figs. 1B,
2, and 10). In addition to these recurrent
collaterals which displayed numerous
varicose-like swellings, often 2 - 5
longer collaterals were observed that
projected outside the NCL. These axonal
arborizations showed less varicosities
and followed one of three general
directions: In most cases 1 - 3
collaterals moved ventrally within the
plane of the slice to terminate within the
underlying archistriatum. Other
collaterals travelled through the NCL
either dorsomedially, towards the
auditory subunit of the NCL, the
neostriatum dorsale (Nd), and possibly
other sensory forebrain areas, or ventromedially, in the direction of the the basal ganglia (c.f.
Metzger et al., 1998; Kröner and Güntürkün, 1999). Ventromedially directed arborizations,
however, could be followed only over relatively short distances before they left the sagittal
plane of the slice.
Figure 2: Morphological features of type I cells. A: Photomicrograph ofa Lucifer Yellow-filled neuron that responded to depolarizing currentpulses with rapidly adapting bursts of action potentials. B: Detail of atype I cell showing a dendrite with numerous spines. C: Dendritic treeand local axon arborizations of a type I neuron. The axon showsnumerous varicose-like swellings in the vicinity of the dendritic field. Thesoma of the cell was separated from the axon and dendrites duringresectioning of the slice. Scale bars are 50 µm (A, C), and 20 µm (B).
Chapter 3: Cell types in the chick neostriatum caudolaterale 81
Type II neurons
Type II cells were characterized by their repetitive firing pattern. They represented 35 of the 90
neurons studied.
Firing properties: Type II had similarly low resting membrane potentials, spike amplitudes
and action potential durations, as well as AHP amplitudes and time-to-peak values as type I
neurons (table 1). In contrast to type I neurons, however, small somatically injected
depolarizing current pulses readily elicited relatively tonic firing in type II cells. Two typical
examples of type II cells are shown in figure 3A. Current pulses that depolarized type II cells
just above threshold usually evoked a single action potential that often had long latencies. With
larger depolarizing currents a pattern of phasic-tonic firing emerged, in which a short burst,
was followed by a longer train of action potentials. This train of action potentials showed
conspicuous frequency adaptation (fig. 3A lower traces, and C), but the repetitive firing pattern
could be sustained at relatively low frequencies (between 5 -15 Hz) over long (1.1 sec)
depolarizing current pulses. This firing behaviour was facilitated by a comparatively low
action potential threshold (table 1). A number of type II cells (n = 8) showed the same sequence
of afterpotentials following an action potential as described for the subset of type I neurons
above.
Membrane rectification: Among the four types identified, type II neurons had intermediate
input resistances and time constants (table 1). Type II cells differed with regard to the existence
of a hyperpolarization-activated time-dependent inward rectification: When hyperpolarizing
current pulses were applied to type II cells usually (n = 22) a fast-activated inward rectifying
conductance appeared at membrane potentials more negative than about -80mV (fig. 3A1). This
inward rectification resulted in a slight upward bend in the hyperpolarized portion of the
current-voltage plot when compared with the extrapolated linear portion (fig. 3D). In another
13 neurons, however, a small sag in the voltage response (2.1 - 6.7 % sag) could be seen at
potentials more negative than about -80 mV. This delayed inward rectifying conductance tended
to push the membrane potential back towards resting values and lead to a rebound overshoot at
the termination of hyperpolarizing current pulses (fig. 3A2, 13B). The amplitude of the sag
increased with more hyperpolarized membrane potentials. These cells in addition seemed to
possess the same fast-activated inward rectification seen in the remaining type II neurons
(above) as their peak voltage responses exhibited inward rectification over the same range of
membrane potentials as the time dependent inward rectification (fig. 3D). At depolarizing
voltage steps the membrane potential often depolarized more than expected from the ohmic
response around resting potential, indicating the contribution of an inward rectification in the
voltage range close to spike threshold (see below).
Chapter 3: Cell types in the chick neostriatum caudolaterale 82
B
A1
-73 mV -68.5 mV
A2
sag
200 ms
20 mV
C
Spi
ke fr
eque
ncy
(Hz)
Time during stimulus (ms)
10
20
30
40
50
60
70
80
0 200 400 600 800 1000 1200
250 pA350 pA450 pA550 pA650 pA750 pA800 pA
D
-120
-100
-140
-80
-60
-40
Current (pA)
Pot
entia
l (m
V)
-400 -200-300 -100 100 200
voltage at peak
voltage at end
0.5 nA 0.5 nA
Figure 3: Intrinsic firing properties and membrane rectification of type II neurons. A: Examples of voltage responses tohyperpolarizing and depolarizing current pulses (-300 - 700 pA) in two neurons that showed a phasic-tonic firing of actionpotentials over long depolarizing current. The cell in A1 displays fast inward rectification to hyperpolarizing current pulses. Thecell in A2 shows the same fast inward rectification and additionally a sag in the voltage response to relatively largehyperpolarizing current pulses, resulting in a rebound overshoot at cessation of current pulses. See text for detail. B: Cameralucida reconstruction of a type II neuron. The soma and dendrites are drawn in black, the local axon collaterals are shown ingrey. Scale bar is 50 µM. C: Relationship between the instantaneous spike frequency and time for different current amplitudesfor the cell shown in A2. After a phasic response the spike frequency rapidly adapts to a steady-state tonic firing. D: Current-
voltage relationship of the cell shown in A2. , voltage response measured at the end of the current pulse; , voltage at thepeak.
Chapter 3: Cell types in the chick neostriatum caudolaterale 83
During long current pulses the
membrane potential sagged back
towards rest (4.8 %- 19.4 % sag)
resulting in a rebound undershoot at the
termination of the current pulse (figs.
3A2, 12B).
Morphology: The gross morphology of
type II cells resembled that of type I
cells (figs. 3B, 4, 10). They had large
oblique somata (diameter 16 - 23µm;
table 2) and long, thick multipolar
dendrites. In 4 neurons that were
thoroughly analyzed, type II cells
displayed the highest number of spines,
but this value was not significantly
different from that obtained for type I
neurons (table 2). Dendrites of type II
neurons had the largest number of
branch points and covered large areas
(diameter of dendritic field 229 - 296
µm; table 2). The axons of type II
neurons often formed a radial axonal
plexus in the vicinity of the soma
giving rise to several local
ramifications as well as long
collaterals that projected to targets
beyond the borders of the NCL. As in
type I cells, most neurons sent 1 - 3
descending collaterals towards the
archistriatum, but also had additional arborizations that seemed to travel ventromedially and
dorsomedially (fig. 5), probably making numerous contacts with other cells within NCL on their
way.
Figure 4: Morphological features of type II cells. A, B:Photomicrographs of two Lucifer Yellow-filled cells that responded withphasic-tonic firing of action potentials in response to depolarizingcurrents. The arrow in A points to where the axon leaves the cell body. Itcourses ventrally and ramifies extensively at the bottom of thephotomicrograph. The cell in B is the same cell as shown in figure 3B. C:Detail of a type II cell showing a dendrite with numerous spines and partof an axon (top left). Scale bars are 50 µm (A, B), and 20 µm (C).
Chapter 3: Cell types in the chick neostriatum caudolaterale 84
Type III neurons:
The key features of type III neurons were the ability to fire action potentials at a high frequency
and a prominent sag in the response to hyperpolarizing current pulses, as well as a short action
potential duration. Type III cells represented 8 of the 90 cells characterized.
Firing properties: In response to suprathreshold depolarizing current pulses all neurons
designated as type III responded with regular tonic firing that showed only relatively little
frequency adaptation. Thus, type III cells were able to initially fire action potentials at
frequencies of about 100 Hz. After several spikes the firing rate usually accomodated to some
degree, but sustained firing over long depolarizing current pulses continued at frequencies much
higher than found in any other cell class (fig. 6C). These cells also showed the most positive
resting membrane potentials, while generally having a very low threshold for the initiation of
action potentials (table 1). The duration of action potentials in type III cells was by far the
shortest among all classes, which was also reflected by short rise- and fall-times (table 1). The
action potential was followed by an AHP that was large in amplitude and had a comparatively
Figure 5: Example of the axonal arborizations of a type II cell within NCL. In addition to recurrent local arborizations mostneurons from all spiny cell classes possessed one or several axon collaterals that left the NCL ventrally towards thearchistriatum, ventromedially, in the direction of the basal ganglia, and/or dorsomedially, possibly to provide feedback to sensoryand associative forebrain areas. The insert shows the location of the cell in the dorsocaudal neostriatum. Scale bars are 500 µM.
Chapter 3: Cell types in the chick neostriatum caudolaterale 85
Table 1: Electrophysiological characteristics of classes of NCL neurons (means ± standard error)
Type I
(n = 34)
Type II
(n = 35)
Type III
(n = 8)
Type IV
(n = 13)
Significant differences*
Resting potential (mV) -75.1 (± 0.9) -73.9 (± 0.9) -63.5 (± 1.4) -68.3 (± 1.8) Type III, IV > type I, II
Input resistance (MΩ) 210.5 (± 12.1) 241.6 (± 13.1) 458.6 (± 28.6) 568.5 (± 28.7) Type IV > type III > type I, II
Membrane time constant (ms) 59.5 (± 2.8) 67.6 (± 5.3) 46.1 (± 6.2) 91.6 (± 8.3) Type IV > type I, II, III
Spike threshold (mV) -25.0 (± 1.2) -30.3 (± 1.0) -33.6 (± 1.7) -27.5 (± 0.7) Type I > type II, III
Max. number of spikes
(1.1s depolarizing pulse)
3.4 (± 0.5) 10.6 (± 0.9) 46.3 (± 5.9) 7.6 (± 0.8 ) Type III > type II > type I, IV
Spike duration at half
amplitude (ms)
1.9 (± 0.07 ) 1.86 (± 0.1) 0.76 (± 0.1 ) 2.32 (± 0.1) Type IV > type I, II > type III
Rise time (10-90%) ms 0.67 (± 0.03) 0.72 (± 0.04) 0.43 (± 0.06) 1.15 (± 0.07) Type IV > type I, II > type III
Fall time (90-10%) ms 1.41 (± 0.1) 1.64 (± 0.05) 0.94 (± 0.1) 1.9 (± 0.08) Type IV > type I > type III;
type II > type III
Spike amplitude (mV) 54.7 (± 2.3) 51.9 (± 2.0) 49.4 (± 6.7) 45.2 (± 2.3) −
AHP amplitude (mV) -17.8 (± 0.7) -17.5 (± 0.7) -20.3 (± 1.7) -15.8 (± 1.1) −
AHP time to peak (ms) 3.4 (± 0.2) 4.1 (± 0.2) 5.6 (± 0.6)= 5.8 (± 0.5) Type III, IV > type I, II
* Significant differences indicated by the use of a Scheffé test for multiple comparisons after analysis of variance (P < 0.05).
long time-to-peak (table 1). Both the amplitude and the time-to-peak could be reduced to a
much larger degree than in the other cell types when increased depolarizing currents drove type
III neurons to high-frequency tonic firing.
Membrane rectification: Type III cells had comparatively high input resistances but relatively
short membrane time constants (table 1). In response to hyperpolarizing current pulses
(membrane potential more negative than -80 mV) a prominent sag in the voltage response (8 -
20.4% sag) occurred that was followed by a rebound depolarization at cessation of the current
pulse. The time dependent inward rectification and the rebound overshoot increased as the
hyperpolarizing current pulses increased. In response to larger negative current pulses the
rebound depolarization could initiate action potentials (fig. 6A). Type III neurons also appear
to possess an additional fast-activated inward rectifier, as the peak voltage responses exhibited
inward rectification over the same range of membrane potentials as the time dependent inward
rectification, (fig. 6D).
Morphology: Type III cells were characterized by small fusiform somata (diameter 10 - 14µm;
table 2) and thin, aspiny or sparsely spiny dendrites (figs. 6B, 7, 10). In fact, in 3 out of 6
neurons that were successfully reconstructed the dendrites were so thin that they could not be
Chapter 3: Cell types in the chick neostriatum caudolaterale 86
BA
- 66.5 mV
200 ms
20 mV
C
Spi
ke fr
eque
ncy
(Hz)
Time during stimulus (ms)
10
20
30
40
50
60
70
0 200 400 600 800 1000 1200
40 pA80 pA
120 pA140 pA180 pA220 pA240 pA
80
90
100
110 D
Pot
entia
l (m
V)
Current (pA)
-120
-100
-140
-80
-60
-40
-400 -200-300 -100 100 200
voltage at peak
voltage at end
0.2 nA
Figure 6: Intrinsic firing properties and membrane rectification of a type III cell. A: Voltage responses to hyperpolarizing anddepolarizing current pulses (-120 - 240 pA). There is a large sag in response to strong hyperpolarizing current pulses, whichresults in a rebound overshoot that is large enough to trigger spikes. Small depolarizing currents elicit continuous firing atcomparatively high frequencies. B: Camera lucida reconstruction of a type III neuron. Scale bar is 50 µM. C: Relationshipbetween the instantaneous spike frequency and time for different current amplitudes for the cell shown in A. There is somefrequency adaptation after the first few spikes, but the cell maintains a high-frequency firing without further attenuation over along current pulse. D: Current-voltage relationship of the cell shown in A. , voltage response measured at the end of the
current pulse; , voltage at the peak.
Chapter 3: Cell types in the chick neostriatum caudolaterale 87
clearly distinguished from the extensive local axonal ramifications. The remaining three cells
had relatively few long multipolar dendrites (diameter of dendritic field 227 - 267 µm). The
dendrites showed a low number of total branch points and especially few higher order (≥ 4th
branches) branchings (table 2). The axon appeared to have a large number of varicosities, and
arborized extensively to form a dense plexus of terminals in the vicinity of the soma (fig. 7).
Axons of type III neurons could never be followed beyond the borders of the NCL. The
diameter of axonal arborizations ranged between 545 - 736 µm and the mean brain area that
was covered by these arborizations was 320,400 µm2 (s.e. ± 35,503 µm2).
Type IV neurons:
The key features of type IV cells are a
high input resistance and a long
membrane time constant, as well as a
long action potential duration. Of the
90 cells characterized, 13 were found
to belong to this class.
Firing properties: Among the classes
identified here, type IV cells had an
intermediate RMP and spike-threshold.
Their bursting spike pattern resembled
that of type I cells (fig. 8), but type IV
neurons were able to initially fire a
larger number of action potentials
(table 1). However, in response to
large depolarizing currents the ability
of type IV neurons to fire repetitively
was markedly attenuated (fig. 8C). A
characteristic of cells in this class was
the long action potential duration,
which was also reflected in the
significantly longest rise- and fall-
times (table 1). Furthermore, the action
potentials of type IV cells showed a
prominent progressive spike
broadening during repetitive firing.
Figure 7: Morphological features of type III cells. A: Photomicrographof a Lucifer Yellow filled sparsely spineous type III cell. B: Detail ofthe cell shown in A. Through the middle of the photomicrograph runs along thin dendrite; parallel to this run axon collaterals above and below.C: Part of the local axonal arborizations of another type III cell. Scalebars are 50 µm (A, B), and 20 µm (C).
Chapter 3: Cell types in the chick neostriatum caudolaterale 88
Type IV cells had comparatively small monophasic AHPs, but long times-to-peak (table 1).
Membrane rectification: The key feature required for type IV cells was a long membrane time
constant (> 60 ms). They also had the highest input resistance (> 350 MΩ) among all classes of
cells (table 1) and showed steep current-voltage relationships (fig. 8C). Only at membrane
potentials more negative than about - 95 mV in most neurons a weak, fast-activated inward
A
- 67 mV
200 ms
20 mV
B
C
Time during stimulus (ms)
0 200 400
10
20
30
40
50
Spi
ke fr
eque
ncy
(Hz)
150 pA210 pA240 pA270 pA300 pA330 pA360 pA
600 800 1000 1200
DP
oten
tial (
mV
)
Current (pA)
-120
-100
-140
-80
-60
-40
-400 -200-300 -100 100 200
0.2 nA
Figure 8: Intrinsic firing properties and membrane rectification of a type IV cell. A: Voltage responses to hyperpolarizing anddepolarizing current pulses (-180 - 360 pA). The cell shows large voltage responses to small hyperpolarizing current pulses withrelatively little inward rectification. Small depolarizing current pulses readily elicit short bursts of action potentials. B: Cameralucida reconstruction of a type IV neuron. The soma and dendrites are drawn in black, the axon collaterals are shown in grey.Scale bar is 50 µM. C: Relationship between the instantaneous spike frequency and time for different current amplitudes for thecell shown in A. D: Current-voltage relationship of the cell shown in A. The input resistance is high, and the voltage responseshows only little inward rectification to large current pulses.
Chapter 3: Cell types in the chick neostriatum caudolaterale 89
inward rectification became evident (fig. 8D). All type IV cells showed a prominent membrane
outward rectification at depolarized potentials.
Morphology: Type IV cells were characterized by small, oblique somata (diameter 12 - 16,5
µm; table 2) and multipolar dendrites that possessed few thin spines. Usually, proximal
dendrites were short and thin, but sometimes 2 or 3 main dendrites were seen that had thick
stems but tapered considerably with distance from the soma (figs 8B, 9). The otherwise
spherical form of the dendritic field (diameter 180 - 225µm) was tilted by these dendrites as
they also extended further than the others. The dendritic tree of type IV neurons showed
relatively few branches and covered the smallest area among the four cell types (table 2).
Axonal arborizations of type IV cells were not as extensive as in type I or type II cells, but
projections followed the same pattern seen in the other classes of spiny neurons. One to three
main collaterals descended ventrally in the direction of the archistriatum, while other
collaterals travelled dorsomedially and/or ventromedially within the NCL.
Table 2: Morphological characteristics of classes of NCL neurons (means ± standard error)
Type I
(n = 15)
Type II
(n = 11)
Type III
(n = 6)
Type IV
(n = 5)
Significant differences*
Soma size (µm²) 217.8 (± 7) 252.6 (± 9) 121.8 (± 7) 142.6 (± 9) Type I, II > type III, IV
Total dendritic length (µm) 4832 (± 281) 5921 (± 354) 2228 (± 48)† 22212 (± 589) Type I, II > type III, IV
Area of dendritic
arborization (µm²)
56449 (± 4251) 59845 (± 4294) 44412 (± 538)† 28782 (± 3133) Type I, II > type IV
Number of branch points 51.7 (± 2.2) 61.9 (± 4.6) 24.6 (± 1.4)† 28.7 (± 0.3) Type I, II > type III, IV
Number of spines (n/10µM)initial segment
1.47 (± 0.59)‡ 2.91 (± 0.89)‡ − † 2.18 (± 0.78)‡ −
2nd branching 5.06 (± 0.22)‡ 6.60 (± 1.08)‡ 0.89 (± 0.32)† 2.87 (± 0.20)‡ Type II > type III, IV;Type I > type III
3rd branching 5.46 (± 0.35)‡ 6.89 (± 0.74)‡ 1.32 (± 0.31)† 2.83 (± 0.41)‡ Type I, II > type III, IV
4th branching 5.11 (± 0.87)‡ 6.59 (± 0.70)‡ 0.55 (± 0.02)† 2.96 (± 0.55)‡ Type II > type III, IV;Type I > type III
5th and 6th branching 5.71 (± 2.32)‡ 7.23 (± 1.03)‡ − † 2.82 (± 0.65)‡ n.a.
Direction of axon ventrally,dorsomedially,
andventromedially
ventrally,dorsomedially,
andventromedially
locally ventrally,dorsomedially,
andventromedially
* Significant differences indicated by the use of a Scheffé test for multiple comparisons after analysis of variance (P < 0.05). †
These values could be obtained in only three type III neurons (see text for details). ‡ Spine frequencies were counted in four
neurons of each class. n.a.: not enough cases for statistical comparison.
Chapter 3: Cell types in the chick neostriatum caudolaterale 90
Mechanisms underlying membrane rectification and firing properties in type I and type II
neurons:
In a number of neurons the mechanisms
underlying membrane rectification and
possibly burst generation were studied.
Due to the relatively low incidence of
recordings that could be obtained from
type III and type IV neurons under blind-
patch conditions these analyses were
restricted to type I and type II cells.
In NCL neurons inwardly rectifying
properties in the hyperpolarized voltage
range seem to depend on K+
conductances. Blockade of K+ channels
by extracellular CsCl (5 mM) abolished
fast inward rectification to
hyperpolarizing current pulses in 5 type
II neurons which showed rapidly
adapting burst firing and 2 regular
spiking neurons classified as type II
cells. Similarly, in a third type II neuron
which showed sag in the voltage
response to hyperpolarizing current
pulses under control conditions, both the
fast and the time-dependent inward
rectifying conductances were blocked by
CsCl (data not shown).
Rectification in the depolarized voltage range was studied in one type I neuron and two type II
neurons. The firing characteristics of these cells were recorded before and after adding 1 µM
tetrodotoxin (TTX) to the perfusate (figure 11). As expected for a Na+ dependent action
potential, TTX completely eliminated the generation of spikes in all cells tested. Under control
conditions the type I cell showed transient depolarizing „hump“ potentials in the subthreshold
range which preceeded the generation of spikes (fig 11A, left column). These hump-potentials
Figure 9: Morphological features of type IV cells. A, B: Twoexamples of Lucifer Yellow filled cells that had high input resistance,small action-potentials of long duration and showed only little inwardrectification to hyperpolarizing current pulses. C: Detail Scale bars are50 µm (A, B), and 20 µm (C).
Chapter 3: Cell types in the chick neostriatum caudolaterale 91
Figure 10: Comparison of the dendritic morphology and the appearance of local axonal arborizations in the 4 cell typesdescribed here. Somata and dendrites are drawn in green, axon collaterals in red. With regard to spine density and dendriticthickness the examples shown here for the type I and type II neurons represent extremes of what appeared to be a continuum.Scale bars are 50 µM.
Chapter 3: Cell types in the chick neostriatum caudolaterale 92
persisted in the presence of TTX (fig 11A, right column), indicating that they might be mediated
by a Ca++ conductance. Like most type I neurons this cell also showed membrane outward
rectification, measured at the end of the current pulse, both under control conditions and in the
presence of TTX. In the type II neurons there was a small anomalous rectification in the
depolarized voltage range under control conditions. The membrane potential tended to „sag“
back towards rest and an undershoot of the potential occured after cessation of the current pulse
(fig. 11B, left column). The underlying current that mediates the inward rectification in the
depolarized voltage range is sensitive to TTX blockade. After bath application of TTX
depolarizing pulses unveiled activation of an outward rectifying conductance (fig 11B, right
column).
In the course of other experiments (data not shown) we had observed spontaneously occuring
all-or-none bursts in type I and type II neurons when picrotoxin (100 µM), a Cl- channel blocker
that eliminates the fast inhibitory effects of GABAergic interneurons, was added to the external
solution. In these spontaneous bursts usually 4 - 6 spikes rode on a depolarizing envelope that
was followed by an afterhyperpolarization of relatively long duration (tens to hundreds of ms)
Type I200 ms
20 mV
A control
- 69.5 mV
1µM TTX
Type II
- 70.5 mV
B control
200 ms
20 mV
1µM TTX
0.5 nA
0.5 nA
Figure 11: Membrane rectification in a type I neuron (A) and a type II neuron (B) before (left traces) and after blockadeof sodium channels by bath-application of tetrodotoxin (TTX, right traces). A: Under control conditions single spikes werefollowed by depolarizing afterpotentials in this type I neuron. Application of TTX abolishes the fast action potentials, butsmall transient depolarizing potentials persist at the beginning of strong depolarizing current pulses. This neuron also showedoutward rectification at depolarized potentials under both conditions. B: Under control conditions this type II neuron showedinward rectification and sag in the response to depolarized current pulses, and a voltage undershoot after the cessation ofcurrent pulses. Application of TTX abolishes the inward rectification and the undershoot.
Chapter 3: Cell types in the chick neostriatum caudolaterale 93
(fig. 12). In addition, when depolarizing
current steps were applied from
hyperpolarized potentials (about -90 mV)
both type I and type II cells responded
with an increase in the number of spikes,
indicating the contribution of a voltage-
gated Ca++ conductance, that is normally
inactive at more positive holding
potentials (e.g. Llinás and Yarom, 1981).
However, changing the cells holding
potential with DC injections never
changed the cell´s firing pattern (c.f.
McCormick and Feeser, 1990; Livingston
and Mooney, 1997).
Taken together, these observations indicated that calcium conductances contribute to the firing
behaviour of cells in the NCL. To test this assumption an additional 7 neurons (8 days post-
hatch) were recorded under conditions suitable to evoke calcium spikes. Electrodes were filled
with CsCl (135 mM) and tetraethylamonium (TEA; 20 mM) to block most K+ conductances.
Immediately following establishment of the whole-cell configuration, a cells firing pattern was
assessed in voltage-clamp. Of the 7 cells
tested, 4 cells showed firing
characteristics of type I cells, while the
remaining 3 neurons maintained repetitive
spiking throughout the duration of the
depolarizing potential step. Following
theses measurements TTX (1 µM) was
bath applied. When K+ conductances were
blocked all neurons tested were able to
generate slow action potentials in the
presence of 1 µM TTX (fig. 13). Such
slow action potentials are most likely
mediated by Ca++ influx (c.f. McCormick
and Prince, 1987).
mAHP
DAP
mAHP
100 ms
20 mV
Figure 12: Spontaneous bursts of action potentials occurred in typeI and type II cells when GABAergic inhibition was diminished byadding picrotoxin (100 µM) to the extracellular solution. In thesebursts 4 - 6 fast action-potentials rode on a slowly developingdepolarizing envelope that was followed by a fastafterhyperpolarization (fAHP) and a medium-durationafterhyperpolarization (mAHP), often with an intercalateddepolarizing afterpotential (DAP), see insert.
Figure 13: Examples of Ca++ spikes that could be evoked in largespiny neurons (n = 7) when K+ conductances were blocked byadding tetraethylamonium (TEA; 20 mM) and CsCl (135 mM) to thepipette solution, and sodium conductances were blocked byextracellular tetrodotoxin (TTX; 1µM).
Chapter 3: Cell types in the chick neostriatum caudolaterale 94
DISCUSSION
We have shown the existence of at least 4 celltypes within the NCL of chicks that differ in their
intrinsic electrophysiological and morphological properties. In general, there was a good
correspondence of physiological and morphological criteria, although the two main celltypes,
type I and II, could not be clearly differentiated by morphological criteria. Neurons from
animals between 2 and 11 days post hatch were studied. As chicks are relatively mature at
hatching and capable of independent action most behavioural studies that test the animals ability
to learn are undertaken within the first two weeks after hatch. As discussed below, several
electrophysiological characteristics that differentiated among neurons between (and within)
celltypes showed some degree of variation related to age, thus probably reflecting
developmental changes. In addition, our finding of 4 main celltypes does not exclude the
existence of further classes of neurons within NCL.
Cell types:
Type I: Type I neurons possessed a high action potential threshold, particularly in relation to
their low resting potential, and they showed prominent outward rectification in response to
depolarizing current pulses. These properties indicate that strong, temporally or spatially
integrated excitatory inputs are neccessary for type I neurons to fire. If sufficiently depolarized,
cells in this class preferentially responded with bursts of action potentials. Interestingly, in the
mammalian cortex it has also been observed that the threshold of bursting neurons is sometimes
higher than that for neurons generating single spikes (Mason and Larkman 1990).
We have used the occurrence of transient „hump“-like depolarizing responses to describe
subsets among the neurons which were grouped as type I cells, according to their burst-firing
behaviour. Neurons that displayed prominent subthreshold „hump“-potentials and those that did
not, differed in several basic parameters, i.e. input resistance, spike amplitude, spike duration
and rise time. In mammals these properties have been shown to be particularly prone to
developmental changes during early postnatal development (McCormick and Prince, 1987;
Kasper et al., 1994b). Thus it is conceivable that the differences among type I neurons reflect at
least in part developmental differences. Cells showing transient „hump“-like depolarizing
responses were indeed recorded in slices from animals with higher mean ages (7.1 days vs. 5.4
days), a difference that was statistically significant (df = 32; t = -2.7; p < 0.05; Student´s t test
for unpaired samples). Similarly, developmental differences might explain the occurrence of a
complex pattern of afterpotentials in some, but not all, type I and type II neurons. Single action
potentials or bursts were sometimes followed by three distinct afterpotentials: an initial fast,
and a late slow after-hyperpolarization, separated by an intercalated depolarizing
Chapter 3: Cell types in the chick neostriatum caudolaterale 95
afterpotential. In mammalian cortical neurons the two phases of the AHP appear to be due to the
activation of Ca++-dependent K+ currents (Lancaster and Adams, 1986; Schwindt et al., 1988)
whereas the DAP results from the activation of a variety of Ca++ dependent currents (Higashi,
1993; Yang et al., 1996; Haj-Dahmane and Andrade, 1997). The expression of the DAP and the
emergence of a tripartionate AHP have been shown to depend on changes during postnatal
development (Kasper et al., 1994b). Similarly, in mammals burst firing does not develop
before the third postnatal week (Kasper et al., 1994b).
A common factor pertinent to the occurrence of either the „humps“ and the DAPs, as well as the
expression of full bursting behaviour in our preparations might be an increase in Ca++
conductances during development. Calcium currents are believed to underlie burst firing in
mammalian central neurons (Wong and Prince, 1981; Llinás and Jahnsen 1982; Friedman and
Gutnick, 1987; Schwindt and Crill, 1999). Notably, the generation of transient depolarizing
potentials by low-threshold Ca++ currents can result in bursts of fast Na+/K+ action potentials
(Llinás and Jahnsen 1982; McCormick and Feeser, 1990; Yang et al., 1996).
When the network activity in the slice was increased by diminishing the inhibitory control of
GABAergic interneurons, spontaneous burst could be seen in type I and II cells that were
clamped at or just below resting potential. In these bursts short-duration action potentials rode
on a slowly activated depolarizing envelope, resembling the plateau potentials generated by
low-threshold Ca++ currents in mammalian central neurons (Llinás and Jahnsen 1982;
McCormick and Feeser, 1990; Yang et al., 1996; Magee and Carruth, 1999). A contribution of a
low threshold Ca++ current to the firing behaviour of the large projection neurons was further
indicated by the fact that preconditioning hyperpolarization of the membrane potential led to an
increased number of spikes in response to a depolarizing current pulse. Also, in addition to
high-threshold Ca++ spikes, transient „hump“-like depolarizing potentials similar to those
occuring in some type I neurons under control conditions could be evoked in the presence of
TTX and TEA. Furthermore, about half of the type I neurons that showed „hump“-like potentials
(6 of 13) also showed prominent DAPs following single spikes. As briefly outlined above the
expression of DAPs depends either directly on low-threshold Ca++ currents (Zhang et al., 1993;
Yang et al., 1996) or the activation of other Ca++ dependent currents (Higashi, 1993; Haj-
Dahmane and Andrade, 1997). Functionally, depolarizing afterpotentials appear to be related to
a neurons ability to fire bursts of action potentials (Wong and Prince, 1981; Chagnac-Amitai et
al., 1990; Mason and Larkman, 1990; Yang et al., 1996; Magee and Carruth, 1999). And it has
been postulated that the Ca++ entry resulting from the DAP is responsible for long-term-
potentation (LTP) because manipulations that eliminate the DAP also block the induction of
LTP (Komatsu and Iwakiri, 1992). While in sum these findings strongly implicate the
Chapter 3: Cell types in the chick neostriatum caudolaterale 96
contribution of Ca++ influx in the emergence of burst firing in type I and type II cells, it should
be noted that other components such as a sodium current (Schwindt et al., 1988; Franceschetti et
al., 1995; Magee and Carruth, 1999) or outward currents (Schwindt et al., 1988: Kang and
Kayano, 1994) might also be involved in the generation of bursts. Taken together, subthreshold
potentials appear to serve an important role in sculpturing a cells final output (Hamill et al.,
1991), and their occurrence in the large spiny neurons of type I and II might reflect the
integrative role these cells have in combining the diverse multimodal synaptic inputs that reach
the NCL from various sensory and associative areas (Metzger et al., 1998; Kröner and
Güntürkün, 1999). Furthermore, a neurons ability to fire bursts of action potentials will
probably enhance its integrative role within a network and aide the plasticity of synaptic
connections: Burst firing increases the probability to drive the postsynaptic cell beyond spike
threshold (Snider et al., 1998). The high frequency firing of action potentials during a burst is
thus thought to amplify a neural signal and to synchronize the activity in a population of
postsynaptic cells, both temporarily and spatially (Hablitz, 1986; Snider et al., 1998).
Type II: An initial tonic firing and a relatively low action potential threshold that characterize
type II neurons may indicate that the firing of these cells is readily elicited by weak excitatory
inputs. The occurrence of spontaneous burst in the absence of GABAergic inhibition and the
phasic-tonic firing pattern that often emerged with large depolarizing currents indicates that
type II neurons will respond strongly but transiently to a brief, new stimulus, yet produce a
sustained response to a maintained input. In contrast to the behaviour of neurons in the burst
firing mode, a pattern of single action potentials can give a more faithful representation of the
characteristics of depolarizing inputs. Functionally, the ability of type II neurons to generate a
tonic firing mode could also enable these cells to retain informations of their input for a short
time period. It is known that NCL-lesions in pigeons cause specific impairments in a variety of
delay tasks (Mogensen and Divac, 1982, 1993, Gagliardo et al., 1996, 1997, Güntürkün, 1997,
Güntürkün and Durstewitz, in press). In these experiments, the animal has to hold online a
specific information provided during a previous cue period to perform a correct response after
delay offset. In mammals, neurons in the deep layers of the prefrontal cortex (PFC) have been
identified as a cellular substrate for short term memory (see Goldman-Rakic, 1996 for review).
When the activity of these cells is recorded in awake rats or primates performing a delayed-
response task, a significant enhancement in their firing rate can be observed during the delay
(e.g. Fuster and Alexander, 1971; Funahashi et al., 1989; Sawaguchi and Yamane, 1999). Thus,
sustained delay activity of PFC neurons may provide the animal with the ability to hold an
internal representation of relevant aspects of the external world which is needed for planning or
Chapter 3: Cell types in the chick neostriatum caudolaterale 97
organizing subsequent responses (Goldman-Rakic, 1996). Neurons with similar delay activities
to those recorded from rat or primate PFC have been observed in vivo in the pigeon’s NCL
during a delayed auditory-visual Go/No-Go task (Kalt et al., 1999). Like their mammalian
counterparts, these cells showed significant alterations of their firing mode during the delay
period. It is conceivable that the type II neurons of the present study may hold the information of
their synaptic inputs within a recurrent network and thus provide a part of the cellular substrate
for the delay activity within the NCL.
At least some type II neurons furthermore showed a small transient sag in the voltage response
to hyperpolarizing currents, which is likely to be mediated by a voltage- and time-dependent
hyperpolarization activated mixed cationic current called Ih (Pape, 1996; see also discussion of
type III below). It has been shown that a time-dependent-inward rectifier produces membrane
resonance (Hutcheon et al., 1996). When resonance is present, neurons fire action potentials
preferentially to rhythmic (oscillatory) inputs. The membrane resonance produced by Ih can be
amplified by a persistent sodium current (Hutcheon et al., 1996). In type II neurons we
observed inward rectification in the depolarized voltage range that may be indicative of a
persistent sodium current (Stafstrom et al., 1985, Yang et al., 1996). The existence of such a
current has already been demonstrated directly in neurons from the caudal forebrain of
songbirds which possess similar firing properties and morphological characteristics as the type
II neurons described here (Kubota and Saito, 1991; Kubota and Taniguchi, 1998). Thus type II
neurons might represent members of a common class of spiny projection neurons in the dorsal
forebrain of birds that can respond preferentially to input of a specific pattern.
Type III: The electrophysiological and morphological characteristics of the type III cells
resemble those of γ-aminobutyric acid-containing inhibitory interneurons found in the
mammalian telencephalon (Schwartzkroin and Mathers, 1978; McCormick et al., 1985;
Kawaguchi, 1995); i.e. little or no accomodation of spike frequency, and beaded aspiny
dendrites. Type III cells offer a wide-band responsiveness and sustained high-frequency firing
if neccessary. They are thus able to perform a very reliable input-output conversion, retaining
the temporal pattern of their synaptic inputs. These firing characteristics are facilitated by the
short rise and fall times of the action potential (table 1). In addition, the relatively pronounced
AHPs of type III cells might also contribute to the ability to fire actions potentials at high
frequencies in that they reduce the accumulation of depolarization-dependent Na+ channel
inactivation, which would otherwise reduce action potential amplitudes during long trains of
high frequency firing (Hamill et al., 1991). It will also reduce the influx of Ca++ into the cell
and thus will diminish the effects of Ca++-activated K+ channels which otherwise might lead to
Chapter 3: Cell types in the chick neostriatum caudolaterale 98
prolonged hyperpolarization and spike frequency adaptation (Lancaster and Adams, 1986;
Storm, 1990).
A prominent feature in all type III cells was the existence of the strong inward-rectifying current
Ih that initiates slow depolarization if the membrane potential has become negative (e.g. by a
GABAB receptor-mediated IPSP or a postspike AHP). In central neurons of mammals Ih is a
mixed Na+/K+ current that has been implicated in the determination of the resting potential and
the generation of „pacemaker“ potentials. It also serves to decrease the propagation of
subthreshold voltage potentials in dendritic trees, thereby regulating the integration of synaptic
inputs (McCormick and Pape, 1990; Magee, 1998, 1999; see Pape, 1996 for review). In
mammalian cortex GABAergic interneurons are long recognized for such various functions as
maintaining the normal integrative properties of the cortex (Sillito, 1984), the control of spread
of activity (Chagnac-Amitai and Connors, 1989) and the synchronization of adjacent projection
neurons via inhibitory phasing (Cobb et al., 1995; Benardo, 1997), which might impose a
rhythm on the activity of the principal neurons, providing a „context“ to the „content“ of their
operation (Buzsáki and Chrobak, 1995). In the prefrontal cortex interneurons also appear to
posses own sensory or „mnemonic“ fields that enable them to fine tune the delay activity of
adjacent pyramidal neurons in a working memory task (Rao et al., 1999). With respect to the
extensive local axonal arborizations of each type III neuron these putative interneurons of the
NCL presumably also can control a large number of adjacent spiny „principal“ neurons (i.e.
types I and II), as is the case in mammalian cortex (e.g. Sik et al., 1995). In sum, given their
basic intrinsic electrophysiological and morphological properties type III neurons of the NCL
are equipped to play a similarly important role for signal integration as their mammalian
counterparts.
Type IV: Type IV neurons were characterized by high input-resistances, long time constants and
a long duration of action potentials. These electrophysiological properties are also
characteristic for developing cortical neurons in mammals (Kasper et al., 1994b; McCormick
and Prince, 1987) and possibly birds (Kubota and Taniguchi, 1998). McCormick and Prince
(1987) have argued that higher input resistances and longer membrane time constants of
immature neurons could increase the magnitude and duration of their responses to imposed
synaptic currents. Such cells should also be electrotonically compact, showing relatively little
cable attenuation of distal dendritic synaptic inputs, thus compensating for similarly poorly
developed synaptic inputs in newborn animals. This is in good agreement with the
morphological appearence of our type IV cells identified here which possesed small somata,
and short, sparsely spineous dendrites with relatively few branchings.
Chapter 3: Cell types in the chick neostriatum caudolaterale 99
Similarly, the long duration of action potentials seen in type IV neurons may serve to facilitate
the output of developing cells: Activity-dependent spike broadening due to a reduction in
specific K+ currents has been correlated with increased efficacy of synaptic transmission onto
the postsynaptic target cell (Mercer et al., 1991; Byrne and Kandel, 1996 for review).
Similarly, the significantly longer action potentials of developing neurons might result in an
enhancement of synaptic transmission onto their postsynaptic cells (Bottjer et al., 1998). An
open question remains whether type IV cells constitute a distinct class of late maturing neurons
or simply represent developing cells of the other spiny celltypes (types I and II, respectively).
However, all physiological and morphological characteristics of type I and II neurons were
already present at the earliest ages recorded. Moreover, type IV cells indeed were recorded in
slices from animals with the highest median age (7 d post hatch). Bradley and coworkers
(1996) have proposed yet another mechanism that may account for developmental and
experience-dependent changes in input resistance that occur in neurons of the chicks IMHV.
They argue that existing large neurons with numerous spines might be morphologically
remodelled to produce cells with significantly higher resistance to render them more
susceptible to presynaptic inputs. It has been shown that in the NCL auditory imprinting reduces
the spine densities on the medial and distal segments of large multipolar neurons, which
correspond most likely to both types I and II of the present study (Bock and Braun, 1999a,b).
While the resulting decrease in membrane surface should lead to a decrease in capacitance and
an increase of apparent resistance, these changes are probably too subtle to account for the
large differences in morphology and electrophysiology observed between type IV neurons and
neurons of the other spiny cell classes.
Connectivity and sensorimotor integration
An important requirement for sensorimotor integration and associative learning is the ability of
a brain structure to combine sensory information from various sources and to affect the animals
behaviour either directly or via motoric and limbic structures downstream. Tracing studies in
vivo have revealed that the NCL receives converging input from the secondary sensory areas of
all modalities, as well as from other associative areas which themselves appear to integrate
information from more than one modality (Metzger et al., 1998; Kröner and Güntürkün, 1999).
Although some degree of regional specialization within NCL might exist, the pattern of afferents
strongly indicates that in large areas of the NCL single neurons might receive multimodal input.
In the pigeon single unit recordings in vivo have shown that neurons in the NCL can encode
Chapter 3: Cell types in the chick neostriatum caudolaterale 100
aspects of a memory task across modalities (Kalt et al., 1999). The same neurons might be
involved in a cell assemblies that „hold“ information over a delay or initiate a motor response
via the archistriatum. In the chick Metzger and coworkers (1998) have shown that single
neurons within the NCL send axon collaterals to the archistriatum and another multimodal
forebrain area, the intermediate, medial part of the hyperstriatum ventrale (IMHV). Our findings
provide further insight into the anatomical arrangement that might underlie the integration of
various sensory features and the propagation of the NCL´s output to other forebrain areas.
Morphologically, single cells in all spiny cell classes of the NCL seem to be able to relay their
transformed input not only to neighbouring cells within NCL, but also to sensorimotor and
limbic areas downstream, namely the archistriatum and the basal ganglia, and possibly also
provide feedback to the sensory areas the original inputs come from. With the exception of the
small type IV cells neurons in the NCL had fairly large dendritic fields (see table 2). The extent
of the local axon collaterals for the spiny cell classes (types I, II, and IV) can be estimated at a
lower limit of about two or three times the area of a given cells dendritic field (c.f. figs. 5, 10).
Type III neurons had relatively symmetric axonal fields and their collaterals did not leave the
NCL. The diameter of the area covered by axonal arborizations of these cells ranged between
545 - 736 µm (mean area 320,400 µm2). A hypothetical basic „circuit“ consisting of a type III
interneuron and two postsynaptic spiny neurons at the rim of the axonal field would thus cover
approximately 650,000 µm2. Such a basic circuit could thus participate in intra-NCL
computations which surpass areas of representation of a single sensory modality. In addition,
most spiny cells were seen to send several long collaterals to targets outside the NCL. On their
way these collaterals often span the whole medio-lateral and/or dorso-ventral extent of the
NCL (c.f. fig. 5). Although the determination of the rostro-caudal extent of these intra-NCL
connections remains a critical topic which was constrained by the thickness of the slices used
here, it appears that these connections enable a stimulus comparison across most, if not all,
possible sensory compartments of the NCL (c.f. Metzger et al., 1998; Kröner and Güntürkün,
1999).
Based on similarities in the pattern of connections and the location in the dorsocaudal forebrain
it has been suggested that the Nd, the auditory subunit of the NCL might be related to the
sensorimotor „high-vocal center“ (HVC) of songbirds (Wild, 1994; Metzger et al., 1998;
Kröner and Güntürkün, 1999). Nucleus HVC is essential for song production and shows
selective responses to complex auditory stimuli. Two pathways arise from within HVC that
converge on the same premotor nucleus of the archistriatum. The first is a direct projection from
HVC to the robust nucleus of the archistriatum (RA), which serves as the primary motor
pathway for song production, and can also carry auditory information to RA. The other
Chapter 3: Cell types in the chick neostriatum caudolaterale 101
projection of HVc begins a pathway through the anterior forebrain that is crucial for song
learning but, although active during singing, is not essential for adult song production. In this
pathway Area X of the lobus parolfactorius, a motor structure of the basal ganglia, is the first
target of the HVC (Nottebohm et al., 1982; Fortune and Margoliash, 1995). In the HVC of zebra
finches Area X and RA projecting neurons constitute two separate populations which resemble
type I and II neurons of the present study with regard to intrinsic electrophysiological
characteristics and morphology (Dutar et al., 1998; Kubota and Taniguchi, 1998). Neurons that
project to RA show inward and outward rectification in the subtreshold range (Dutar et al.,
1998; Kubota and Taniguchi, 1998) and possibly a strong accomodation in the firing pattern
(Dutar et al., 1998; but see Kubota and Taniguchi, 1998), comparable to that seen in at least
some type I neurons described here. Area X projecting neurons show a time-dependent inward
rectification and regular spiking pattern very similar to that described for type II cells here
(Dutar et al., 1998; Kubota and Taniguchi, 1998). However, our finding that almost all spiny
neurons of the NCL sent at least one axon collateral in the direction of the archistriatum and
sometimes also in the direction of the basal ganglia makes it unlikely that a similar distinction
between cell types exists as in the songbird forebrain. These differences might reflect the
specialization of the HVC relevant for singing and the need to exert control over specific
aspects of singing related motor behaviour. Yet in the songbird brain parts of the lateral
neostriatum and the „shelf“ region which adjoin the HVC share most afferents with the HVC
(Fortune and Margoliash, 1995; Vates et al., 1996; Foster et al., 1997; Iyengar et al., 1999) and
also show a similar pattern of projections (Fortune and Margoliash, 1995; Mello et al., 1998;
Iyengar et al., 1999), therefore these areas might resemble the NCL more closely.
In sum, our findings provide a framework for further study into sensorimotor integration and
associative learning in chicks and possibly other birds.
Acknowledgement: The authors would like to thank Markus Hausmann, Burkhard Hellmann, Thomas Kalt, Ralf
Mohrmann, Simon Rumpel and Markus Werner, who all gave valuable comments and technical support at
various stages of this study. This work was supported by grants of the DFG (GU 227/5 - 1 and SFB 509).
Chapter 4: Dopaminergic innervation of the avian telencephalon 102
The Dopaminergic Innervation of the Avian Telencephalon
Daniel Durstewitz, Sven Kröner and Onur Güntürkün
Table of contents
1. Introduction
2. Overview over the neuroanatomy of the avian telencephalon
3. Origin of dopaminergic fibers in the avian telencephalon: Mesencephalic A8-A10 cell groups
4. General features of the dopaminergic innervation of the avian telencephalon
4.1. 'Basket'- and 'en passant'-type of dopaminergic innervation
4.2. Tyrosine hydroxylase as a marker of dopaminergic fibers in the avian telencephalon
4.3. DARPP-32 as a marker of D1-receptors and dopaminoceptive neurons
4.4. Ultrastructural features of the dopaminergic innervation and of dopaminoceptive neurons
5. Distribution of dopaminergic fibers and dopaminoceptive elements in the avian telencephalon
5.1. Primary sensory areas
5.1.1. Dopaminergic fibers and projections
5.1.2. D1 receptors
5.1.3. D2 receptors
5.1.4. DARPP-32
5.1.5. Colocalizations
5.2. Secondary sensory and multimodal areas
5.2.1. Dopaminergic fibers and projections
5.2.2. D1 receptors
5.2.3. D2 receptors
5.2.4. DARPP-32
5.2.5. Colocalizations
5.3. Septum, hippocampal complex, archistriatum
5.3.1. Dopaminergic fibers and projections
5.3.2. D1 receptors
5.3.3. D2 receptors
5.3.4. DARPP-32
5.3.5. Colocalizations
5.4. Basal ganglia
5.4.1. Dopaminergic fibers and projections
5.4.2. D1 receptors
5.4.3. D2 receptors
Chapter 4: Dopaminergic innervation of the avian telencephalon 103
5.4.4. DARPP-32
5.4.5. Colocalizations
6. Comparative aspects of the dopaminergic system in mammalian and avian species
6.1. Basal ganglia
6.2. Cortex
6.3. Laminae-specific distribution of dopaminergic fibers
6.4. Ultrastructural features and postsynaptic targets of the dopaminergic innervation
7. Behavioral studies and functional implications
7.1. Dopaminergic modulation of (unconditioned) motor functions
7.2. Dopaminergic involvement in arousal and wakefulness
7.3. Dopaminergic modulation of appetitive and consummatory behavior
7.4. Dopaminergic modulation of learning and conditioned behavior
7.5. Dopaminergic modulation of working memory
7.6. The possible function of dopamine in sensory-motor processes
Abstract
The present review provides an overview of the distribution of dopaminergic fibers and dopaminoceptive
elements within the avian telencephalon, the possible interactions of dopamine with other biochemically
identified systems as revealed by immunocytochemistry, and the involvement of dopamine in behavioral
processes in birds. Primary sensory structures are largely devoid of dopaminergic fibers, dopamine receptors,
and the D1-related phosphoprotein DARPP-32, while all these dopaminergic markers gradually increase in
density from the secondary sensory to the multimodal association and the limbic and motor output areas.
Structures of the avian basal ganglia are most densely innervated but, in contrast to mammals, show a higher D2
than D1 receptor density. In most of the remaining telencephalon D1 receptors clearly outnumber D2
receptors. Dopaminergic fibers in the avian telencephalon often show a peculiar arrangement where fibers coil
around the somata and proximal dendrites of neurons like baskets, probably providing them with a massive
dopaminergic input. Basket-like innervation of DARPP-32-positive neurons seems to be most prominent in the
multimodal association areas. Taken together, these anatomical findings indicate a specific role of dopamine in
higher order learning and sensory-motor processes, while primary sensory processes are less affected. This
conclusion is supported by behavioral findings which show that in birds, as in mammals, dopamine is
specifically involved in sensory-motor integration, attention and arousal, learning, and working memory. Thus,
despite considerable differences in the anatomical organization of the avian and mammalian forebrain, the
organization of the dopaminergic system and its behavioral functions are very similar in birds and mammals.
Progress in Neurobiology 59:161-195 (1999)
Chapter 4: Dopaminergic innervation of the avian telencephalon 104
1. Introduction
The neuromodulatory dopaminergic system of mammals and birds is critically involved in
numerous cognitive and behavioral functions, including appetitive and aversive behaviors
(Balthazart et al., 1997; Bertolucci-D'Angio et al., 1990b; Deviche, 1984; Salamone, 1992,
1994; Sokolowski et al., 1994), states of arousal and wakefulness (Ferrari and Giuliani, 1993;
Ongini, 1993), motor control (Goodman et al., 1983; Rieke, 1981, 1982; Salamone, 1992;
Sokolowski & Salamone, 1994; Waddington and Daly, 1993), learning and memory (Beninger,
1993; Gruss and Braun, 1997; McDougall et al., 1987; Schultz et al., 1995), as well as working
memory and attention (Güntürkün & Durstewitz, in press; Montaron et al., 1982; Roberts et al.,
1994; Sawaguchi and Goldman-Rakic, 1991, 1994; Schultz et al., 1993; Seamans et al., 1998;
Zahrt et al., 1997). Since structure and function are tightly coupled in neural systems,
information about the neuroanatomical organization of the dopaminergic system is required to
unravel the functional role of dopamine (DA) in these various cognitive and behavioral
processes. A large body of evidence indicates that the principal function of dopamine is very
similar in mammals and birds. Since members of these two classes of vertebrates have highly
developed cognitive skills (e.g., Epstein et al., 1984; von Fersen et al., 1990; Lanza et al.,
1982; Lubow, 1974) but radically different forebrain organizations, comparative studies may
uncover the invariant structural features which enable the dopaminergic system to exert its
specific functions.
The present article provides an overview over the structural organization of the dopaminergic
system in avian species, compares it to mammals, and discusses behavioral findings and
possible functional implications. However, this review will be restricted in two ways: First,
almost all studies that dealt specifically with markers of the dopaminergic system in the avian
brain (i.e., DA, DA receptors, DARPP-32) have been conducted within the last decade, and the
present review will focus mainly on these studies. Older literature on the general distribution of
markers of the monoaminergic or catecholaminergic systems are covered in the excellent
review by Reiner et al. (1994). Second, most of our knowledge on the dopaminergic
innervation of the bird brain derives from studies in pigeons and chicks. However, the few
available studies from other avian species make it likely that the results obtained in these avian
species generalize to other birds as well.
Chapter 4: Dopaminergic innervation of the avian telencephalon 105
2. Overview over the neuroanatomy of the avian telencephalon
The avian telencephalon consists of nuclear structures, and mostly lacks the layered
organization that is characteristic of mammalian isocortex (Figure 1). This lack of lamination
led early investigators to assume that most of the avian telencephalon consists merely of a
hypertrophied striatum, and accordingly most forebrain areas were termed with the suffix '-
striatum', and this nomenclature remains still in use (Ariëns-Kappers et al., 1936; Edinger,
1903). On the basis of embryological (Källén, 1953, 1962; Kuhlenbeck, 1938; see also
Striedter et al., 1998) cytoarchitectonic (Rehkämper et al., 1984, 1985, Rehkämper and Zilles,
1991), hodological (Karten, 1969; Karten and Dubbeldam, 1973; Veenman et al., 1995) and
Figure 1. Morphology of the avian telencephalon (brain of a pigeon, Columba livia). (A-C) A series of hemispheres stained forcresyl-violet, indicating the location of several major structures. Rostro-caudal positions of the slices shown are approximately A4.25 (A), A 7.25 (B), and A 10.00 (C) according to the pigeon brain atlas of Karten and Hodos (1967). (D) Detail from C,showing part of the basal ganglia (PA, PP), the primary recipient area of the visual tectofugal pathway (ectostriatum), and apresumed multimodal area (NIM). (E-G) Prototypical examples of cells from the pigeon telencephalon that were labeled byintracellular filling (E) or Golgi impregnation (F, G). All cells display multipolar dendritic fields, a feature that seems to beassociated with the organization of the avian forebrain in nuclei rather than layers. Scale bars represent 1 mm (A, B, C), 50 µm(D), 25 µm (E, F, G). See list for abbreviations.
Chapter 4: Dopaminergic innervation of the avian telencephalon 106
histochemical (Juorio and Vogt, 1967) criteria, however, it became clear that large parts of the
telencephalon could be regarded as pallial, and as such might constitute possible cortex
equivalents.
The flow of sensory information in the avian telencephalon follows a common pattern for all
modalities: The primary sensory areas which receive subtelencephalic input, relay their
information to adjacent secondary sensory structures, which in turn project in parallel to
presumed multimodal areas as well as to motor and limbic structures, including the basal
ganglia (Figure 2).
As in all amniotes, two main visual pathways convey visual information from the retina to the
telencephalon in birds. These are the thalamofugal and the tectofugal pathway. The tectofugal
pathway may correspond to the mammalian colliculo-thalamo-extrastriate pathway, whereas the
thalamofugal pathway is comparable to the mammalian geniculostriate pathway (review by
Güntürkün, 1991). The ectostriatum (E) is the primary sensory structure of the tectofugal
pathway (Benowitz and Karten, 1976; Kondo, 1933). From there intratelencephalic projections
lead to the surrounding ectostriatal belt (Karten and Hodos, 1970; Ritchie, 1979). The
thalamofugal visual pathway consists of a bilateral projection from a retinorecipient nucleus in
the dorsal thalamus onto a thin band of granule cells, the intercalated nucleus of the
hyperstriatum accessorium (IHA), as well as to the lateral part of the hyperstriatum dorsale
(HD), which together constitute the primary sensory areas within the so-called Wulst. The IHA
and HDl in turn project to secondary sensory structures within the Wulst, mainly to the
hyperstriatum accessorium (HA; Karten et al., 1973; Shimizu et al., 1995).
The same organization applies to a part of the somatosensory system which occupies the rostral
extent of the Wulst, and that seems to be comparable to the mammalian primary somatosensory
cortex (Karten, 1971; Medina et al., 1997): Somatosensory fibers from the dorsal thalamus
terminate in the HD and the IHA (Delius and Bennetto, 1972, Funke, 1989, Wild, 1997) which
in turn project to the rostral HA (Wild, 1987). Another primary somatosensory telencephalic
area is located within the nucleus basalis (Bas). The Bas mainly processes trigeminal
information (Schall et al., 1986), but also receives additional somatosensory and auditory input
(Arends and Zeigler, 1986; Wild and Farabaugh, 1996; Wild et al., 1997). It relays this
information to the overlying frontal neostriatum (NF; Wild and Farabaugh, 1996; Wild et al.,
1985).
The main telencephalic area reached by auditory input from the thalamus is the so-called Field
L complex in the caudomedial neostriatum (Karten, 1968). Field L2 is the primary sensory
Chapter 4: Dopaminergic innervation of the avian telencephalon 107
structure of the auditory system, which projects to adjacent fields L1 and L3 (Bonke et al.,
1979; Karten, 1968; Wild et al., 1993).
The secondary sensory areas of all modalities are reciprocally connected with a region in the
caudolateral neostriatum, which in the pigeon is referred to as NCL (Kröner and Güntürkün,
1999; Leutgeb et al., 1996; Metzger et al., 1998). Other multimodal areas probably exist in the
intermediate aspect of the hyperstriatum ventrale (IMHV) and the intermediate neostriatum
(NIM; or mediorostral neostriatum/hyperstriatum ventrale, MNH, in chicks). Both these areas
have been studied extensively in chicks in the context of imprinting (Bradley et al., 1985;
Bredenkötter and Braun, 1997; Metzger et al., 1996; reviews: Horn, 1998; Scheich, 1987). In
addition, a system of "cortico"-striatal projection neurons which occupies the most dorsal and
lateral extent of the external pallium (PE; Brauth et al., 1978; Veenman et al., 1995) includes,
from rostral to caudal, the lateral frontal neostriatum (NFL), the lateral intermediate neostriatum
(NIL), the area temporo-parieto-occcipitalis (TPO) and the area corticoidea dorsolateralis
(CDL).
Figure 2. Schematic overview of the functional anatomy of the pigeon telencephalon as described in Section 2. Brain areas thatare still poorly understood with regards to their connections and functions were left blank. It should be noted, however, that thehyperstriatum intercalatus superior (HIS) probably receives secondary and, to a limited extent, possibly also primary visual input.The hyperstriatum ventrale (HV) is a highly heterogeneous region which probably encompasses multiple polysensorysubdivisions.
Chapter 4: Dopaminergic innervation of the avian telencephalon 108
At the next level of information processing in the avian brain, sensory information is funneled in
parallel from both, the tertiary as well as the secondary sensory areas to the basal ganglia and,
in addition, to the ventrolaterally located archistriatum (e.g. Bradley et al., 1985; Csillag et al.,
1994; Kröner and Güntürkün, 1999; Leutgeb et al., 1996; Metzger et al., 1998; Phillips, 1966;
Shimizu et al., 1995; Wild et al., 1985, 1993). Functionally, the avian archistriatum has been
divided into two main subdivisions (Davies et al., 1997; Dubbeldam et al., 1997; Zeier and
Karten, 1971). These are a somatic sensorimotor part and a viscerolimbic division that is
considered to be equivalent to the mammalian amygdala (Davies et al., 1997; Dubbeldam et al.,
1997; Zeier and Karten, 1971).
The connectivity, neurotransmitter content, and cytoarchitecture of the avian basal ganglia are
highly similar to that in mammals (Anderson and Reiner, 1991b; Karten and Dubbeldam, 1973;
Reiner and Anderson, 1990; Reiner et al., 1983, 1984a; Veenman and Reiner, 1994; Veenman
et al., 1995). Based on the palliostriatal connections, a functional segregation of the avian
striatum into associative, sensorimotor and limbic territories has recently been suggested
(Veenman et al., 1995), similar to the situation in mammals (Parent, 1990). The paleostriatum
augmentatum (PA) and lobus parolfactorius (LPO) make up the dorsal (somatomotor) striatum.
Ventral striatal structures include the nucleus accumbens (Acc), the bed nucleus of the stria
terminalis (BNST), and the olfactory tubercle (TO) which constitute the ’visceral-limbic’ parts
of the avian striatum. The pallidal parts of the avian basal ganglia consist of the paleostriatum
primitivum (PP), which is homologous to the globus pallidus of mammals, and the ventral
pallidum (VP) which is comparable to the "limbic" ventral pallidum of mammals (Karten and
Dubbeldam, 1973; Medina and Reiner, 1997).
The description of the dopaminergic innervation of the avian telencephalon given in chapter 5
will follow the functional classification outlined above. It will turn out that the distribution of
DA and DA receptors is closely related to these functional subdivisions. However, certain
exceptions exist, as, for example, components of the song system of songbirds subserve sensory
as well as motor functions.
3. Origin of dopaminergic fibers in the avian telencephalon: Mesencephalic A8-A10 cell
groups
Comparable to the situation in mammals, the dopaminergic innervation of the avian
telencephalon arises mainly from three mesencephalic dopaminergic cell populations located in
the n. tegmenti pedunculopontinus pars lateralis and the area ventralis tegmentalis of Tsai
Chapter 4: Dopaminergic innervation of the avian telencephalon 109
(AVT) (Figure 3; Kitt and Brauth, 1986; Metzger et al., 1996; Waldmann and Güntürkün, 1993).
The former nucleus is homologeous to the mammalian substantia nigra pars compacta (SNC;
Brauth et al., 1978; Karten and Dubbeldam, 1973; Kitt and Brauth, 1986; Reiner et al., 1983;
Rieke, 1981, 1982), where according to Reiner et al. (1994) the rostro-ventral part comprises
the A9 group and the caudo-dorsal part the A8 group. The medial part of the n. tegmenti
pedunculopontinus is considered to be equivalent to the mammalian substantia nigra, pars
reticulata (SNR; Kitt and Brauth, 1981; Reiner et al., 1983). The avian AVT is homologeous to
the ventral tegmental area of mammals (A10 group) (Kitt and Brauth, 1981, 1986; Reiner et al.,
1983, 1994; Waldmann and Güntürkün, 1993). In the AVT and SNC, intense perikaryal
immunoreactivity (ir) for tyrosine hydroxylase (TH) and DA (Figure 3A-C) but not for
dopamine-beta-hydroxylase (DBH), noradrenaline (NA), or phenylethanolamine-N-methyl
transferase (PNMT) can be observed (Bailhache and Balthazart, 1993; Moons et al., 1995;
Reiner et al., 1994; Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995). TH is the
rate-limiting enzyme in the synthesis of all catecholamines and, with a few exceptions (Smeets
and Gonzales, 1990), must be present in dopaminergic neurons. The presence of DBH which
converts DA to noradrenaline, or of PNMT which converts noradrenaline to adrenaline, would
indicate non-dopaminergic catecholaminergic neurons and fibers. An A8-A10 group has been
recognized in many avian species, including zebra finches (Bottjer, 1993; Lewis et al., 1981),
quail (Bailhache and Balthazart, 1993; Bons and Olivier, 1986), warbling grass parakeet
(Takatsuki et al., 1981), chicks (Metzger et al., 1996; Moons et al., 1994), and pigeons (Kitt
and Brauth, 1981, 1986; Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995). The
SNR, on the other hand, exhibits comparatively little perikaryal but high neuropil labeling for
DA (Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995), as it is the case in
mammals where the SNR mainly contains GABAergic neurons (Fallon and Loughlin, 1995).
From the AVT and SNC, fibers ascend mainly ipsilaterally within two tracts of the mediolateral
part of the lateral forebrain bundle (Karten and Dubbeldam, 1973; Kitt and Brauth, 1986;
Metzger et al., 1996; Wynne and Güntürkün, 1995). In general, there is substantial overlap in
the telencephalic projection zones of the SNC and AVT (Kitt and Brauth, 1986; Metzger et al.,
1996; Waldmann and Güntürkün, 1993), although some degree of specialization and
topographical organization seems also to be present (see, e.g., Section 5.4.1).
Chapter 4: Dopaminergic innervation of the avian telencephalon 110
Vice versa, the A8-A10 group receives pallidal inputs from the PP and striatal inputs from the
LPO, PA, and Acc (Karten and Dubbeldam, 1973; Kitt and Brauth, 1981; Reiner et al., 1994).
By far most of the striatonigral projection neurons co-contain substance P and dynorphin, while
the remainder contain enkephalin (Anderson and Reiner, 1990, 1991b; Reiner et al., 1983,
1984b; Reiner and Anderson, 1990). These two apparently disjunctive groups of neurons
project to different populations of dopaminergic and overlapping populations of non-
dopaminergic tegmental and nigral neurons (Reiner et al., 1994; Smeets, 1991), where
Figure 3. Dopaminergic neurons in the avian midbrain. (A-C and inset) Cells in the nucleus tegmenti pedunculopontinus and thearea ventralis tegmentalis of Tsai constitute the avian homologues of the substantia nigra, pars compacta (SNC) and reticulata(SNR), and the ventral tegmental area (AVT), respectively. (D) shows a similar view as seen in the right part of C labeled forDARPP-32. In contrast to dopamine-immunoreactivity as shown in C, only fibers are labeled with DARPP-32ir. Scale barsrepresent 1 mm (A, B) and 500 µm (C, D).
Chapter 4: Dopaminergic innervation of the avian telencephalon 111
substance P-ir fibers make symmetric synaptic contacts predominantly on thin dendrites and
spines (Anderson et al., 1991), whereas enkephalinergic fibers contact dopaminergic somata as
well (Medina et al., 1995).
Both, D1 and D2 receptor densities seem to be quite low in the pigeon SNC compared to the
'striatal' parts of the avian basal ganglia (Dietl and Palacios, 1988; Richfield et al., 1987).
Thus, D2 receptors seem not to play such a prominent role as autoreceptors in avian
dopaminergic midbrain neurons (at least not within the local SNC/VTA circuit) as they do in
mammals (Brock et al., 1992; Bunney et al., 1987; Cooper et al., 1996; Yung et al., 1995).
Furthermore, DARPP-32 neuropil labeling (indicating the presence of D1 receptors, see
Section 4.3) but no soma labeling could be detected in the VTA, SNC, and SNR (Figure 3D).
This observation agrees well with the mammalian situation (Ouimet et al., 1984, 1992).
containing a small number of dopaminergic neurons around and below the glomeruli (Wynne
and Güntürkün, 1995), which probably correspond to the mammalian A16 group described by
Björklund and Lindvall (1984).
4. General features of the dopaminergic innervation of the avian telencephalon
Before getting into a detailed description of the distribution of dopaminergic fibers and
dopaminoceptive elements in the avian telencephalon, some general remarks on specific
features of the dopaminergic innervation of the avian brain, and on markers of the dopaminergic
system seem to be in place.
4.1. 'Basket'- and 'en passant'-type of dopaminergic innervation
The dopaminergic innervation in the avian telencephalon takes two different, and possibly
discrete, forms. The first form, which is found also in the mammalian cortex (Oades and
Halliday, 1987; Williams and Goldman-Rakic, 1993), is characterized by dopaminergic fibers
contacting the somata and dendrites of neurons 'en-passant' while passing through their target
region. These dopaminergic axons often travel in close vicinity along the somata and dendrites
of target-neurons while forming a large number of bouton-like axonal swellings. The second
one, called the 'basket'-type, is a very peculiar arrangement of dopaminergic fibers in the avian
telencephalon. In this case, single fibers densely coil around the somata and intial dendrites of
postsynaptic targets, enwrapping them in basket-like structures (Figure 4). In some
telencephalic regions, this type of innervation can be so dense that unlabeled postsynaptic
neurons and their initial dendrites can virtually be seen by labeling of catecholaminergic fibers
Chapter 4: Dopaminergic innervation of the avian telencephalon 112
alone (Figure 4A, B). These fibers exhibit many varicosities in the vicinity of the soma and
proximal dendrites, which have been shown to contain large numbers of round clear vesicles
and also a few dense core vesicles (Metzger et al., 1996; Karle et al., 1992, 1994; see 4.4.).
Baskets seem to contact predominantly bigger neurons, whereas smaller neurons are more
likely innervated en-passant (Wynne and Güntürkün, 1995). However, basket-type structures
are not a speciality of the dopaminergic system, as they can also be demonstrated with
antibodies against NA and DBH (Moons et al., 1995).
4.2. Tyrosine hydroxylase as a marker of dopaminergic fibers in the avian telencephalon
Many studies have been carried out using THir as a marker of dopaminergic fibers in the avian
telencephalon. As noted above, TH is the rate-limiting enzyme in the synthesis of all
catecholamines and, with few exceptions (Smeets and Gonzales, 1990), must be present in
dopaminergic neurons. In general, the distribution of THir fibers closely follows that of DAir
fibers and might be a better indicator for the distribution of dopaminergic fibers than for the
much sparser noradrenergic innervation in the avian telencephalon, which predominantly
distributes along the VP, septum, HA, and the hippocampal region (Bailhache and Balthazart,
1993; Karle et al., 1996; Moons et al., 1994, 1995; Reiner et al., 1994; Wynne and Güntürkün,
1995). However, there are also some exceptions like the area parahippocampalis (APH) or the
n. taeniae (Tn) which are quite high in THir but low in DAir fibers (Wynne and Güntürkün,
1995). Hence, THir might be a good marker of dopaminergic fibers in most but not all
telencephalic areas.
4.3. DARPP-32 as a marker of D1-receptors and dopaminoceptive neurons
DARPP-32 is a Dopamine- and cAMP-Regulated PhosphoProtein of molecular weigth 32,000
that plays a role as a "third messenger" in the intracellular cascade induced by D1-receptor
stimulation (Hemmings et al., 1987a, 1987b, 1995). Via activation of the adenylyl cyclase, D1-
receptor action stimulates cAMP synthesis, which through protein kinase A leads to
phophorylation of DARPP-32. Phosphorylated DARPP-32 in turn inhibits protein phosphatase-
1, and thus affects the state of various target proteins. In rat striatal neurons for instance, DA
affects via phosphorylation of DARPP-32 the gating of Na+ and various high-voltaged-activated
Ca2+ channels (Hemmings et al., 1987a, 1995). DARPP-32 is itself dephosphorylated by
Ca2+/calmodulin-dependent protein phosphotase 2B (calcineurin), thus opening possibilities for
the interaction of glutamatergic transmission and Ca2+ influx with
Chapter 4: Dopaminergic innervation of the avian telencephalon 113
DA-induced effects (Hemmings et al., 1987a, 1995; Nishi et al., 1997). In mammals the
distribution of DARPP-32 has been shown to be closely related to that of D1-receptors (Berger
et al., 1990; Hemmings and Greengard, 1986; Ouimet et al., 1984; Walaas and Greengard,
1984). In general, this is also the case in the pigeon (Durstewitz et al., 1998) and chick
(Schnabel et al., 1997) brain.
Figure 4. Modes of dopaminergic innervation in the avian telencephalon. (A, B) Examples of the ‘basket-type’ of dopaminergicinnervation. (C-G) Examples illustrating various densities and relative contributions of en-passant and basket-type innervations.Photomicrographs were taken from the NCL (A, C), PP (B), Aid (D), Ai (E), NIM (F), and Av (G). Scale bars represent 100µm (C, D, E), 50 µm (F, G) and 25 µm (A, B).
Chapter 4: Dopaminergic innervation of the avian telencephalon 114
The use of antibodies against DARPP-32 as a marker of dopaminoceptive structures has the
advantage that postsynaptic neural targets of dopaminergic fibers are often labeled to a large
extent (Figure 5). In some cases even fine dendritic branches and spines are clearly visible at
the light-microscopic level (Figure 5D-F), revealing additional information about the
morphological properties of dopaminergic targets which are not apparent using D1 receptor-ir
(see Schnabel et al., 1997). The clear perikaryal labeling obtained with DARPP-32-ir also
enables morphometric approaches since cell counts can easily be performed.
Figure 5. DARPP-32 immuno-labeling in the pigeon forebrain. (A) Frontal section at level A 9.00, illustrating the differentintensities of DARPP-32-positive neuropil staining. (B, C) Most DARPP-32-positive cells displayed only labeling of the somaand initial dendritic segments. (D-F) In some neurons also fine dendritic processes and spines are visible. Photomicrographswere taken from the n. basalis (B), neostriatum frontolaterale (C), ventromedial hippocampus (D), border of ectostriatum andectostriatal belt (E), and medial septum (F). Scale bars represent 250 µm (A), 50 µm (B, D, E), 20 µm (C), 15 µm (F).Scalebars represent 250 µm (A), 50 µm (B, D, E), 20 µm (C), 15 µm (F). Taken from Durstewitz et al. (1998).
Chapter 4: Dopaminergic innervation of the avian telencephalon 115
4.4. Ultrastructural features of the dopaminergic innervation and of dopaminoceptive
neurons
At the ultrastructural level, dopaminergic and THir fibers in the avian basal ganglia and in the
neostriatum form many varicosities which often exhibit multiple active zones and contain many
round clear small to medium-sized vesicles together with some large dense core vesicles
(Karle et al., 1992, 1994, 1996; Metzger et al., 1996), indicating the co-release of DA and
neuropeptides as it is the case in mammalian dopaminergic neurons (Cooper et al., 1996;
Seroogy et al., 1988a, 1988b). Some of these varicosities (about 50% in the avian basal ganglia
according to Karle et al., 1996) show small and flat synaptic specializations, most often of the
symmetrical type, but occasionally also asymmetrical ones (Karle et al., 1996; Metzger et al.,
1996). Dopaminergic synapses within the neostriatum, LPO, and PA are most often found on
thin dendritic shafts (about 50% in the basal ganglia), less frequently on dendritic spines, and
occasionally (< 20% in the basal ganglia) on thick dendritic shafts or perikarya (Karle et al.,
1996; Metzger et al., 1996). Thus, in the vicinity of the soma, unspecialized varicosities seem
to prevail. On dendritic spines, dopaminergic synapses are sometimes engaged in 'triadic
complexes' with other unlabeled symmetric or asymmetric synapses terminating on the same
spine, or where they make axo-axonic contacts on these unlabeled synapses (Metzger et al.,
1996). Thus, in the avian brain as in mammals (Calabresi et al., 1987; Hernández-López et al.,
1997; Law-Tho et al., 1994; Pralong and Jones, 1993; Yang and Seamans, 1996), DA might
control both, synaptic transmission via pre- and/or postsynaptic mechanisms as well as
postsynaptic somatic and dendritic membrane properties. Furthermore, both these aspects of
neural functioning may be modulated via D1 receptors, as D1 receptor-ir and DARPP-32ir
have been observed not only within the dendrites but occasionally also in axons and axon
terminals (Schnabel et al., 1997).
5. Distribution of dopaminergic fibers and dopaminoceptive elements in the avian
telencephalon
The distribution of dopaminergic fibers is closely related to functional subdivisions of the
avian telencephalon. Most prominently, structures of the avian basal ganglia receive by far the
densest dopaminergic input, whereas all primary sensory structures seem to be devoid of DA,
L-DOPA, DA receptors, and DARPP-32 (Dietl and Palacios, 1988; Durstewitz et al., 1998;
Juorio, 1983; Juorio and Vogt, 1967; Metzger et al., 1996; Moons et al., 1994; Schnabel and
Braun, 1996; Wynne and Güntürkün, 1995). All other telencephalic areas fall somewhere in
Chapter 4: Dopaminergic innervation of the avian telencephalon 116
Figure 6. Schematic illustration of the distribution of dopaminergic fibers in the avian telencephalon.
between, however, with clear regional differences. In the following, we will describe the
distribution of dopaminergic fibers and dopaminoceptive elements according to functional
subdivisions, following the pathway of sensory information from the primary sensory to the
motor output structures as outlined in Section 2. Figures 6 to 9 give an overview over the
distribution of dopaminergic fibers, D1 receptors, D2 receptors, and DARPP-32 neuropil and
soma labeling throughout the avian telencephalon. In addition, findings on colocalizations of
markers of the dopaminergic system with other biochemically identified systems will be
discussed.
However, before going into details, it should be mentioned that although the general pattern may
be the same across avian species, some strain and species differences exist. Thus, Divac et al.
(1988) observed an about two-fold higher DA concentration in the posterior telencephalon of
mixed breed than of white Carneaux pigeons, while in the anterior telencephalon the DA
concentration was only slightly higher. Nevertheless, the relative DA concentration in six
telencephalic regions examined remained qualitatively the same in both strains. Similarly,
none - very low
high
very highmedium
low
Hp
SM
PAPP
CDL
HV
APH
SL
TPO
A 7.75
HA
LPOBas
NFL
NF
HVLFS
A 12.50
CDL
APH
NCL
AvAp
Hp
NC
A 5.25
A 9.25
HD
Hp
HA
ENIM
LPO PA
PP
HV
Ep
INP
NIL
VP
A 6.75
APH
HpCDL
NCL2
L1L3
AvAi
Aid
Tn
PA
NCL
HV
A 10.50
HA
NIL
Hp
Ep
HDHV
E
PALPO
NIM
NC
COS
AccAcc
LFSLFS
Chapter 4: Dopaminergic innervation of the avian telencephalon 117
telencephalic DA concentrations were noted to vary by factors of up to five between chicks,
ducks, finches, pigeons, fowls, and quails (Juorio and Vogt, 1967; Juorio, 1983).
5.1. Primary sensory areas
5.1.1. Dopaminergic fibers and projections
Primary sensory areas of the pigeon and chick telencephalon display by far the lowest DAir and
L-DOPAir (Figure 10A; Metzger et al., 1996; Wynne and Güntürkün, 1995; Moons et al.,
1994), and no projections from the VTA or SNC to these areas have been demonstrated, except
for a restricted input to the HD (Kitt and Brauth, 1986). Whereas the E (visual tectofugal), Field
L2 (auditory), and the Bas (trigeminal) are devoid of dopaminergic fibers, some DAir fibers
are present in the rostral (somatosensory) and caudal (visual thalamofugal) IHA, and lateral
HD, the two input laminae of the Wulst. Interestingly, the specific sensory nuclei of the thalamus
(e.g., the n. rotundus which projects to the E) also contain little or no THir (Reiner et al., 1994).
Figure 7. Schematic illustration of the distribution of D1 receptors in the avian telencephalon.
none - very low
low
medium
high
very high
A 12.50
A 6.75
CDL
NCL2
HV
L3 NCL
Hp
PAAid
AvAi
Tn
APH
L1
A 10.50A 9.25
NC
AvAp
CDLHp
APH
NCL
A 5.25
ENIM
HDHA
Hp
PALPO
PP
Ep
HV
NIL
INP
A 7.75
SMTPO
Hp
APH CDL
HV
PAPPSL
NC
COS
LPO
LPOBas
NFLHV
NF
HA HIS
HV
HD
HA
Ep
ENIL
Hp
NIM
PALPO
Chapter 4: Dopaminergic innervation of the avian telencephalon 118
Thus, primary sensory processes do not seem to be modulated, at least not to a significant
extent, by DA in the adult avian brain.
5.1.2. D1 receptors
Given the virtual absence of a dopaminergic innervation, it comes with no surprise that the D1
receptor density in the primary sensory structures is very low, with the E exhibiting the lowest
D1-specific binding in the whole telencephalon (Dietl and Palacios, 1988; Schnabel et al.,
1997; Schnabel and Braun, 1996; Stewart et al., 1996). However, as it is the case for DAir
labeling, the IHA and HD are higher in D1-specific binding, although still significantly lower
than the respective secondary projection zones in the HA and most of the neostriatum (Schnabel
et al., 1997).
5.1.3. D2 receptors
In general, D2 receptor densities as measured by [3H]spiperone and [3H]CV205-502 are low
and homogeneously distributed throughout the whole avian telencephalon, except for the basal
ganglia and the hippocampal complex as discussed below (Dietl and Palacios, 1988; Schnabel
and Braun, 1996; Stewart et al., 1996).
5.1.4. DARPP-32
The distribution of DARPP-32ir confirms the extremely low or absent dopaminergic input to
the primary sensory structures. The E and Field L2 are largely devoid of DARPP-32ir (Figure
10B; Durstewitz et al., 1998; Schnabel et al., 1997). The IHA and HD display low levels of
neuropil labeling and a low percentage of labeled neurons, while, however, scoring in both
respects again much lower than the respective secondary sensory zones in the HA and most of
the neostriatum (Durstewitz et al., 1998; Schnabel et al., 1997). Hence, primary sensory regions
in the avian forebrain could be sharply delineated and differentiated from bordering areas by
virtue of their low DARPP-32ir (Figure 5A; Figure 10B).
The only primary sensory forebrain area that might deviate from this general pattern, at least in
pigeons, is the trigeminal Bas. Although it is very low in terms of DARPP-32ir neuropil
labeling, the pigeon’s Bas contains a high proportion of labeled perikarya, many of them
exhibiting clear neuronal properties (Figure 5B; Durstewitz et al., 1998; but see Schnabel et al.,
1997, for chicks). This finding might be related to other peculiarities of the Bas and to the
specific demands imposed on the neural circuit controlling pecking behavior, in which the Bas
Chapter 4: Dopaminergic innervation of the avian telencephalon 119
Figure 8. Schematic illustration of the distribution of D2 receptors in the avian telencephalon.
is critically involved (Schall, 1987; Wild et al., 1985). The Bas is the only avian forebrain
structure with direct sensory inputs bypassing the thalamus (Schall et al., 1986). Moreover,
within the trigeminal neural circuit controlling pecking, sensory signals possibly must be
integrated very fast and relayed rather directly to motor outputs. As the dopaminergic system
probably plays a central role in sensory-motor integration and learning (see Section 7), the
dopaminergic modulation might start earlier in this trigeminal somatosensory circuit than within
other forebrain systems. This notion is furthermore underpinned by the fact that the Bas receives
auditory and possibly also vestibular input, in addition to trigeminal information (Schall et al.,
1986; Schall and Delius, 1986; Wild et al., 1997), and might thus actually be regarded as a
multimodal structure. Furthermore, behavioral evidence supports the hypothesis of a
dopaminergic modulation within the pigeon Bas: Lindenblatt and Delius (1988) showed that
local apomorphine-injections into the Bas induce pecking bouts, while Wynne and Delius
(1996) made it likely that Bas lesions decrease pecking fits induced by peripheral
apomorphine-injections. However, the high number of DARPP-32ir neurons in the pigeon Bas
CDL
TPO
Hp HV
APH
SMSL
PAPP
A 7.75
A 10.50 A 12.50LPO
HA
LPOBas
NFL
NF
HV
high
very highmedium
low
very low
A 9.25
Av
Hp
NC
APH
CDL
NCL
Ap
A 5.25
HD
Hp
HA
ENIM
LPO PA
PP
HV
Ep
INP
NIL
VP
A 6.75
APH
Hp
CDL
NCL2
L3
AvAi
Aid
Tn
PA
NCL
HV
L1
HA
NIL
Hp
EpNIM
HD
HV
E
PALPO
NC
Chapter 4: Dopaminergic innervation of the avian telencephalon 120
is clearly at odds with the lack of D1-receptors and dopaminergic fibers reported so far, as
well as with the lack of DARPP-32 in the chick Bas (Schnabel et al., 1997). In this context, it
should be pointed out that DARPP-32 is not exclusively involved in the D1-induced cascade
but is regulated by other neuromodulator/neurotransmitter pathways as well, e.g. by D2- or
NMDA-receptor stimulation via Ca2+ influx (Hemmings et al., 1995; Nishi et al., 1997). Thus,
DARPP-32 in the Bas might be linked to intracellular pathways other than the one coupled to
the D1 receptor, and differences between pigeons and chicks might exist in this respect.
5.1.5. Colocalizations
Durstewitz et al. (1998) found that glutamate decarboxylase (GAD) ir neurons were never
colocalized with DARPP-32 or located in THir baskets throughout the whole pigeon
telencephalon, including the primary sensory areas.
5.2. Secondary sensory and multimodal areas
5.2.1. Dopaminergic fibers and projections
In general, the dopaminergic innervation of both, the secondary sensory and tertiary association
areas is clearly higher than that of the primary areas (Figures 10, 11). In addition, most of the
multimodal areas score higher than the secondary sensory areas due to an increasing
rostrocaudal and mediolateral gradient of DAir structures (Figure 6; Metzger et al., 1996;
Wynne and Güntürkün, 1995). Thus, in the secondary visual Ep and the trigeminal NF, DA
concentration is very low. The HA which includes the secondary projection fields of the
thalamofugal visual and the somatosensory system receives a moderate dopaminergic input
(Metzger et al., 1996; Moons et al., 1994; Wynne and Güntürkün, 1995).
Among the presumed multimodal forebrain structures, the rostrally located NFL is only
moderate and thus lowest in its DA content (Metzger et al., 1996; Moons et al., 1994; Wynne
and Güntürkün, 1995), while the medial portion of the lamina frontalis superior seems to be
most densely innervated by dopaminergic fibers (Figure 11A), with the fibers arising mainly
from the SNC (Bottjer, 1993; Kitt and Brauth, 1986; Wynne and Güntürkün, 1995). High DAir
and basket-like structures are also present in the MNH (Figure 11A; Metzger et al., 1996;
Moons et al., 1994; Wynne and Güntürkün, 1995), an area that has been implicated in filial
imprinting (Gruss and Braun, 1996, 1997). Metzger et al. (1996) also demonstrated a prominent
input to these areas from VTA by retrograde tracing, and to a lesser extent from SNC.
Chapter 4: Dopaminergic innervation of the avian telencephalon 121
Considerable numbers of basket-like structures and dopaminergic fibers are also present in
multimodal areas comprising the pallium externum, especially in the TPO, which receives its
major input from SNC (Kitt and Brauth, 1986). Within the caudal neostriatum which is
generally very high in DA input, the NCL has received special attention because of its
presumed equivalency to the prefrontal cortex (PFC) of mammals. This assumption was first
derived from behavioral, radioenzymatical, and histofluorescence studies conducted by Divac,
Mogensen and Björklund (Divac et al., 1985; Divac and Mogensen; 1985; Mogensen and
Divac, 1982). These authors demonstrated that the NCL contains a high DA-to-NA-ratio and an
especially high number of catecholaminergic fibers of presumed dopaminergic origin, as it is
characteristic for the PFC of mammals (Berger et al., 1988, 1991; Björklund et al., 1978; Divac
et al., 1978; Glowinski et al., 1984; Van Eden et al., 1987). Using immunocytochemical
methods, Waldmann and Güntürkün (1993) and Wynne and Güntürkün (1995) confirmed that
DARPP-32neuropil labeling
medium - high
none - very lowlow
very high
Inserts: Percentage of DARPP-32+ neurons < 10 %20 - 40 %40 - 70 %> 70 %
Hp
SL
HV
TPO
CDL
PAPP
SM
A 9.25
A 7.75
A 10.50 A 12.50LPO
HA
LPO
NFLHV
Bas
NF
HAHD
Ep
HV
NILE
LPO
PPPA
Acc INP
VP
NIM
CDL
NC
AvAp
NCL
APH
Hp
A 5.25 A 6.75
NC NCL
APHCDL
L2
L1
HV
TnAv
Ai
AidPA
L3
Hp
HV
HD
HA
NILEp
NIM
LPO
E
PA
HA
NC
Figure 9. Schematic illustration of the distribution of the D1-receptor related phosphoprotein DARPP-32 in the aviantelencephalon.
Chapter 4: Dopaminergic innervation of the avian telencephalon 122
the NCL could be differentiated from the surrounding caudal neostriatum by its denser
dopaminergic innervation and by the higher number of DAir fibers which constitute a basket,
while the number of baskets per se does not increase in the NCL (Figure 11B, C; see also
Divac et al., 1994). The dopaminergic input to the NCL arises from the dopaminergic midbrain
nuclei AVT and SNC (Waldmann and Güntürkün, 1993).
In chicks, a region in the dorsocaudal neostriatum shares the location, connections and dense
dopaminergic innervation with the pigeon´s NCL (Metzger et al., 1996, 1998). This tertiary
area has also been studied in the context of imprinting (Bock et al., 1997; Schnabel and Braun,
1996). Its dopaminergic innervation is also characterized by a higher occurrence of basket-like
structures and by a dopaminergic input from AVT and SNC (Metzger et al., 1996). However, it
appears that the area of densest DAir and THir is shifted somewhat ventromedially from the
ventricle compared to pigeons (Metzger et al., 1996; own unpublished observations).
5.2.2. D1 receptors
In general, the distribution of D1 receptors closely mimics that of dopaminergic fibers, although
D1 receptors seem to be more homogeneously distributed throughout the entire neostriatum and
hyperstriatum than dopaminergic fibers, which seem to be more locally restricted (Figure 7).
The HA, HD, HV, and neostriatum contain comparable, medium concentrations of D1
receptors, with the hyperstriatal areas scoring a bit higher than at least the rostral and
intermediate neostriatum (Ball et al., 1995; Dietl and Palacios, 1988; Schnabel and Braun,
1996; Schnabel et al., 1997; Stewart et al., 1996). In addition, two obvious tendencies in
distribution are apparent (Schnabel and Braun, 1996; Schnabel et al., 1997): First, the number
of D1 receptors increases from rostral to caudal in both, hyperstriatal and neostriatal areas.
Second, in the neostriatum, the number of D1 receptors increases from medial to lateral. Thus,
the NCL and the caudal HV are the telencephalic areas highest in D1-specific binding outside
the basal ganglia. This finding also implies that secondary sensory structures in the frontal
neostriatum, like the Ep and the NFL, but also the secondary auditory fields L1 and L3, are less
dense in D1 receptors than most tertiary telencephalic regions. Hence, in general, and possibly
with some species differences (Ball et al., 1995), there is an increase of D1-receptor density
proceeding from primary sensory to tertiary multimodal structures. This pattern might be related
to an increase in sensory-motor integration and complex learning processes that take place
primarily in higher-order structures.
Chapter 4: Dopaminergic innervation of the avian telencephalon 123
Figure 10. Distribution of dopaminergic fibers and dopaminoceptive elements in primary and secondary sensory areas asexemplified for the auditory Field L complex. (A) The primary sensory area L2 is almost devoid of dopaminergic fibers and (B)of DARPP-32 immunoreactivity, while the surrounding secondary sensory “belt” regions L1 and L3 show considerably morelabeling for dopamine and DARPP-32. Scale bars represent 500 µm.
Figure 11. Dopaminergic innervation and DARPP-32 immunoreactivity in higher order sensory and multimodal structures. (A)Distribution of dopaminergic fibers in the rostromedial forebrain. The neostriatum intermedium (NIM) and hyperstriatumventrale (HV), and possibly also the lamina frontalis superior (LFS), constitute multimodal areas. Note that in the NIM and HVthe density of dopaminergic fibers increases slightly from lateral to medial. (B) Localization of the neostriatum caudolaterale(NCL), the presumed prefrontal cortex of birds, in the caudal telencephalon. (C) Detail of the dopaminergic innervation of theNCL and the posterior archistriatum. (D) DARPP-32 immunoreactivity in the caudal neostriatum is evenly distributed atmedium levels, but is low in the archistriatum. Scale bars represent 1 mm (B) and 50 µm (A, C, D).
Chapter 4: Dopaminergic innervation of the avian telencephalon 124
5.2.3. D2 receptors
D2 receptor densities in the neostriatum and hyperstriatum are low and homogeneously
distributed (Figure 8; Dietl and Palacios, 1988; Schnabel and Braun, 1996; Stewart et al.,
1996). For example, Schnabel and Braun (1996) found the density of D2 receptors to be about
one third or less (< 25 fmol/mg) of that of D1 receptors in these areas. Hence, outside the basal
ganglia, dopaminoceptive neurons in the avian telencephalon seem to be modulated
predominantly via receptors of the D1 class, although it should be kept in mind that even low
densities of D2 receptors might play an important physiological role.
5.2.4. DARPP-32
DARPP-32ir is distributed with medium to high densities throughout the entire neostriatum, HA,
CDL and TPO (Figure 9; Durstewitz et al., 1998; Schnabel et al., 1997). Thus, DARPP-32ir
reaches much higher levels in the secondary and tertiary areas than in the primary sensory
structures (Figures 5A, 10B). In addition, Schnabel et al. (1997) reported a rostrocaudal and a
mediolateral trend in the DARPP-32 distribution in the chick neostriatum as it has been
described for D1 receptors. However, Durstewitz et al. (1998) were not able to discriminate
between secondary sensory and multimodal pigeon forebrain structures based on DARPP-32ir
neuropil labeling and cell countings, although a slight trend in neuropil labeling was also
observed by these authors. Thus, differences between secondary sensory and multimodal areas
with regards to their dopaminergic input may not be so apparent in DARPP-32ir as they are in
D1 receptor and dopaminergic fiber density.
A relatively high percentage of neurons (up to about 60 %) exhibits DARPP-32ir in the
secondary sensory and multimodal areas, about the same as in structures of the basal ganglia
and the ventral archistriatum, and significantly higher than in the HD (receiving primary visual
input) and the dorsal archistriatum (Figure 11D; Durstewitz et al., 1998). The NCL has already
been introduced as the possible avian equivalent of the mammalian PFC, and as such receives a
prominent dopaminergic input. However, this structure cannot be differentiated from the
surrounding neostriatal areas by DARPP-32 neuropil labeling or by its relative number of
DARPP-32-positive neurons (Figure 11D; Durstewitz et al., 1998). In contrast, the percentage
of DARPP-32-positive neurons seems even to decrease proceeding from rostral to caudal.
Similar discrepancies in the distribution of dopaminergic fibers, D1 receptors and DARPP-32
have also been oberserved in the mammalian neocortex (Berger et al., 1990; see Section 6.2).
One of the most obvious discrepancies between the distributions of DARPP-32ir and D1
Chapter 4: Dopaminergic innervation of the avian telencephalon 125
receptors, however, is the significantly higher amount of DARPP-32 in the neostriatum than in
the HV, while just the opposite is the case for the density of D1 receptors (Durstewitz et al.,
1998; Schnabel et al., 1997). Another possible discrepancy is the considerable DARPP-32ir in
the CDL where Ball et al. (1995) reported very low concentrations of D1 receptors. These
discrepancies might be partly related to the fact that ligand-binding autoradiographic
demonstrations of D1 receptors have different properties than the immunocytochemical
demonstration of a receptor-related phosphoprotein. Thus, while receptorautoradiography might
provide objective means to demonstrate receptor densities, immunocytochemistry reveals more
about the morphology and possible colocalizations of the cells under study. However, it should
be stressed that DARPP-32 generally is a good marker for the distribution of D1 receptors in
avian species as it is in mammals (Berger et al., 1990; Hemmings and Greengard, 1986; Ouimet
et al., 1992).
5.2.5. Colocalizations
Figure 12A presents examples from the caudal neostriatum of DARPP-32ir neurons which are
located in TH-baskets. A quantitative examination of the number of DARPP-32ir neurons
located in TH-baskets showed that this percentage differs considerably between various areas
(Durstewitz et al., 1998). The highest percentage (15-30 %) of DARPP-32/TH-basket
colocalizations was observed in the multimodal lateral and caudal aspects of the neostriatum,
including the NCL, i.e. exactly in those regions, which expressed the highest numbers of D1
receptors according to Schnabel et al. (1997). Less DARPP-32/TH-basket colocalizations
were found in the rostral and intermediate neostriatum (5-15 %), including most of the
secondary sensory areas. The lowest percentage of DARPP-32/TH-basket colocalizations
occurred in the hyperstriatal and hippocampal regions (< 5 %), although the HA is strongly
innervated by dopaminergic fibers and exhibits a high concentration of D1 receptors. That some
areas are dense in D1 receptors and dopaminergic input but display only few basket-like
structures is a further hint to a functional dissociation between an 'en passant'-mode of
dopaminergic innervation and the basket-like DA input. According to the functional
considerations outlined in Section 6.3, one might speculate that the higher level lateral and
caudal neostriatal areas have a special role in synchronizing and coordinating network activity.
In the male quail telencephalon, especially within the neostriatum, colocalizations of THir
baskets and varicosities and aromatase-ir cells were observed (Balthazart et al., 1998). The
enzyme aromatase converts testosterone into 17β-estradiol which is involved in the activation
Chapter 4: Dopaminergic innervation of the avian telencephalon 126
of male sexual behavior. These colocalizations may thus represent an anatomical basis for the
regulation of male sexual behavior by DA (see Section 7.3.) DA probably stimulates aromatase
activity and is, vice versa, possibly influenced itself by aromatase (Balthazart and Absil, 1997;
Pasqualini et al., 1995).
From a functional point of view, it is of great importance to identify the postsynaptic targets of
the dopaminergic innervation. Baskets provide a clear and convenient morphological marker to
do this. GABAergic inhibitory neurons constitute one of the prominent potential candidates
which might be dopaminergically modulated via baskets. Veenman and Reiner (1994) estimate
the fraction of GABAergic neurons to be about 10-12 %, homogenously distributed throughout
most of the telencephalon. Antibodies directed against GAD, the enzyme which converts
glutamate to GABA, labels approximately the same population of cells, and thus represents a
useful indicator for GABAergic neurons (Veenman and Reiner, 1994). Like in other
telencephalic structures, throughout the neostriatum and hyperstriatum no GADir neurons were
observed that were located in THir baskets (Figure 12B) or were colocalized with DARPP-32
(Figure 12C; Durstewitz et al., 1998). Thus, GABAergic neurons in the pigeon telencephalon
might not express D1 receptors and might not receive a strong dopaminergic input. As in
addition D2 receptors are very low in these areas, GABAergic neurons possibly do not receive
a significant dopaminergic input at all. Hence, an interesting functional conclusion might be that
mainly or solely excitatory neurons are the targets of the dopaminergic innervation in the pigeon
telencephalon. However, it has to be emphasized that D2 receptors despite their low densities
might nevertheless modulate the physiological activity of dopaminoceptice neurons in a
significant manner. Thus, it still has to be investigated whether GABAergic neurons express D2
receptors.
5.3. Septum, hippocampal complex, archistriatum
5.3.1. Dopaminergic fibers and projections
Prominent motor and limbic structures in the avian telencephalon include the septum, the
hippocampal complex, and the archistriatum. The lateral septum exhibits a high number of
DAir, L-DOPAir, and THir baskets and fibers (Bailhache and Balthazart, 1993; Balthazart et
al., 1998; Bottjer, 1993; Moons et al., 1994; Wynne and Güntürkün, 1995), whereas the medial
septum displays a high density of THir and NAir fibers (Moons et al., 1995) but only moderate
to low amounts of DAir and L-DOPAir (Figure 13A). The lateral septum, and especially the
ventrolaterally located region, receives most of its dopaminergic input from the VTA, while the
Chapter 4: Dopaminergic innervation of the avian telencephalon 127
medial septum is more densely innervated by axons from the SNC (Kitt and Brauth, 1986). In
the hippocampus (Hp) and APH only a low number of DAir and L-DOPAir structures was
detected (Metzger et al., 1996; Moons et al., 1994; Wynne and Güntürkün, 1995), which mainly
stem from the SNC (Kitt and Brauth, 1986).
Fig. 12. Fluorescence double-labeling against (A) DARPP-32 and TH, (B) GAD and TH, and (C) DARPP-32 and GAD. Thephotomicrographs of each pair were taken from the same site in caudal neostriatum of the pigeon telencephalon. Two of threeDARPP-32ir neurons (A1) are surrounded by THir baskets (A2). GADir neurons (B1) do not receive input from THir baskets(B2). GADir neurons (C2) do not show DARPP-32 immunoreactivity (C1), although GADir and DARPP-32ir neurons mayoccur in close vicinity to each other. Arrows mark corresponding positions in each pair. In (C), open and filled arrows indicatepositions of DARPP-32ir and some GADir cells, respectively. Scale bars represent 30 µm (A, B), 40 µm (C). Taken fromDurstewitz et al. (1998).
Chapter 4: Dopaminergic innervation of the avian telencephalon 128
Hence, the dense THir in these areas may be mainly due to a noradrenergic innervation, which
has also been demonstrated by DBHir and NAir (Bailhache and Balthazart, 1993; Moons et al.,
1995; Reiner et al., 1994).
The archistriatum displays a very heterogenous innervation by dopaminergic fibers (Figures
11C, 14A). A particular dense dopaminergic input, reaching levels as high as in the basal
ganglia, was described for the most dorsal archistriatum intermedium (Aid), which constitutes
part of the sensorimotor archistriatum, as well as for the posterior 'limbic-visceral'
archistriatum (Ap) (Bailhache and Balthazart, 1993; Balthazart et al., 1998; Metzger et al.,
1996; Wynne and Güntürkün, 1995). This input stems mainly from SNC, and to a lesser extent
from VTA (Kitt and Brauth, 1986). However, whereas Metzger et al. (1996) reported the rest
of the archistriatum in the chick to be low in dopaminergic structures, Wynne and Güntürkün
(1995) demonstrated a relatively high number of dopaminergic structures throughout the whole
pigeon archistriatum, with a decreasing gradient of dopaminergic fiber density from dorsal to
ventral. The ventromedially located Tn is devoid of DAir, but is relatively high in THir, NAir
and DBHir (Bailhache and Balthazart, 1993; Moons et al., 1995). Interestingly, despite the very
low DAir fiber density in the ventral archistriatum, there is nevertheless a high number of DAir
baskets present in this structure, possibly surrounding the large projection cells which give rise
to the descending tractus occipitomesencephalicus (Wynne and Güntürkün, 1995). Projections
to the intermediate archistriatum from VTA and SNC have also been demonstrated by Kitt and
Brauth (1986).
5.3.2. D1 receptors
In general, the medial and lateral septum seem to be very low in D1 receptor binding, at least in
the quail (Ball et al., 1995). However, in accordance with its relatively dense input from SNC
(Kitt and Brauth, 1986), the n. commissuralis of Baylé et al. (1974) within the medial septum is
relatively high in D1 density (Ball et al., 1995), and this seems also to be the case in the
domestic chick as judged from Figure 1 of Stewart et al. (1996). Both, the Hp and the APH are
very low in D1-specific binding (Schnabel and Braun, 1996; Schnabel et al., 1997; Stewart et
al., 1996). The pattern of [3H]SCH23390 labeling in the archistriatum in general follows that
described for dopaminergic fibers above. In particular, the dorsal archistriatum intermedium
contains a very high number of D1 receptors, while the density strongly drops in the ventral
archistriatum (Dietl and Palacios, 1988; Schnabel and Braun, 1996; Schnabel et al., 1997;
Stewart et al., 1996).
Chapter 4: Dopaminergic innervation of the avian telencephalon 129
5.3.3. D2 receptors
The dorsal Hp and medial APH are the only
telencephalic regions besides the basal ganglia
(see below) which display considerable
amounts of D2 receptors (Schnabel and Braun,
1996; Stewart et al., 1996). As it is the case in
the basal ganglia, D2 receptors even outnumber
D1 receptors in the avian hippocampal areas.
5.3.4. DARPP-32
The septum is devoid of DARPP-32ir except
for the n. commissuralis of Baylé et al. (1974)
and a circumscribed region in the lateral
septum (Figure 13B), both of which contain
DARPP-32ir somata as well as fibers
(Durstewitz et al., 1998).
DARPP-32ir neuropil labeling in the Hp is heterogenous, with the medial part being completely
blank, while the ventromedial part and the lateral border show considerable neuropil labeling
and a high number of DARPP-32ir somata (Durstewitz et al., 1998). These regions probably
correspond to the V-shaped region and area 7 of Krebs et al. (1991) and Erichsen et al. (1991),
respectively. The APH displays moderate to high levels of DARPP-32ir neurons and neuropil
labeling.
With the exception of the Tn, which is virtually devoid of DARPP-32, the pigeon archistriatum
is homogenously stained by DARPP-32ir with densities comparable to the neostriatum and the
APH (Figure 14B; Durstewitz et al., 1998). Only the ventral but not the dorsal archistriatum in
addition contains a high percentage of DARPP-32ir neurons, while just the opposite is the case
for the distribution of DA fibers and receptors (Metzger et al., 1996; Wynne and Güntürkün,
1995; see Section 5.3.1). However, the finding of many large DARPP-32ir neurons in the
ventral archistriatum is consistent with the relatively high number of dopaminergic baskets in
this region (Wynne and Güntürkün, 1995), and may thus further establish a functional
dissociation between a ‘basket‘- and an ‘en passant’-mode of dopaminergic innervation. In
addition, a small, dense population of DARPP-32ir cells was observed at the dorsomedial
Figure 13. (A) Dopamine- and (B) DARPP-32immunoreactivity in the septum. (C) Dopamine- and (D)DARPP-32 immunoreactivityin the nucleus accumbens. Scalebars represent 50 µm (A, B, C) and 25 µm (D).
Chapter 4: Dopaminergic innervation of the avian telencephalon 130
border of the intermediate archistriatum (Figure 14B; Durstewitz et al., 1998), lying within the
dorsal archistriatum intermedium dorsale which is also densely innervated by dopaminergic
fibers and is dense in D1-receptor content (see Sections 5.3.1 and 5.3.2). As judged from figure
3 in Schnabel et al. (1997), the situation seems to be comparable in chicks, with quite high
numbers of DARPP-32ir somata and fibers in the dorsal intermediate and most ventral
archistriatum, but low staining in between.
5.3.5. Colocalizations
In the archistriatum, and especially in its ventral component, a large proportion of DARPP-32ir
neurons are located within THir baskets (15-40 %; Durstewitz et al., 1998). Since axons which
constitute baskets very likely exert a massive effect on their postsynaptic targets, it is
conceivable that many D1-positive archistriatal cells are to an important extent under
modulatory control of the dopaminergic system. Since in the quails archistriatum intermedium,
THir fibers have also been found to enwrap aromatase-ir neurons, it is possible that D1-
positive archistriatal cells are jointly modulated by DA and steroids and thus play an important
role in the motor control of sexual behavior (Balthazart et al., 1998). In contrast, in the
hippocampal complex, the percentage of DARPP-32ir neurons located in THir baskets is very
low (< 5%), further supporting the notion that most if not all THir baskets in this region may be
of non-dopaminergic and thus probably noradrenergic origin (Moons et al., 1995; Reiner et al.,
1994; Wynne and Güntürkün, 1995). In the archistriatal and hippocampal regions GADir
neurons were never observed to be located within TH baskets or to be colocalized with
DARPP-32 (Durstewitz et al., 1998). Thus, in these regions like in most other telencephalic
areas the avian dopaminergic system might mainly modulate non-GABAergic and thus
excitatory forebrain neurons. The situation may be different in the hippocampal region,
Figure 14. (A) Dopaminergic fibers and (B) DARPP-32 immunoreactivity in the archistriatum. DARPP-32 immunoreactivitywithin the archistriatum is largely restricted to the Av and a small rim of the Aid (arrows). Scale bars represent 500 µm.
Chapter 4: Dopaminergic innervation of the avian telencephalon 131
however, where a higher density of D2 receptors has been observed (see Section 5.3.3). In the
mammalian cortex, mainly GABAergic interneurons seem to be modulated via D2 receptors,
while the activity of pyramidal cells seems to be mainly modulated via D1 receptors (Godbout
et al., 1991; Law-Tho et al., 1994; Pirot et al., 1992; Rétaux et al., 1991; Yang and Seamans,
1996). If this would hold true also in the dorsal HP and medial APH, where D2 receptors are
even more numerous than D1 receptors, then one might speculate that in the hippocampal areas
mainly the network of inhibitory interneurons is modulated by DA, in contrast to the situation in
other telencephalic areas.
5.4. Basal ganglia
5.4.1. Dopaminergic fibers and projections
The avian basal ganglia can be subdivided into four major functional regions: 1) the LPO and
the PA which together are comparable to the dorsal, somatomotor striatum, 2) the Acc, TO, and
BNST, which form the ventral, visceral-limbic striatum, 3) the PP, and 4) the VP, which
constitute the dorsal and ventral pallidum, respectively (Karten and Dubbeldam, 1973; Reiner
et al., 1984a; Veenman et al., 1995). Whereas the dorsal striatum contains small, densely
packed cells, the PP mainly contains sparsely scattered large projection neurons.
Comparable to the mammalian situation, anterograde and retrograde tracing studies
demonstrated that structures of the avian basal ganglia are the ones receiving the densest input
from the dopaminergic midbrain nuclei SNC and VTA, with some degree of topographical
organization (Bons and Oliver, 1986; Brauth et al., 1978; Karten and Dubbeldam, 1973; Kitt
and Brauth, 1986, Metzger et al., 1996). Thus, the LPO and the rostromedial PA receive input
from VTA and the central core of SNC (but see Metzger et al., 1996, in chicks), the medial and
caudal PA receive inputs mainly from the medial SNC, and the lateral PA from the lateral SNC
(Bons and Oliver, 1986; Brauth et al., 1978; Kitt and Brauth, 1986). Structures of the ventral
striatum also receive projections from the SNC, and to a somewhat lesser degree from VTA
(Balthazart and Absil, 1997; Kitt and Brauth, 1986). In contrast, notable mesencephalic
projections to the PP and n. intrapeduncularis (INP), i.e. pallidal parts of the avian basal
ganglia, were not observed (Brauth et al., 1978; Kitt and Brauth, 1986; Medina and Reiner,
1997).
The results from tracing studies have been confirmed by immunocytochemical investigations in
several avian species using antibodies directed against DA, L-DOPA, or TH (Figure 15;
Chapter 4: Dopaminergic innervation of the avian telencephalon 132
Bailhache and Balthazart, 1993; Balthazart et al., 1998; Bottjer, 1993; Karle et al., 1996;
Metzger et al., 1996; Moons et al., 1994; Wynne and Güntürkün, 1995). The 'striatal' parts of
the avian basal ganglia are characterized by an extremely dense meshwork of dopaminergic
fibers (Figure 15A, C), which exhibit many bouton-like axonal swellings. Actually, this
meshwork is so dense that basket- and non-basket-types of dopaminergic innervation can hardly
be differentiated in these regions, except for the BNST and the Acc, where the innervation is
less dense (Karle et al., 1996; Wynne and Güntürkün, 1995). In the Acc, a high number of DAir
(but not THir) basket-like structures are observed in addition to DAir and THir fibers (Figure
13C). In contrast, DA- and TH-positive labeling in the 'pallidal' parts of the avian basal ganglia
is comparatively weak (Figure 15A, C; Bailhache and Balthazart, 1993; Balthazart et al., 1998;
Bottjer, 1993; Karle et al., 1996; Metzger et al., 1996; Moons et al., 1994; Wynne and
Güntürkün, 1995). Moons et al. (1994) were not able to detect any DAir or L-DOPAir in the PP
and INP in the chick brain at all. However, Wynne and Güntürkün (1995) observed that many
unlabeled large neurons in the pigeon PP are enwrapped by basket-like DA-positive structures
(c.f. Figure 4B). The light labeling of the PP may thus in part be due to the sparse distribution of
the large projection neurons in this area.
Considerable THir occurs in the VP, which must be largely due to labeling of noradrenergic
fibers (Bailhache and Balthazart, 1993; Karle et al., 1996; Moons et al., 1995; Wynne and
Güntürkün, 1995). In contrast, by far the most TH-positive elements in the PA and LPO seem to
be of dopaminergic origin, as only little DBHir and NAir were observed in these regions
(Karle et al., 1996; Moons et al., 1995). Furthermore, in the PA and LPO, DAir and THir
terminals make up for about 15-20% of all synaptic terminals in these areas (Karle et al.,
1996). A substantial decrease of DA concentration within the basal ganglia from rostral to
caudal has been noted by Juorio and Vogt (1967).
5.4.2. D1 receptors
Autoradiographic D1-receptor binding studies, all using [3H]SCH23390 as ligand, in chicks
(Schnabel et al., 1997; Stewart et al., 1996), pigeons (Dietl and Palacios, 1988; Richfield et
al., 1987), Japanese quails (Ball et al., 1995), and European starlings (Casto and Ball, 1994)
consistently demonstrated that D1 receptors are most abundant in structures of the avian basal
ganglia (Figure 7). However, the absolute binding of [3H]SCH23390 varied by several orders
of magnitude between studies, ranging in the LPO from 3.8 fmol/mg protein (Stewart et al.,
1996) to 2800 fmol/mg (Ball et al., 1995). This might partly be due to species and strain
Chapter 4: Dopaminergic innervation of the avian telencephalon 133
differences (see Divac et al., 1988; Juorio and Vogt, 1967), but as large variations occur even
within a given species (e.g., compare studies in chicks of Schnabel et al., 1997, and Stewart et
al., 1996), differences in technical procedures are probably the main factor. Nevertheless, the
consistent result of all of these studies was an about 5- to 10-fold lower concentration of D1
receptors in the pallidal parts (PP and VP) than in the striatal parts (LPO, PA, BNST, Acc, TO)
of the avian basal ganglia.
With regards to subdivisons within the avian striatum, some authors reported higher D1
concentrations in the LPO than in the PA (Ball et al., 1995; Schnabel et al., 1997), while others
could not observe notable differences (Dietl and Palacios, 1988; Richfield et al., 1987; Stewart
et al., 1996). In addition, Schnabel et al. (1997) reported a decreasing rostro-caudal gradient of
D1 receptors in the LPO, in accordance with the rostro-caudally decreasing DA concentration
noted above (Juorio and Vogt, 1967; Juorio, 1983). In a developmental study by Schnabel and
Braun (1996) in chicks, D1 receptors showed a gradual but non-significant increase during the
first post-hatch week. Thus, the D1 receptor system seems to be largely developed in chicks at
the time of hatching, consistent with their precocial nature.
Figure 15. (A, C) Dopamine-immunoreactive fibers and (B, D) DARPP-32 immunoreactivity in the basal ganglia. Scale barrepresents 1 mm.
Chapter 4: Dopaminergic innervation of the avian telencephalon 134
5.4.3. D2 receptors
The density of D2 receptors (Figure 8) in striatal parts of the avian basal ganglia (LPO, PA,
BNST, TO, Acc) seems to be even higher (about 1.5-fold) than that of D1 receptors (Dietl and
Palacios, 1988; Richfield et al., 1987; Stewart et al., 1996), although this was not consistently
found (Schnabel and Braun, 1996; Stewart et al., 1996). The lack of a higher D2 receptor
density in the Schnabel and Braun (1996) study is possibly due to a significant developmental
increase in the striatal parts of the basal ganglia during the first post-hatch week as reported by
these authors. Stewart et al. (1996), on the other hand, found that the relation of D1:D2 receptor
densities in the basal ganglia depended on the learning history of the animals, since after one-
trial avoidance learning the density of D1- but not D2-receptors was highly increased in the
LPO. The density of D2 receptors in the LPO is approximately the same as (Dietl and Palacios,
1988; Richfield et al., 1987; Schnabel and Braun, 1996) or slightly lower than in the PA
(Stewart et al., 1996). In contrast, D2 densities are 5- to 10-fold lower in the PP and INP than
in the LPO/PA (Dietl and Palacios, 1988; Richfield et al., 1987; Stewart et al., 1996).
5.4.4. DARPP-32
In pigeons (Durstewitz et al., 1998) and chicks (Schnabel et al., 1997), the striatal parts of the
basal ganglia exhibit very dense DARPP-32 labeling (Figure 15B, D), consistent with the
abundance of D1 receptors in these structures. In addition, and in contrast to the
autoradiographic D1-receptor binding studies, the pallidal PP (but not the INP) is as dense in
its DARPP-32 labeling as the striatal components (Figure 15B, D). However, DARPP-32-
positive labeling in the PP is almost exclusively constrained to fiber labeling, whereas a high
number of DARPP-32ir somata shows up in the neighbouring PA and LPO. It is conceivable
that the dense neuropil labeling in PP stems from descending D1-positive axons, as it is the
case in the globus pallidus of rats. In these animals, D1- and DARPP-32- but not D2-positive
fibers which probably belong to the striatonigral and striatoentopeduncular tracts have been
described surrounding unlabeled somata and dendrites in the globus pallidus (Ouimet and
Greengard, 1990; Yung et al., 1995). Finally, the avian Acc is relatively low in DARPP-32
content (Figure 13D; Durstewitz et al., 1998; Schnabel et al., 1997), in contrast to the high
densities of dopaminergic fibers (Wynne and Güntürkün, 1995) and D2 receptors (Dietl and
Palacios, 1988), but consistent with the low density of D1 receptors in the quail Acc according
to Ball et al. (1995).
Chapter 4: Dopaminergic innervation of the avian telencephalon 135
5.4.5. Colocalizations
Anderson and Reiner described two major populations of striatonigral projection neurons, one
that co-contains substance P and dynorphin and makes up for about 85-95 % of all projection
neurons, and the other that contains enkephalin and makes up for only 1-4 % of all projection
neurons (Anderson and Reiner, 1990, 1991b; Reiner et al., 1984b; Reiner and Anderson, 1990).
Projections from both of these neuronal populations in the medial striatum terminate on DAir
and THir, but also - to an about equal degree - on unlabeled cells in the SNC (Anderson et al.,
1991; Medina et al., 1995). Vice versa, both populations of striatonigral projection neurons
receive input from THir fibers as demonstrated by EM, targeting perikarya, dendritic shafts,
and spines (Karle et al., 1992, 1994). Furthermore, a subpopulation of substance P-ir
projection neurons (39%) also contained somatostatin, whereas neurons containing only
somatostatin or neuropeptide Y were not observed to project to the SNC (Anderson and Reiner,
1990). These findings demonstrate that in the avian like in the mammalian basal ganglia (Song
& Harlan, 1994; Takagi, 1986) subpopulations of striatal output neurons which interact with the
dopaminergic midbrain neurons could be differentiated by means of the neuropeptides co-
released by these neurons.
Reiner and Anderson (1990; Reiner et al., 1994) suppose that both, substance P containing and
enkephalin containing neurons are also colocalized with GABA. However, until now there is
no convincing evidence confirming this hypothesis. According to immunocytochemical studies,
the percentage of GADir and GABAir neurons in the avian basal ganglia seems to be low (< 15
%), even after pre-treatment with colchicine, and no significant regional variations could be
observed throughout the entire telencephalon (Durstewitz et al., 1998; Veenman and Reiner,
1994). If this pattern should hold true in the light of other methods such as in situ hybridization,
this would stand in striking contrast to what is known from the mammalian basal ganglia, where
the vast majority of neurons are of the medium spiny type which utilizes GABA (Chesselet et
al., 1987; Kita and Kitai, 1988; Surmeier et al., 1988). This apparent difference between the
mammalian and avian basal ganglia might also explain why no colocalizations of GAD and
DARPP-32 could be detected in the PA by Durstewitz et al. (1998). In contrast, in the
mammalian striatum, the very high percentages of GABAergic and DARPP-32ir neurons per se
implicate a high degree of overlap (Anderson and Reiner, 1991a; Ouimet et al., 1984, 1992).
However, it has to be pointed out that the extremely dense meshwork of THir and DAir fibers
and the very high concentration of DARPP-32 in the avian basal ganglia make the study of
possible colocalizations at the light-microscopic level extremely difficult if not impossible in
Chapter 4: Dopaminergic innervation of the avian telencephalon 136
this structure. For two other reasons the apparent lack of DARPP-32/GAD colocalizations in
the paleostriatum does not exclude that GABAergic neurons in these regions are
dopaminoceptive: First, Veenman and Reiner (1994) reported a subpopulation of small
GABAergic neurons not detected by GAD immunochemistry. Second, as discussed above, D2
receptors are even more abundant than D1 receptors in the avian basal ganglia, so that the
possibility remains that GABAergic cells in these regions are equipped with receptors of this
class. Hence, the issue whether GABAergic neurons in the avian basal ganglia receive a
significant dopaminergic input awaits further investigation.
In the VP and the BNST of the male quail brain, aromatase-ir cells were found to be located in
THir baskets, and THir varicosities were found in close vicinity of almost all of these cells
(Balthazart et al., 1998). In fact, due to the particular high concentration of both of these
markers in the VP and BNST, these structures might be major loci of interaction between
catecholaminergic systems and forebrain steroid hormone activity.
5.5. Dopaminergic innervation of song nuclei in song birds
The brain of song birds contains several sexually dimorphic nuclei involved in the song system
(Nottebohm et al., 1976, 1982) which are adapted to the specific sensorimotor requirements of
song perception and production. In the context of the dopaminergic system, these specific nuclei
deserve a special discussion, because, interestingly, they receive a much denser dopaminergic
input as measured by THir and catecholamine histofluorescence than the surrounding tissue
(Bottjer, 1993; Lewis et al., 1981; Sakaguchi and Saito, 1989; Soha et al., 1996). Among these
nuclei are a nucleus in the dorsolateral LPO, the so-called Area X, the robust n. of the
archistriatum, the lateral magnocellular n. of the anterior neostriatum, the n. interfacialis, and
the high vocal center located in the caudal neostriatum. In addition to the higher THir exhibited
by these nuclei compared to the surrounding areas, a higher density of D1-specific binding has
been demonstrated in area X of male starlings compared to the surrounding LPO (Casto and
Ball, 1994). Furthermore, a dense input to area X from VTA and SNC has been shown by
retrograde tracing (Lewis et al., 1981). In addition, the sexual dimorphism of the song nuclei is
also reflected in their dopaminergic innervation as these nuclei in male birds which sing
receive a much denser dopaminergic input than in the non-singing females. In contrast, sex
differences with regards to DA receptor densities have not been observed in non-singing birds
like quails (Ball et al., 1995).
What makes these nuclei even more interesting in terms of their dopaminergic innervation is that
Chapter 4: Dopaminergic innervation of the avian telencephalon 137
the density of the dopaminergic input to these nuclei as well as DA levels and turnover rates are
clearly correlated with the phases of song learning. Thus, in young zebra finches (< 30 d), THir
in the song nuclei is even less dense than in the surrounding tissue (Sakaguchi and Saito, 1989;
Soha et al., 1996), while a strong developmental increase occurs with the onset of the
sensorimotor phase of song learning around day 35. In accordance with these findings, Harding
et al. (1998) observed highly significant increases in DA levels and turnover rates in the song
nuclei within the LPO, the n. robustus of the archistriatum, the lateral magnocellular n. of the
anterior neostriatum, and the n. interfacialis in the neostriatum in zebra finches between
postnatal days 35-55, which strongly declined again at day 90. A similar peak around day 35 at
least in DA turnover was observed in the auditory Field L. Thus, the dopaminergic system
probably has a prominent role in song learning and production that might be related to the
requirement that sensory and motor aspects have to be integrated over time during song learning
and production (see 7.6).
6. Comparative aspects of the dopaminergic system in mammalian and avian species
From the description above, readers familiar with the dopaminergic system of mammals may
have recognized that the avian dopaminergic system shares many important organizational
features with its mammalian counterpart. Like in mammals (Berger and Gaspar, 1994;
Björklund and Lindvall, 1984; Fallon and Loughlin, 1995; Swanson, 1982), the dopaminergic
innervation of the avian telencephalon arises mainly from a small population of dopaminergic
midbrain nuclei in the VTA and SNC, where only a small percentage of fibers crosses into the
contralateral hemisphere (Metzger et al., 1996; Kitt and Brauth, 1986). The following
description focuses on some very prominent and interesting similarities and differences in the
dopaminergic innervation of the telencephalon of birds, rodents, and primates.
6.1. Basal ganglia
Like the avian 'striatum', the caudate-putamen in rats and primates receives the densest
dopaminergic fiber input and displays the highest concentrations of D1- as well as D2-
receptors (Ariano, 1997; Björklund and Lindvall, 1984; Brock et al., 1992; Camps et al., 1990;
Joyce et al., 1993; Ouimet et al., 1984; Yung et al., 1995). In addition, in mammals and birds
the caudate-putamen, or dorsal striatum, is higher in its dopaminergic innervation, DARPP-32
content, and DA receptor density (at least in D2-specific binding; Richfield et al., 1987) than
Chapter 4: Dopaminergic innervation of the avian telencephalon 138
the n. accumbens/ ventral striatum (Ball et al., 1995; Brock et al., 1992; Durstewitz et al., 1998;
Hemmings and Greengard, 1986; Joyce et al., 1993; Karle et al., 1996; Yung et al., 1995;
Wynne and Güntürkün, 1995). In contrast, 'pallidal' structures in both amniote classes are much
weaker innervated. Furthermore, despite the lower D1 receptor densities in pallidal compared
to striatal structures, a high amount of DARPP-32 is present in the mammalian globus pallidus
(Hemmings and Greengard, 1986; Ouimet et al., 1984, 1992) and its avian equivalent PP (see
5.4.4). However, as discussed for the PP in Section 5.4.4, the high DARPP-32ir in the globus
pallidus of rats is almost exclusively due to neuropil labeling, in contrast to the high number of
DARPP-32ir neurons in the striatum (compare Figure 5B in Durstewitz et al., 1998, to Figure 8
in Ouimet et al., 1984), and may stem from intense staining of descending striatonigral and
striatopallidal fibers. Finally, a rostro-caudal gradient in DA receptor densities and DA
concentration as observed in birds (see Section 5.4) has also been noted in mammals (Bockaert
et al., 1976; Boyson et al., 1986; Richfield et al., 1987; Tassin et al., 1976)
However, some differences between birds and mammals are also apparent. First, direct
comparisons between pigeons, rats, and cats revealed that the concentration of D1 receptors in
the avian 'striatum' is about 5- to 10-fold lower, and that in the avian 'pallidum' is about 20-fold
lower than in the mammalian striatum (Dietl and Palacios, 1988, Richfield et al., 1987). In
contrast, the density of D2 receptors in the pigeons 'striatum' is only about half of that in the
mammalian striatum, while, however, this difference is larger for the pallidum (Dietl and
Palacios, 1988, Richfield et al., 1987). Second, the first point implies that the ratio of D1:D2
receptors is different in the avian and the mammalian basal ganglia. Whereas in mammals D1
receptors are much more prevalent than D2 receptors (Joyce et al., 1993), the opposite seems to
be the case in birds (see 5.4.3). As D1- and D2-receptors are linked to different G-proteins and
influence adenylate cyclase activity in opposite ways (Hemmings et al., 1987b; Robinson and
Caron, 1997), the different D1:D2 receptor ratios may imply important differences in basal
ganglia function between birds and mammals.
The possible lack of DARPP-32/GAD colocalizations in the pigeon PA has already been
discussed (Section 5.4.5), and has been related to other particularities of the avian basal
ganglia, namely to the fact that the number of GAD- and GABA-positive neurons in the avian
basal ganglia as assessed by immunocytochemical techniques (Durstewitz et al., 1998;
Veenman & Reiner, 1994) seems to be much lower than in the mammalian basal ganglia (Kita
and Kitai, 1988; Surmeier et al., 1988).
Chapter 4: Dopaminergic innervation of the avian telencephalon 139
6.2. Cortex
Like in birds, in rodents and primates the dopaminergic input to cortical areas is clearly weaker
than that to the striatum, and the densities of dopaminergic receptors and of DARPP-32 sharply
decline outside the basal ganglia (Ariano, 1997; Berger et al., 1988, 1990, 1991; Berger and
Gaspar, 1994; Björklund and Lindvall, 1984; Brock et al., 1992; Hemmings and Greengard,
1986; Joyce et al., 1993; Ouimet et al., 1984, 1992). In the frontal cortices of rats and primates,
the density of D1 receptors has been estimated to be about five- to tenfold higher than that of D2
receptors (Joyce et al., 1993; Lidow et al., 1991), whereas this ratio seems to be in the range of
3:1 throughout most of the bird telencephalon (Dietl and Palacios, 1988; Schnabel and Braun,
1996; Stewart et al., 1996). In contrast, in the avian Hp the density of D2 receptors outnumbers
that of D1 receptors (Stewart et al., 1996). In the mammalian Hp, the D1:D2 ratio seems to be
at least lower than in the neocortex although in general D1 receptors may still be more
numerous (Dewar and Reader, 1989; Joyce et al., 1993). However, D1 and D2 receptors
distribute differentially across hippocampal layers (Köhler et al., 1991).
With respect to the finctional organization of the dopaminergic innervation of the avian and
mammalian 'neocortex', strong equivalencies can be observed. Like in birds, the primary visual,
auditory, and somatosensory cortices in mammals are only weakly innervated by dopaminergic
fibers, at least much weaker than the respective secondary areas (Berger et al., 1988, 1991;
Berger and Gaspar, 1994; Joyce et al., 1993). These areas also express lower levels of
DARPP-32 (Berger et al., 1990). In addition, layer IV in the granular cortices in rats and
primates, which is the major target zone of specific thalamic input, lacks a significant
dopaminergic input (Berger et al., 1988, 1991; Berger and Gaspar, 1994; Joyce et al., 1993;
Phillipson et al., 1987).
With respect to the 'higher order' neocortical areas, considerable differences between rodents
and primates have been pointed out by Berger and coworkers (Berger et al.,1991; Berger and
Gaspar, 1994). In general, the dopaminergic innervation of the primate neocortex is more
widespread than that of the rat neocortex, and may thus compare better to the dopaminergic
innervation of the avian neostriatal and hyperstriatal areas. In both, rodents and primates, the
prefrontal areas and the anterior cingulate cortex are densely innervated (Berger et al., 1991;
Berger and Gaspar, 1994; Joyce et al., 1993). Therefore, the dense dopaminergic innervation of
the NCL in pigeons was taken as an indication that this multimodal area may be comparable to
the mammalian PFC (Divac et al., 1985; Divac and Mogenson, 1985; Waldmann and
Güntürkün, 1994; Wynne and Güntürkün, 1995). However, the PFC of mammals is not a
Chapter 4: Dopaminergic innervation of the avian telencephalon 140
homogeneous structure. In primates, only the orbitofrontal and ventrolateral portions receive a
dense dopaminergic input, while the dorsolateral part is lower in this respect (Berger et al.,
1988; Lewis et al., 1992; Williams and Goldman-Rakic, 1993), and is also very low in
DARPP-32 content in adult animals (Berger et al., 1990). In rats, however, the medial
prefrontal areas, in particular the pre- and infralimbic region, which are assumed to be
equivalent to the primate dorsolateral PFC, are the neocortical areas highest in DA fiber
density (Berger et al., 1991; Berger and Gaspar, 1994; Joyce et al., 1993). In fact, this was one
of the reasons for Preuss (1995) to question the equivalency between the rat pre- and
infralimbic cortex and the primate dorsolateral PFC. Similar subdivisions of the avian
'prefrontal cortex' were not identified yet. Given the dense dopaminergic input of the NCL, one
would compare this area to the prelimbic/infralimbic area of rats, or to the orbitofrontal or
ventrolateral PFC of primates. On the other hand, the percentage of DARPP-32ir neurons in the
NCL was lower than in other, more rostrally located neostriatal structures, reminiscent of the
relatively low DARPP-32ir in the adult primate dorsolateral PFC (Berger et al., 1990). The
NCL would also compare better to the primate dorsolateral PFC with respect to its functional
characteristics, as both areas seem to be involved especially in spatial working memory
(Funahashi and Kubota, 1994; Gagliardo and Divac, 1993; Gagliardo et al., 1996; Goldman-
Rakic, 1988; Güntürkün, 1997; Mogenson and Divac, 1982, 1993; Wilson et al., 1993; but see
Petrides, 1995).
Furthermore, in primates, in contrast to rats, the premotor and motor cortices are very densely
innervated by the dopaminergic system (Berger et al., 1991; Berger and Gaspar, 1994; Joyce et
al., 1993). Insofar, pigeons and chicks compare better to monkeys than to rats (see also Reiner
et al., 1994), as major parts of the avian 'motor cortex', in particular the dorsal archistriatum
intermedium, also receive a rich dopaminergic input. The comparison holds also for the
distribution of DARPP-32ir neurons: Despite its exceptionally high dopaminergic input, the
primate motor cortex displays only a sparse distribution of DARPP-32ir neurons (Berger et al.,
1990), similar to the very low number of DARPP-32ir neurons throughout most of the
intermediate archistriatum (Durstewitz et al., 1998). In addition, Berger et al. (1990) pointed
out a mismatch between the distribution of D1 receptors and DARPP-32ir neurons in the
primate motor cortex similar to that described for the dorsal archistriatum (Sections 5.3.2 and
5.3.4).
Finally, moderate to high numbers of dopaminergic fibers are present in the secondary sensory
and association areas of the primate neocortex (Berger et al., 1988, 1991; Berger and Gaspar,
Chapter 4: Dopaminergic innervation of the avian telencephalon 141
1994). In this respect, the dopaminergic innervation of the avian telencephalon might also be
more similar to that of primates than to that of rats.
6.3. Laminae-specific distribution of dopaminergic fibers
An additional feature of the dopaminergic innervation of the mammalian neocortex is its
laminae-specific distribution. In the rat medial and orbital (lateral) prefrontal areas, the deep
layers V-VI are the major targets of dopaminergic fibers (Berger et al., 1991; Björklund and
Lindvall, 1984; Joyce et al., 1993). In the primate granular cortices, the superficial layers I-III
are generally even more densely innervated than the deep layers V-VI, whereas in the motor and
anterior cingulate (agranular) cortices all layers receive a dense dopaminergic input (Berger et
al., 1988, 1991; Berger and Gaspar, 1994; Goldman-Rakic et al., 1992; Lewis et al., 1992). As
outlined in Section 2, a cortical lamination is absent in the avian brain. Nevertheless, based on
hodological, histochemical, and functional criteria, some authors have compared subdivisions
of the avian telencephalon to specific laminae of the mammalian neocortex. It was argued that
the primary sensory areas (E, IHA,, Field L2, Bas) are equivalent to the thalamic input layer IV
of the respective neocortical areas (extrastriate and striate visual, auditory, somatosensory),
and the secondary sensory belts surrounding these primary areas and the HD to layers II-III
(Karten and Shimizu, 1989; Reiner and Karten, 1983; Veenman et al., 1995). Furthermore, the
sensorimotor archistriatum, the HA, structures of the PE, and possibly the NCL, which either
give rise to the major descending pathways or directly project onto the sensorimotor avian
striatum, have been identified with the neocortical output layers V-VI. This conception would
again render the dopaminergic innervation of the avian brain more similar to that of primates
than to that of rats, as both, the 'deeper layers' and the 'superficial layers' of the avain
'neocortex' would be high in DA and DARPP-32.
From a functional perspective, another interesting parallel between birds and mammals might
lie in the fact that DA-baskets seem to contact predominantely bigger neurons in the avian brain
(Ø > 15 µm), whereas smaller neurons are more likely innervated en-passant (Wynne and
Güntürkün, 1995). Likewise, in the mammalian neocortex, the bigger pyramidal neurons reside
in the deeper layers and are thus the ones probably most heavily innervated by DA fibers within
their deep proximal and, in primates in addition, upper layer distal apical dendrites. Moreover,
the bigger pyramidal cells are the ones which most likely exhibit repetitive oscillatory bursting-
characteristics in the neocortex (Mason and Larkman, 1990; Yang et al., 1996a), an
electrophysiological feature that - theoretically - could be exclusively due to their bigger
Chapter 4: Dopaminergic innervation of the avian telencephalon 142
somata and dendrites (Mainen and Sejnowski, 1996). Thus, dopaminergic baskets may
innervate neurons in the avian telencephalon with specific functional characteristics as
dopaminergic fibers possibly do in the mammalian neocortex.
6.4. Ultrastructural features and postsynaptic targets of the dopaminergic innervation
With respect to the postsynaptic targets of the dopaminergic input to the mammalian neocortex,
pyramidal cells seem to be the predominant dopaminoceptive population, and most DARPP-
32ir neurons are of the pyramidal type (Berger et al., 1990; Goldman-Rakic et al., 1989; Smiley
and Goldman-Rakic, 1993). Thus, the finding that GADir neurons in the avian telencephalon do
not express DARPP-32 and are never located in THir baskets (Durstewitz et al., 1998), on a
first glance, fits well into the mammalian schema. However, dopaminergic fibers in the PFC
have also been observed to terminate on smooth, presumably GABAergic, stellate cells (Smiley
and Goldman-Rakic, 1993), and the activity of GABAergic neurons in the mammalian PFC has
been shown to be modulated by DA in vivo and in vitro (Penit-Soria et al., 1987; Pirot et al.,
1992; Rétaux et al., 1991; Yang et al., 1997). The dopaminergic effect on GABAergic activity
can be antagonized by D2- but not by D1-receptor blockers (Godbout et al., 1991; Pirot et al.,
1992; Rétaux et al., 1991, but see Yang et al., 1997), although D2 receptors are present in much
lower densities in the mammalian neocortex than D1 receptors (see above). Hence, GABAergic
neurons may be affected mainly via D2 receptors, and this might also be the case in the avian
brain. Thus, despite their low densities, D2 receptors might play an important functional role
also in the avian telencephalon, and further investigation of this subject is certainly very
important.
Dopaminergic fibers in the avian telencephalon also have most of their ultrastructural features
in common with their mammalian counterparts. In both animal classes, dopaminergic synapses
are relatively small, are mainly of the symmetric type, contact predominantly dendritic arbors
and spines, and sometimes converge with other unlabeled synapses on the same spine (i.e., form
triadic complexes) (Goldman-Rakic et al., 1989; Karle et al., 1996; Metzger et al., 1996;
Séguéla et al., 1988; Smiley and Goldman-Rakic, 1993; Yung et al., 1995; see 4.4). In addition
to a specialized synaptic release-mode, much of the neuromodulator seems to be released via
unspecialized axonal varicosities in both amniote classes (Cooper et al., 1996).
In conclusion, the general pattern of the distribution of dopaminergic fibers and receptors in the
avian telencephalon, as well as the laminar and biochemical characteristics of the
dopaminergic target neurons, are quite similar to that of mammals. Some specific differences
Chapter 4: Dopaminergic innervation of the avian telencephalon 143
seem also to exist, but may partly be due to the fact that our knowledge about DA receptor
subtypes in the avian brain is still incomplete. Finally, considerable differences in the cortical
organization of the dopaminergic system have also been observed within the class of mammals.
7. Behavioral studies and functional implications
From the neuroanatomical features of the dopaminergic innervation in the avian brain, some
functional clues could be derived. For example, the fact that primary sensory areas are devoid
of DA, while higher sensory, associative, and motor areas that have a direct link to structures of
the avian basal ganglia receive a dense dopaminergic input and are high in DA receptors makes
it likely that DA plays a special role in sensory-motor integration and associative learning.
In fact, the dopaminergic system in mammals is implicated especially in motor, associative, and
higher order functions like aversive and appetitive learning, and working memory (Beninger,
1993; Salamone, 1992, 1994; Sawaguchi and Goldman-Rakic, 1991, 1994; Schultz et al., 1993,
1995; Seamans et al., 1998; Sokolowski et al., 1994; Zahrt et al., 1997). Although much less is
known about the involvement of DA in behavioral and cognitive functions in birds, from the
studies in avian species that do exist so far a similar functional involvement of the
dopaminergic system as in mammals is apparent. The next sections will deal with these studies.
7.1. Dopaminergic modulation of (unconditioned) motor functions
Dopamine in the avian brain has been shown to be critically involved in motor functions. Rieke
(1980, 1981) observed that unilateral kainic acid lesions of the paleostriatum, or its source of
dopaminergic input, the SNC, in pigeons induced persistent turning in one direction, postural
problems, arhythmic movements, and head or whole body tremors. These behavioral
dysfunctions were reproduced by unilateral injections of GABA agonists into the SNC (Rieke,
1982). Furthermore, the turning behavior elicited by unilateral GABA-agonistic injections into
the SNC or 6-hydroxydopamine (6-OHDA) destruction of dopaminergic neurons in this area
could be enhanced by apomorphine but was suppressed by haloperidol administration (Rieke,
1982; Yanai et al., 1995).
Apomorphine has also been shown to facilitate stereotypic pecking bouts in a dose-dependent
manner, alone or induced by feeding stress, and the avian basal ganglia is a likely site for these
dopaminergic effects (Cheng and Long, 1974; Deviche, 1985; Goodman et al., 1983; Nisticò
and Stephenson, 1979; Rieke, 1982; Zarrindast and Amin, 1992). In contrast, DA antagonists
Chapter 4: Dopaminergic innervation of the avian telencephalon 144
like haloperidol, chlorpromazine, or clozapine inhibit behavioral stereotypies (Cheng and
Long, 1974; Goodman et al., 1983; Kostal and Savory, 1994), and could cause sedation and
tonic immobility that could be reversed by paleostriatal but not by neostriatal lesions (Sanberg
and Mark, 1983). The effects of apomorphine and amphetamine on pecking behavior can
furthermore be antagonized by specific D1- (SCH23390) as well as by high doses of specific
D2-receptor blockers (sulpiride), with a combination of both being most effective, while D1-
or D2-specific agonists enhance apomorphine induced pecking (Dehpour et al., 1998;
Zarrindast and Amin, 1992; Zarrindast et al., 1992; Zarrindast and Namdari, 1992). Low doses
of sulpiride, however, enhanced pecking, presumably by blocking D2 autoreceptors, and low
doses of apomorphine, on the other hand, might reduce pecking also via an autoreceptor
mechanism (Deviche, 1985). It is important to point out that DA antagonists like haloperidol do
not prevent all aspects of motor behavior although they reduce pecking bouts and turning, and
may lead to sedation. Hence, even after haloperidol administration, Goodman et al. (1983)
observed pigeons engaging in some normal behaviors like feeding, and Rieke (1982) was still
able to elicit orienting and escape responses, including flying, by stressful stimuli.
Stereotypic motor behavior and hyperactivity on the one hand as induced by supranormal DA
receptor stimulation or DA activity, and sedation on the other hand as induced by, e.g.
neuroleptic drugs, are also typically observed in mammals including humans (Bo et al., 1988;
Clark and White, 1987; Gessa et al., 1985; Le Moal and Simon, 1991; Ridley, 1994;
Sokolowski and Salamone, 1994; Waddington and Daly, 1993; Wickens, 1990). However, like
in birds, behavioral reactions to intense or significant stimuli may be left intact after DA
depletion or subnormal DA receptor stimulation in the basal ganglia (Salamone, 1992). Thus,
Salamone (1992) concluded that DA is not that much involved in motor processes per se but
more in sensory-motor integration that requires novel motor sequences or in processes that
extend significantly in time (see also Taylor and Saint-Cyr, 1995). DA might also be especially
important for motor behavior that is driven by internal states without being directly controlled
by external stimuli.
Lindenblatt and Delius (1988) demonstrated that apomorphine-induced pecking bouts could
also be elicited by local injections into the Bas, and could be reversed by 6-OHDA injections
into this structure. However, since dopaminergic fibers and receptors seem to be lacking in the
Bas, the behavioral effects of DA-agonistic drugs might also be attributed to ligand diffusion
into the adjacent PA and PP. Consistent with this interpretation, apomorphine injections into the
Bas also produced contralateral turning (Lindenblatt and Delius, 1988), a typical symptome of
Chapter 4: Dopaminergic innervation of the avian telencephalon 145
altered DA activity in the avian basal ganglia (Rieke, 1981, 1982). Wynne and Delius (1996)
reported that apomorphine-induced pecking could be reduced by lesions of the Bas, thus
demonstrating that the Bas is involved in apomorphine-induced stereotypies. However, this
result does not necessarily imply that the Bas is in fact a site of apomorphine action.
Apomorphine might increase pecking rates via actions in the basal ganglia, while Bas lesions
could reduce pecking rates independently. On the other hand, the finding of Durstewitz et al.
(1998) that the Bas had high number of DARPP-32ir neural cell bodies may hint to some
anatomical basis for the apomorphine induced pecking bouts. Hence, the neuroanatomical site
of the apomorphine-induced pecking bouts remains unclear.
7.2. Dopaminergic involvement in arousal and wakefulness
Ferrari and Giuliani (1993) found that DA in the chick brain might also participate in the
regulation of states of arousal and wakefulness. Selective D2 agonists induced hypomotility and
sleep-like states. However, for some D2 agonists (like lisuride) or the mixed D1/D2 agonist
apomorphine, this effect reversed at higher doses, leading to behavioral excitation and
increased spontaneous pecking instead. Similar dose-dependent patterns were also observed in
rodents, where the sedatory effects of the drugs were attributed to predominant stimulation of
presynaptic D2 autoreceptors at lower doses (Ferrari and Giuliani, 1993; Ongini, 1993). In
accordance with this interpretation, microinfusion of apomorphine into the rat VTA, but not into
the Acc, caudate n., or PFC, induces behavioral and EEG signs of sleep, which could be
antagonized by sulpiride (Bagetta et al., 1988a, 1988b). In general, in mammals dopaminergic
drugs increase alertness, wakefulness and exploratory acitvity, with accompanying indications
in the EEG, while lesions of the dopaminergic system, DA antagonists, or subnormal DA
activity after preferential D2 autoreceptor stimulation have the opposite effect (Bagetta et al.,
1988a, 1988b; Gessa et al., 1985; Montaron et al., 1982; Ongini, 1993). Dopaminergic effects
on arousal and wakefulness on the one hand, and on behavioral excitation and stereotypies
(Section 7.1) on the other, may be mediated by the same neural mechanism (Section 7.6).
7.3. Dopaminergic modulation of appetitive and consummatory behavior
DA in the mammalian brain is well known to regulate aspects of food and water intake
(Bertolucci-D’Angio et al., 1990a, 1990b; Cooper and Al-Naser, 1993; Wilson et al., 1995),
and of sexual behavior (Pfaus and Phillips, 1989). Likewise, apomorphine and DA
administration in pigeons reduces food consumption (Deviche, 1984; Ravazio and Paschoalini,
Chapter 4: Dopaminergic innervation of the avian telencephalon 146
1992), possibly because apomorphine and DA could substitute for the rewarding value of food.
Alternatively, the low doses of apomorphine used by Deviche (1984) might have reduced food
intake by very much the same mechanism that has been proposed for apomorphine-induced
sedation (see Section 7.2), i.e. primarily via D2 autoreceptors. In fact, Deviche (1984) noted
that the doses of apomorphine used in his study were too low to elicit pecking bouts, or even
inhibited pecking.
Apomorphine also reduces appetitive and consummatory aspects of sexual behavior in the male
quail (Absil et al., 1994; Castagna et al., 1997). These apomorphine-induced effects are
probably due to D2-receptor stimulation as D2 agonists reduce both appetitive and
consummatory sexual behavior, while D1 agonists do just the reverse, i.e. increase both aspects
of sexual behavior (Balthazart et al., 1997). Again, these actions of the dopaminergic system
may be related to a rewarding function of DA which has been proposed by several authors
(Schultz et al., 1993, 1995; see Beninger, 1993; see Salamone, 1992, 1994; Wickens, 1990;
Wickens and Kötter, 1995). According to studies in rats, part of the dopaminergic effects on
food consumption and sexual behavior might be regulated by dopaminoceptive hypothalamic
nuclei (Cooper and Al-Naser, 1993). However, additional structures are probably also
involved since increases of DA metabolites during food consumption or upon presentation of
appetitive stimuli have also been observed in the Acc, medial PFC, and in the amygdala in rats
(Bertolucci-D’Angio et al., 1990b; Wilson et al., 1995).
The ‘reward theory’ of DA function partly derived from studies on intracranial self-stimulation
and self-administration of dopaminergic drugs (Fibiger, 1978; Le Moal and Simon, 1991; Wise,
1978; Yokel & Wise, 1975). Self-administration of cocaine was also observed in pigeons,
where it could be antagonized by haloperidol (Winsauer and Thompson, 1991), pointing to a
similarly ‘rewarding’ action of DA in the avian as in the mammalian brain.
7.4. Dopaminergic modulation of learning and conditioned behavior
Dopamine in the avian telencephalon is involved in various forms of learning. Stewart et al.
(1996) found that after a one-trial taste aversion avoidance learning, D1- but not D2-specific
binding increased highly significantly in the LPO. A role for DA in parts of the striatum, namely
in the Acc, in passive avoidance training (and, more generally, in aversive situations) is also
well established in mammals (Beninger, 1993; Bertolucci-D’Angio et al., 1990a, 1990b;
Salamone, 1994). In an in vivo microdialysis study, Gruss and Braun (1997) demonstrated
decreased levels of the DA metabolite homovanillic acid in the MNH after auditory imprinting
Chapter 4: Dopaminergic innervation of the avian telencephalon 147
in young chicks, while significant increased levels were observed after exposing the chicks to
handling stress. Stressful situations induce increases of DA metabolites also in rats in the Acc
and medial PFC (Abercrombie et al., 1989; Bertolucci-D’Angio et al., 1990b). The studies by
Soha et al. (1996) and Harding et al. (1998) reported in Section 5.5 suggest that DA is
additionally involved in the sensorimotor stage of song learning in zebra finches as the
postnatal development of the innervation of various forebrain song nuclei by THir fibers as
well as DA levels and turnover in these nuclei are correlated with this period. A direct
involvement of DA in learning mechanisms is made likely by a study by Lindenblatt and Delius
(1987) who showed that apomorphine (and thus probably DA) might serve as a positive
reinforcer when coupled with a conditioned stimulus. Furthermore, DA antagonists like
haloperidol reduce the rate of conditioned responding without affecting discrimination
performance (Tombaugh, 1981), while conditioned responding of young chicks for heat
reinforcement was shown to be enhanced especially after combined D1- and D2-agonist
injection (Dose and Zolman, 1994). Finally, following apomorphine administration, chicks
were found to be unable to suppress formerly rewarded but now punished responses, while this
can be reversed by haloperidol pretreatment (McDougall et al., 1987).
Although these studies show that DA is somehow involved in various learning processes, the
nature of the underlying mechanism is still far from clear. Thus, often it may not be easy to
decide whether the DA-induced effects can be attributed to a specific role of DA in learning, to
its motivational effect, to its role in motor behavior, or to its impact on attention and arousal
(see Salamone, 1992). This is especially true when DA agonists are administered systemically,
thus acting on many structures. For example, McDougall et al. (1987) hinted to the fact that
apomorphine in their experiments generally enhanced the response rate but that this effect may
have been obscured due to a ceiling effect as long as the response was associated with reward
only. Only when punishment was introduced, the behaviorally exciting effect of apomorphine
might have become apparent. Thus, although it is interesting to note that increased pecking rates
prevail even in the presence of punishment, the main effect of apomorphine may not be to inhibit
response suppression learning but to induce behavioral excitation.
7.5. Dopaminergic modulation of working memory
A failure to inhibit inadequate responses as demonstrated by McDougall et al. (1987) is also
often observed after lesions of the rat and primate PFC, or lesions of its dopaminergic afferents
(Dias et al., 1997; Fuster, 1989; Sokolowski and Salamone, 1994). A failure to suppress
Chapter 4: Dopaminergic innervation of the avian telencephalon 148
irrelevant response options may also play a role in the inability of pigeons with NCL lesions -
depending on the extent to which the NCL was destroyed - to perform a reversal learning
(Hartmann and Güntürkün, 1998) or a go/no-go task (Güntürkün, 1997). In addition, lesions of
the NCL or its main source of thalamic afferents, the n. dorsolateralis posterior thalami, lead to
diminished delayed alternation performance (Gagliardo et al., 1996; Güntürkün, 1997;
Mogensen and Divac, 1982, 1993). Tasks which involve a delay are thought to be indicative of
working memory functions, as in these tasks an animal has to actively hold information in
memory for the guidance of responding in forthcoming situations (Fuster, 1989). Reduced
delayed alternation performance is also a characteristic deficit observed after PFC lesions in
mammals (Fuster, 1989). With regards to the transient nature of the spatial working memory
deficits after NCL lesions, the NCL compares better to the pre-/infralimbic region of rats than
to the dorsolateral PFC of primates, where lesions lead to more serious and longer-lasting
working memory deficits (Preuss, 1995).
DA and D1 receptors within the PFC are well known to be involved in working memory
functions in primates and rats (Brozoski et al., 1979; Müller et al., 1998; Sawaguchi and
Goldman-Rakic, 1991, 1994; Seamans et al., 1998; Simon et al., 1980; Zahrt et al., 1997). Also
in pigeons, blockade of dopaminergic transmission or receptors by systemic administration of
various neuroleptic drugs like chlorpromazine and clozapine has been shown to decrease
delayed matching to sample performance in a dose-dependent fashion (Picker and Massie,
1988; Watson and Blampied, 1989). Interestingly, both these drugs increased accuracy when
administered at low doses, possibly via predominant actions on D2 autoreceptors at these doses
(Picker and Massie, 1988).
Only recently our laboratory has gathered evidence that D1 receptors specifically in the pigeon
NCL might be involved in response inhibition and working memory. Thus, Diekamp et al.
(1998) presented evidence that local D1 receptor blockade by SCH23390 in the NCL impairs
go/no-go performance. After local SCH23390 application, pigeons tended to respond to all
stimuli irrespective of their reward value. Güntürkün and Durstewitz (in press) trained pigeons
in a labyrinth task which had a spatial working memory and a non-spatial reference memory
component. At the beginning of a daily session pigeons were randomly placed at one of two
starting positions in a labyrinth with 24 chambers, in which 12 arms contained red cups that
were never baited, while the remaining 12 chambers contained white cups that were baited only
at the onset of a session. Thus, as a reference memory component of the task, pigeons had to
learn to discriminate red from white cups as only the latter ones contained food pellets.
Chapter 4: Dopaminergic innervation of the avian telencephalon 149
Moreover, they had to learn never to return to an arm visited previously as each white cup only
contained food once at the onset of a session. This was the working memory component of the
task as pigeons mentally had to keep track of all arms already visited, or - complementary - had
to keep in mind all arms still worth visiting. After local SCH23390 injections into the NCL,
pigeons had no problems with their reference memory, but were significantly impaired
compared to pigeons which received saline control injections in the working memory part. In
addition to true working memory errors, which were defined as re-entrances into arms after the
animal had already visited one or more other arms, there was also a significant increase in
'perseveration' errors. Perseveration in this task was defined as an immediate re-entrance into
an arm, most often due to the fact that a pigeon stayed in an arm and tried to access the same
food cup a couple of times in close temporal succession. Perseveration is also a well known
phenomenon in prefrontal mammals and patients with prefrontal disorders or lesions (Dias et
al., 1997; Iversen and Mishkin, 1970; Fuster, 1989; Milner and Petrides, 1984).
7.6. The possible function of dopamine in sensory-motor processes
Summarizing Sections 7.1-7.5, the dopaminergic system in birds - consistent with the
anatomical data - plays a role in motor functions, in arousal, in learning, and probably in
working memory, while DA levels do not change with visual experience (Davies et al., 1983)
and DA receptor antagonists, in general, do not seem to affect sensory discrimination per se,
although they might interfere with sensory-motor integration and instrumental responding
(Güntürkün and Durstewitz, in press; Mogensen and Divac, 1993; Tombaugh, 1981). Thus, in
the avian telencephalon the dopaminergic system is involved in very much the same motor and
cognitive functions as it is in mammals, supporting the notion of a tight link between structure
and function in the nervous system. That is, the similar structural/anatomical organization of the
dopaminergic system in birds and mammals seems to give rise to a similar functional
organization, despite the facts that the evolution of birds and mammals diverged more than 200
million years ago and that the organization of avian telencephalic areas is quite different from
that of mammals.
As already noted in Section 7.4, some of the behavioral functions of DA might be difficult to
discern. According to Salamone (1992), the apparent dopaminergic involvement in motor
functions and in motivation might actually refer to common neural processes, related to sensory-
motor integration evolving in time, and to instrumental or 'intentional' behavior. To unravel the
possible function of DA in various motor and cognitive processes, and to explicate the detailed
Chapter 4: Dopaminergic innervation of the avian telencephalon 150
neural mechanisms by which DA achieves these functions, knowledge about DA-induced
manipulations of neural and synaptic biophysical properties is essential. However, to our
knowledge, until now nothing is known about dopaminergic modulation of electrophysiological
and biophysical properties of neurons in avian forebrain regions, neither in vivo nor in vitro.
In the PFC of rats, DA enhances a persistent Na+ current (Gorelova and Yang, 1997; Shi et al.,
1997; Yang and Seamans, 1996), reduces a slowly inactivating K+ and a dendritic high-voltage-
activated Ca2+ current (Seamans et al., 1997; Yang and Seamans, 1996; Yang et al., 1996b),
reduces synaptic currents through AMPA and NMDA channels (Law-Tho et al., 1994; Pralong
and Jones, 1993), and increases spontaneous activity of GABAergic interneurons (Godbout et
al., 1991; Penit-Soria et al., 1987; Pirot et al., 1992; Rétaux et al., 1991; Yang et al., 1997). It
could be shown by computational modeling that DA by inducing these biophysical changes
could act to stabilize active neural representations, and to protect them against interfering
stimulation and noise (Durstewitz et al., 1999). That is, DA ensures that delay-activity of PFC
neurons in working memory tasks, which is believed to represent the active holding of goal-
related information (Funahashi and Kubota, 1994; Fuster, 1989; Goldman-Rakic, 1995), is
sustained until the goal has been achieved. The finding that mainly or exclusively excitatory
neurons are equipped with D1 receptors (see Section 5) fits well into this functional schema,
as, at least in mammals, the D1 receptor-mediated effects on pyramidal cells are the ones
contributing most to the stabilizing effect of DA (Durstewitz et al., 1999).
The proposed function of DA may not just be important in working memory situations, but in
fact in any sensory-motor process, where a representation of the current goal-state of the
movement has to be kept upright and to be compared with incoming sensory information.
Indeed, sustained delay-activity has also been observed in the primate premotor and motor
cortices (Di Pellegrino and Wise, 1991), the striatum (Apicella et al., 1992), and the posterior
parietal cortex (Constantinidis and Steinmetz, 1996), which all receive a dense dopaminergic
input in primates. In addition, as pointed out by Salamone (1992), temporally extended sensory-
motor processes and sequences are especially susceptible to interference with the
dopaminergic system while brief responses are less easily disrupted. Sustained delay-activity
may be furthermore important in various, especially operant, learning processes where a
temporal gap between related stimuli has to be bridged in order to detect the relation. Thus,
various forms of learning, selective attention, working memory, and sensory-motor integration
may all depend on the stability (and thus maintenance) of neural representations, which might be
critically regulated by DA. Hence, some of the seemingly very different effects that DA excerts
Chapter 4: Dopaminergic innervation of the avian telencephalon 151
in different anatomical structures may in fact be related to the same basic neural mechanism.
However, active maintenance of representations may be less important in primary sensory
processes, and this could explain why primary sensory areas are not or only weakly innervated
by DA in birds as well as in mammals.
The here proposed function of DA might also explain the peculiar finding that many DARPP-
32ir neurons show up in the Bas, although it is a primary sensory center. The Bas is the only
region of the avian telencephalon that receives direct sensory inputs without thalamic relay, and
it is involved in a forebrain circuit for the control of pecking where sensory and motor signals
have to be integrated within extremely short time spans. Thus, the Bas might be directly
involved in sensory-motor processes where sustained delay-activity plays an important role.
In conclusion, the structural and functional organization of the telencephalic dopaminergic
system in birds and mammals suggests that DA might have a common function across animal
classes and possibly within different telencephalic regions. DA seems to be particularly
involved in sensory-motor and associative processes, where it might act to stabilize and sustain
neural activity related to behavioral or motor goal states, or to linkage of temporal
discontingent stimuli.
Acknowledgements
Supported by grants of the Alfried Krupp-Stiftung (D.D. and O.G.) and by the Deutsche
Forschungsgemeinschaft through its Sonderforschungsbereich NEUROVISION (S.K. and O.G.).
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 152
The Dopaminergic Innervation of the Pigeon Telencephalon: Distribution of
DARPP-32 and Co-occurrence with Glutamate Decarboxylase and
Tyrosine Hydroxylase
Daniel Durstewitz, Sven Kröner, Hugh Hemmings JR, and Onur Güntürkün
Abstract
Dopaminergic axons arising from midbrain nuclei innervate the mammalian and avian telencephalon with
heterogeneous regional and laminar distributions. In primate, rodent, and avian species, the neuromodulator
dopamine is low or almost absent in most primary sensory areas and is most abundant in the striatal parts of the
basal ganglia. Furthermore, dopaminergic fibers are present in most limbic and associative structures.
Herein, the distribution of DARPP-32, a phosphoprotein related to the dopamine D1-receptor, in the pigeon
telencephalon was investigated by immunocytochemical techniques. Furthermore, co-occurrence of DARPP-
32-positive perikarya with tyrosine hydroxylase-positive pericellular axonal ‘baskets’ or glutamate
decarboxylase-positive neurons, as well as co-occurrence of tyrosine hydroxylase and glutamate decarboxylase
were examined. Specificity of the anti-DARPP-32 monoclonal antibody in pigeon brain was determined by
immunoblotting. The distribution of DARPP-32 shared important features with the distribution of D1-
receptors and dopaminergic fibers in the pigeon telencephalon as described previously. In particular, DARPP-
32 was highly abundant in the avian basal ganglia, where a high percentage of neurons were labeled in the
‘striatal’ parts (paleostriatum augmentatum, lobus parolfactorius), while only neuropil staining was observed in
the ‘pallidal’ portions (paleostriatum primitivum). In contrast, DARPP-32 was almost absent or present in
comparatively lower concentrations in most primary sensory areas. Secondary sensory and tertiary areas of the
neostriatum contained numbers of labeled neurons comparable to that of the basal ganglia and intermediate
levels of neuropil staining. Approximately up to one third of DARPP-32-positive neurons received a basket-
type innervation from tyrosine hydroxylase-positive fibers in the lateral and caudal neostriatum, but only about
half as many did in the medial and frontal neostriatum, and even less so in the hyperstriatum. No case of
colocalization of glutamate decarboxylase and DARPP-32 and no co-occurrence of glutamate decarboxylase-
positive neurons and tyrosine hydroxylase-basket-like structures could be detected out of more than 2000
glutamate decarboxylase-positive neurons examined, although the high DARPP-32 and high tyrosine
hydroxylase staining density hampered this analysis in the basal ganglia.
In conclusion, the pigeon dopaminergic system seems to be organized similar to that of mammals. Apparently,
in the telencephalon, dopamine has its primary function in higher level sensory, associative and motor
processes, since primary areas showed only weak or no anatomical cues of dopaminergic modulation.
Dopamine might exert its effects primarily by modulating the physiological properties of non-GABAergic and
therefore presumably excitatory units.
Neuroscience 83:763-779 (1998)
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 153
INTRODUCTION
The mesotelencephalic dopaminergic system of mammals (Berger et al., 1991; Björklund and
Lindvall, 1984; Joyce et al., 1993) and birds (Kitt and Brauth, 1986; Moons et al., 1994;
Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995) consists of a relatively small
population of dopaminergic midbrain neurons in the ventral tegmentum and substantia nigra
(cell groups A8, A9 and A10) which project diffusely to various limbic, motor and association
areas. Primary sensory structures receive much weaker innervation (Berger et al., 1991; Berger
et al., 1988; Joyce et al., 1993; Lidow et al., 1986; Moons et al., 1994; Wynne and Güntürkün,
1995). In mammals, the most prominent telencephalic target areas of the dopaminergic
innervation are parts of the basal ganglia (caudate-putamen, n. accumbens), the prefrontal
cortex, and the olfactory, entorhinal and anterior cingulate cortex. There are considerable
differences in the distributions of dopaminergic fibers in the cortex of rodents and primates
(Berger et al., 1991). For example, in primates the cortical motor areas (supplementary motor,
premotor and motor cortex) are also densely innervated whereas in rats they are not. In addition
to these area-specific differences in dopaminergic intensity, differential patterns of laminar
distribution have also been observed (Berger et al., 1991).
The dopaminergic system plays an important role in instrumental learning and working memory
(Apicella et al., 1992; Baunez et al., 1995; Beninger, 1993; Brozoski et al., 1979; Salamone,
1992; Sawaguchi and Goldman-Rakic, 1994; Schultz et al., 1995; Shu et al., 1988; Sokolowski
and Salamone, 1994; Sokolowski et al., 1994). For example, blockade of D1-receptors in the
primate prefrontal cortex produces severe deficits in a spatial delayed response task
(Sawaguchi and Goldman-Rakic, 1994). There is evidence that dopamine is more involved in
the acquisition of instrumental responses than in their performance (Beninger, 1993).
Dopaminergic midbrain neurons are active in new situations where reward or reward
predicting stimuli occur (Ljungberg et al., 1992; Schultz and Romo, 1993; Schultz et al., 1993).
However, after acquisition of the operant, stimulus-triggered activity disappears in most
dopaminergic units. In accordance with these data, Montaron et al. (1982) showed that lesions
of the ventral tegmentum in the cat disrupt periods of explorative behavior and focal attention.
Also, the 35-45 Hz EEG rhythms normally associated with these states disappear in ventral
tegmentum-lesioned animals. Computational models of dopaminergic function suggest that
dopamine may enable fast learning in new or unexpected and biologically significant situations
by suppressing interference with previously learned associations and aiding ‘relearning’ of
connections. (Durstewitz and Güntürkün, 1996).
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 154
Although extensive data have been accumulated on the anatomy and functional relevance of the
dopaminergic system, detailed comparative information concerning the neural architecture
within which dopaminergic fibers interact with other chemically identified systems is lacking.
To gain further insight into the structural basis of dopaminergic function, we extended previous
studies on the dopaminergic innervation of pigeon telencephalon, which demonstrated
substantial similarities to mammals (Divac et al., 1985; Divac et al., 1994; Juorio and Vogt,
1967; Kitt and Brauth, 1986;Moons et al., 1994; Waldmann and Güntürkün, 1993; Wynne and
Güntürkün, 1995). Here, the distribution of DARPP-32 throughout the pigeon telencephalon
was analyzed and estimates of the percentage of DARPP-32-positive (DARPP-32+) neurons in
selected telencephalic regions are provided. Furthermore, co-occurrence of DARPP-32,
tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD) was investigated. DARPP-32
is a dopamine- and cyclic AMP-regulated phosphoprotein (Mr = 32,000), which is closely
associated with cells expressing the dopamine D1-receptor (Berger et al., 1990; Hemmings et
al., 1984; Quimet et al., 1992; Qimet et al., 1984;Walaas and Greengard, 1984). The D1
receptor is the principal dopamine receptor subtype in mammalian cortex (Joyce et al., 1993)
and bird telencephalon outside the basal ganglia (Dietl and Palacios, 1988). It has been
implicated in working memory and associative learning (Beninger, 1993; Sawaguchi and
Goldman-Rakic, 1994). TH, the rate-limiting enzyme in the synthesis of catecholamines, is
present in catecholaminergic neurons and fibers. A special feature of the dopaminergic
innervation of pigeon telencephalon is the presence of ‘basket’-like structures (Veenman and
Reiner, 1994; Wynne and Güntürkün, 1995) in which dopaminergic fibers densely coil around
single somata and thereby provide a massive input to them (Metzger et al., 1996). Baskets make
possible the observation of postsynaptic targets of dopaminergic innervation at the light
microscopic level. We therefore investigated co-occurrence of DARPP-32+ neurons and TH
baskets. More interestingly from a functional perspective, we determined whether GABAergic
neurons, as assessed by GAD-immunoreactivity, receive this type of dense catecholaminergic
input. In addition, colocalization of GAD and DARPP-32 was analyzed, which would indicate
the expression of D1-receptors in GABAergic neurons.
EXPERIMENTAL PROCEDURES
Animals and tissue preparation. A total of 14 adult pigeons (Columba livia) from local stock were used.
Animals were injected with 1,000 IU heparin 15 min prior to perfusion and deeply anaesthetized with 0.3 - 0.4
ml Equithesin (Güntürkün et al., 1993) per 100 g body weight. Pigeons were then perfused intracardially with
400 ml 0.9 % saline (40°C) followed by 1,000 ml of a fixative consisting of 4 % paraformaldehyde in 0.12 M
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 155
phosphate buffer (PB; 4°C, pH 7,4). After perfusion, brains were dissected and stored for one hour in the same
fixative to which 30 % (w/v) sucrose was added, and then transferred to 30 % (w/v) sucrose in PB for 12 h at
4°C. Brains were cut in frontal slices of 35-40 µm and collected in PB containing 0.05 % (w/v) NaN3 as a
preservative. Representative sections were then processed either for a peroxidase-antiperoxidase (avidin-
biotin-conjugate, ABC, or Vector VIP) or the indirect immunofluorescence technique. Treatment of animals
conformed to the specifications of the German law for the prevention of cruelty to animals.
Immunocytochemistry: Avidin-biotin-peroxidase procedure. For the ABC technique, slices were treated
according to the following procedure: Free floating sections were incubated overnight at 4°C in anti-DARPP-
32 (C24-6a) from mouse (Hemmings and Greengard, 1986) (working dilution 1:10,000) or anti-TH from
mouse (Boehringer; working dilution 1: 200) in phosphate buffer containing 0.9 % (w/v) NaCl (PBS; pH 7,4)
and 0.3 % (v/v) Triton X-100 (Sigma). The following steps were carried out at room temperature, separated by
three washes in PBS of 10 min each. Slices were preincubated in 10 % (w/v) bovine albumin fraction V (Serva)
or sheep (Sigma) serum in PB. After washing, slices were incubated in the secondary antibody directed against
mouse from sheep (Boehringer) diluted 1:200 in PBS containing 0.3 % Triton X-100 for 1 hour. After three
washes, slices were put for 1 h in Vectastain ABC (Vector) in PBS and 0.3 % Triton X-100. Three washes in
PBS were followed by two additional washes in 0.12 M acetate buffer (pH 6). Staining was achieved by the
3,3'-diaminobenzidine-(DAB) technique with heavy-metal amplification (Adams, 1981; Shu et al., 1988) by
adding H8N2NiO8S2 (2.5 g/ 100 ml), NH4Cl and CoCl2 (both 40 mg/ 100 ml) or the VIP-(Vector) technique,
yielding a black or violet signal, respectively. For labeling with DAB, 400 mg/ 100 ml β-D-glucose were added
to the solution. After 15 min of preincubation the reaction was catalyzed with 100-200 U/ mg glucose-oxidase
(Sigma, type VII). In some cases, instead of β-D-glucose and glucose-oxidase, a solution of 0.3% H2O2 was
used to catalyze the reaction. Finally, slices were washed three more times for 5 min in 0.12 M acetate buffer
and two times in PBS. For labeling with VIP, 110 µl of reagent 1 of the VIP Substrate Kit were added to 50 ml
PBS and mixed well. Then 70 µl of reagent 2 were added and mixed well. Finally 70 µl of reagent 3 were added
and mixed well. The reaction was started by a 0.3% H2O2 solution. Washing of slices in PBS stopped the
reaction. Slices were then mounted and coverslipped.
Immunocytochemistry: Indirect immunofluorescence procedure. For indirect immunofluorescence, the
following basic procedure was used: Slices were incubated overnight at 4°C in the first primary antibody in
PBS. For DARPP-32 and TH, but not for GAD, 0.3 % (v/v) Triton X-100 was added; Triton X-100 yields
poorer staining with GAD-antibodies. As described above, slices were washed three times (per 10 min) in PBS,
preincubated in 10 % (w/v) bovine serum albumin, and then washed three more times. Slices were then
incubated for 1 h with fluorescence dye-coupled secondary antibody and the reaction was stopped by three
washes in PBS. This procedure was repeated for the second primary antibody. The primary antibodies were
diluted as follows (the respective dilutions of the secondary antibodies are given in brackets): anti-DARPP-32
from mouse 1:1,000 (1:200), anti-TH from mouse (Boehringer) 1:100 (1:50), anti-GAD from rabbit
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 156
(Chemicon) 1:500 (1:100). Fluoresceine FITC, Texas Red TRSC and Rhodamine LRSC or TRITC labeled
donkey antibodies directed against mouse or rabbit IgG (all Jackson) were used as secondary antibodies.
In some cases, the following procedure was used to amplify the DARPP-32, TH, or GAD immunosignal:
Instead of the respective fluorescence dye-coupled secondary antibody, a biotinylated secondary antibody
directed against mouse IgG from sheep (Boehringer) was used. Slices were then incubated in avidin-coupled
fluoresceine or Texas Red (avidin DCS; Vector) for 1h, followed by incubation in a biotinylated tertiary
antibody directed against avidin (anti-avidin D; Vector) for 1 h, and again fluoresceine or Texas Red avidin DCS
for 1h. All these steps were carried out in PBS with Triton X-100 omitted, and were separated by three washes
in PBS of 10 min each.
For immunofluorescence the following filters were used: Olympus BH - IB block with additional short-pass
emitter filter G520 (FITC). Olympus BH-G with additional long-pass exciter filter EO 530 (TRITC/LRSC).
Chroma exciter HQ 577/10 with dichromic beamsplitter Q 585 LP and emitter HQ 645/75 (TRSC).
In general, fluorescence methods yielded poorer staining with fewer cells labeled compared to the avidin-
biotin-peroxidase techniques.
Quantitative analysis. To estimate the percentage of DARPP-32+ neurons in different brain regions,
consecutive sections of 35 µm were stained alternately for DARPP-32 as described above or Nissl-stained
with cresyl violet. 20 anatomical structures, which are listed in the results section, were selected due to
functional considerations. For each of these structures, a center region was defined as illustrated in Fig. 3,
divided into thirds, and from each third one sample area was drawn randomly. These sample areas had a size of
108 × 87 µm2
, which was the maximum size processed by our image analysis system (analySIS, Münster) using
a 100 × 2.5 × 1.6 magnification. All DARPP-32 labeled neurons in this area were counted. By use of
landmarks, the corresponding area at the same location was selected in the succeeding Nissl-stained section,
and here also all neurons were counted. DAB sections were shrunk by a factor of approximately 1.43 compared
to Nissl preparations and cell countings were adjusted accordingly. The percentage of DARPP-32+ neurons in
a region was estimated by dividing the corrected mean DARPP-32+ count by the respective mean of Nissl-
stained neurons.
For calculating percentages of DARPP-32+ neurons located in TH-baskets, we used sections with TH+ fibers
labeled with DAB, and DARPP-32+ neurons labeled with VIP, with anti-TH used first. As both antibodies are
derived from mouse, cross-reactions might be expected. However, since TH labels only fibers in the
telencephalon, with the sole exception of a number of cell bodies in the olfactory bulb, (Wynne and Güntürkün,
1995) staining patterns of DARPP-32 and TH could easily be separated. Proceeding in steps of 500 µm from
rostral A 13.0 to caudal A 4.5, a frame of size 95 × 80 µm2
was moved across the whole latero-medial and
dorso-caudal extent of the telencephalon with a constant step size of 800 µm in vertical and horizontal
direction. All labeled neurons within the area enclosed by the frame were checked for being enwrapped by a
TH-basket. The same basic procedure was applied to the investigation of GAD/TH and GAD/DARPP-32
colocalizations. However, in these analysis, indirect immunofluorescence was used, since double labeling
against GAD and TH with ABC produced a high background such that the GAD signal could not be well
discriminated.
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 157
Control experiments. For GAD, a blocking experiment was performed. Slices were incubated in the primary
antibody together with the GAD antigen (10-3 M, Worthington) and then processed according to the ABC-
technique. This resulted in an unspecific light brown staining. The specificity of the antibody directed against
DARPP-32 was assessed by immunoblotting (see below). As controls for the fluorescence double-labeling
studies, in different trials either one of the two primary or one of the secondary antibodies was omitted. In all
of these cases, no specific staining or no signal at all could be detected for the respective target protein.
Immunoblotting. Samples of pigeon brain cerebellum and neostriatum were rapidly dissected and frozen.
Frozen brain samples were thawed and homogenized in 1% (w/v) sodium dodecyl sulfate (SDS) and 10 µg/ml
leupeptin (Chemicon). Aliquots (100 µg of total protein determined by the bicinchoninic acid method (Smith et
al., 1985) with bovine serum albumin as standard) were subjected to SDS/polyacrylamide gel electrophoresis
(SDS/PAGE) on gels cast from 13% (w/v) acrylamide and transferred to 0.2 µm nitrocellulose membrane
(Schlicher and Scheull) as described (Hemmings and Adamo, 1997). Immunoblotting with anti-bovine DARPP-
32 antibody C24-6a (Hemmings and Greengard, 1986) at a 1:500 dilution was performed as described
(Hemmings and Adamo, 1997) using prestained molecular weight markers (Amersham).
RESULTS
Immunoblotting
The specificity of the antibody to DARPP-32 was demonstrated by the selective labeling of a
single protein of Mr = 32,000 in pigeon neostriatum (Fig. 1). No immunoreactive protein was
detected in the cerebellum by immunoblotting or by immunocytochemistry.
Distribution of DARPP-32
The antibody directed against DARPP-32 stained a number of neurons to a large extent so that
fine dendritic processes with spines were clearly visible (Figs 2D-F, 4D). More often,
however, only somata with the first segments of stem dendrites were labeled (Fig. 2B-C).
Neurons of various sizes and shapes were stained, with soma diameters ranging from about 5 -
25 µm. Neurons also differed considerably in their degree of staining intensity.
Telencephalic regions differed with respect to the ratio of soma to neuropil staining. Whereas,
for example, in the paleostriatum primitivum the neuropil was densely stained and neurons
seemed to be selectively spared (Fig. 5B), in the n. basalis there was almost no neuropil
staining while a large number of somata were clearly DARPP-32+ (Fig. 4A). To acknowledge
the different contributions of neuropil and soma labeling, telencephalic subdivisions were
classified according to two different schemes with four categories each. The results are shown
in Fig. 3.
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 158
The first categorization was based on neuropil staining intensity. Structures were defined as "1"
when being virtually devoid of any labeling, as "2" for a light neuropil staining, as "3" for
considerably stronger staining, and as "4" for extremely dense staining (Fig. 2A). The second
categorization was based on the percentage of DARPP-32+ neurons. Percentage estimates were
obtained as described in Experimental Procedures for a subset of 20 areas, selected for
functional reasons.
Figure 1. Characterization of the specificity of anti-DARPP-32 monoclonal antibody C24-6a by immunoblotting. Samples ofpigeon cerebellum (left 2 lanes) and neostriatum (right 2 lanes) were dissected from the brain, homogenized in SDS, subjectedto SDS/PAGE (13% acrylamide), and electrophoretically transferred to a nitrocellulose membrane as described inExperimental Procedures. The nitrocellulose membrane was labeled with a 1:500 dilution of monoclonal antibody C24-6a,followed by 1:500 rabbit anti-mouse IgG and 125I-labeled protein A, and analyzed by autoradiography. Pigeon DARPP-32 (Mr= 32,600) is indicated by the arrow. Samples contained 100 µg of total brain protein.
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 159
Figure 2. DARPP-32 immuno-labeling in the pigeon forebrain. (A) Frontal section at level A 9.00 according to the pigeon brainatlas of Karten and Hodos (1967), illustrating the four categories of DARPP-32+ neuropil staining (see text). Paleostriatumaugmentatum (PA) and paleostriatum primitivum (PP), displaying the densest neuropil labeling, were classified as ‘4’,ectostriatum (E), showing almost no labeling, as ‘1’, neostriatum (N) as ‘3’, and hyperstriatum ventrale (HV) as ‘2’. (B, C) MostDARPP-32+ cells displayed only labeling of the soma and initial dendritic segments. (D-F) In some neurons, however, also finedendritic processes and spines were visible. Photomicrographs were taken from the n. basalis (B), neostriatum frontolaterale (C),ventromedial hippocampus (D), border of ectostriatum and ectostriatal belt (E), and medial septum (F). Scale bars represent 250µm (A), 50 µm (B, D, E), 20 µm (C), 15 µm (F).
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 160
Primary sensory forebrain areas included in our analysis were the ectostriatum of the visual
tectofugal, (Benowitz and Karten, 1976) the hyperstriatum dorsale pars lateralis of the visual
thalamofugal, (Karten et al., 1973) field L2 of the auditory, (Bonke et al., 1979) and n. basalis
of the trigeminal system (Schall et al., 1986). The intercalated nucleus of the hyperstriatum
accessorium, a further termination area of the visual thalamofugal pathway, (Güntürkün and
Remy, 1990) was not included in the analysis since percentage estimates of labeled neurons
could not be obtained reliably in this extremely thin structure. The secondary sensory forebrain
areas included in our analysis were the ectostriatal belt, which receives afferents from the
ectostriatal core, (Ritchie, 1979) the hyperstriatum accessorium as the projection zone of the
lateral hyperstriatum dorsalis, (Shimizu et al., 1995) field L1, which receives afferents from
L2, (Wild et al., 1993) and the neostriatum frontale, pars trigeminale, which is innervated by
the primary trigeminal n. basalis (Wild et al., 1985). Multimodal (tertiary) areas were the
neostriatum caudolaterale as defined by Waldmann and Güntürkün (Veenman and Reiner, 1994)
with its afferents from ectostriatal belt, (Ritchie, 1979) hyperstriatum accessorium, (Shimizu et
al., 1995) L1, (Rehkämper and Zilles, 1991) and neostriatum frontale trigeminale (Wild et al.,
1985). Three further multimodal structures, which comprise the pallium externum of Veenman
et al., (1995) were included in our analysis: the neostriatum frontolaterale as defined by
Shimizu et al (1995). with its afferents from hyperstriatum accessorium/hyperstriatum dorsale
(Shimizu et al., 1995) and ectostriatal belt; (Ritchie, 1979) the temporo-parieto-occipital area,
and the area corticoidea dorsolateralis. The latter two are known to receive visual afferents
from hyperstriatum dorsale (Shimizu et al., 1995) and ectostriatal belt, (Veenman et al., 1995)
as well as trigeminal afferents from neostriatum frontale trigeminale (Dubbeldam and Visser,
1987). The archistriatum intermedium with its descending projections to various brainstem
structures was considered as a motor output area (Zeier and Karten, 1971). Limbic structures
included in our analysis were the archistriatum ventrale and archistriatum posterior (Veenman
et al., 1995; Zeier and Karten, 1971). The lobus parolfactorius, the paleostriatum augmentatum,
and the paleostriatum primitivum were analyzed as parts of the avian basal ganglia (Karten and
Dubbeldam, 1973; Reiner et al., 1984; Veenman et al., 1995). The description of DARPP-32
distribution follows this functional classification. Figure 3 depicts the topographical
distribution of neuropil staining density and the DARPP-32+ neuron percentage classification.
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 161
Primary sensory areas. Primary sensory regions showed the lowest DARPP-32
immunoreactivity of all telencephalic areas (Figs 3, 4). The ectostriatum and field L2 appeared
almost completely blank, containing only few broadly scattered cells (Fig. 2A; Figs
4B, D). The lateral hyperstriatum dorsale had more neuropil staining and labeled neurons, but
was considerably lower in both respects compared to most secondary sensory and tertiary
areas, particularly its secondary projection zone, the hyperstriatum accessorium (Fig. 4C). The
LPOINP
PAPP
E
Ep
HVHD
HA
A 6.75
A 7.75
Hp
SLSM
HV
TPO
CDL
PA
CDL
NCNCL
AvAp
APH/Hp
A 9.25
A 10.50
A 12.50
A 5.25
Acc
PP
1
2
3
4
DARPP-32neuropil labeling
medium - high
none - very lowlow
very high
Percentage of DARPP-32+ neurons< 10 %20 - 40 %40 - 70 %> 70 %
E
PALPO
LPO
HA
NFL
BasNFT
LPO
Ep
HA
HVHD
N
Aid AivTn
PA
NCL
APH/Hp
HV
Av
CDL
L2L1
Figure 3. Distribution of DARPP-32 in pigeon telencephalon (levels according to the atlas of Karten and Hodos43). The left sideof each section shows the relative density of DARPP-32+ neuropil staining; on the right side shading codes for the percentageof DARPP-32+ neurons. Telencephalic regions, for which no percentage estimates were obtained (see text), appear blank.Frames indicate the areas from which samples for counting were actually drawn as described in Experimental Procedures.
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 162
primary sensory area of the trigeminal system, the n. basalis, was an exception. Although it
showed very light neuropil staining, it actually contained the highest percentage of DARPP-32+
neurons of all selected areas (Figs 2B, 4A).
Secondary sensory areas. The secondary sensory areas of the visual-tectofugal (ectostriatal
belt), visual-thalamofugal (hyperstriatum accessorium) and auditory (L1) system ranked clearly
higher in both neuropil labeling and the relative number of DARPP-32+ neurons when
compared to the respective primary sensory areas (Fig. 3). As mentioned above, the trigeminal
system was an exception with the neostriatum frontale trigeminale having a much denser
neuropil staining than the n. basalis, but containing a lower percentage of labeled neurons than
the latter one (Fig. 4A). In general, secondary structures displayed approximately equal relative
numbers of DARPP-32+ neurons. Only the caudally located field L1 scored lower than the
more rostral structures.
Figure 4. DARPP-32 immunoreactivity in primary and adjacent secondary sensory areas of the pigeon telencephalon. (A) Thetrigeminal n. basalis (Bas) displays a high number of DARPP-32+ neurons, although neuropil staining is very low in this area.(B) Primary auditory Field L2 shows almost no labeling in contrast to the adjacent secondary areas. (C) Hyperstriatum dorsale(HD), from which the lateral portion receives thalamic input and is part of the primary thalamofugal visual system, shows clearlyless soma and neuropil labeling than its secondary projection zone, the hyperstriatum accessorium (HA). (D) Single DARPP-32-positive cell in the otherwise unstained ectostriatum (E), surrounded by the ectostriatal belt and the neostriatum. Scale barsrepresent 500 µm (A-C), 200 µm (D).
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 163
Tertiary areas. The tertiary structures neostriatum frontolaterale, area temporo-parieto-
occipitalis, corticoidea dorsolateralis, neostriatum caudolaterale, and the caudal neostriatum
were stained homogeneously at medium to high densities. Based on the intensity of neuropil
labeling as well as on the percentage of DARPP-32+ neurons, these areas could not be
differentiated from the secondary sensory structures (Fig. 3). Among the tertiary regions, the
more rostral areas of the neostriatum (neostriatum frontolaterale, area temporo-parieto-
occipitalis, corticoidea dorsolateralis) showed more DARPP-32+ neurons than the caudal
components (neostriatum caudale, neostriatum caudolaterale).
Motor and limbic structures. According to neuropil staining intensity, structures of the avian
basal ganglia (paleostriatum augmentatum, paleostriatum primitivum, lobus parolfactorius)
were most heavily labeled and could not be distinguished from each other (Figs 2A, 3, 5A).
Lobus parolfactorius and paleostriatum augmentatum also contained a high percentage of
DARPP-32+ neurons, whereas in the paleostriatum primitivum only a very small number of
cells was labeled (Fig. 5B). On the basis of cell counting alone, no difference between lobus
Figure 5. DARPP-32 immunoreactivity of pigeon motor and limbic structures. (A) Avian basal ganglia at level A 8.50. (B)Border between the paleostriatum augmentatum (PA), in which both neuropil and cell bodies were extensively labeled, and thepaleostriatum primitivum (PP), which showed only neuropil labeling. (C) Archistriatum at level A 7.50. While the ventralarchistriatum (Av) contains many DARPP-32+ neurons, the dorsal archistriatum (Ad) displays only neuropil labeling, with theexception of a dorsomedial rim where also high perikaryal labeling could be observed (arrowhead). Unstained fibers of thetractus occipito-mesencephalicus travel through the archistriatum ventrale. (D) Septum, showing two DARPP-32+ cellpopulations in the lateral (SL) and ventromedial (SM) part. Scale bars represent 1000 µm (A), 250 µm (B), 500 µm (C, D).
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 164
parolfactorius/paleostriatum augmentatum and secondary or tertiary areas could be observed
(Fig. 3). The n. accumbens and bed nucleus of the stria terminalis, according to the definition of
Veenman et al., (1995) also showed a considerable number of labeled cells.
Neuropil labeling in the archistriatum was homogeneous, comparable to the neostriatal regions
and considerably lower than in the basal ganglia (Figs 3, 5C). The ventral portions of the
‘motor’ as well as of the ‘limbic-visceral’ archistriatum demonstrated a high percentage of
DARPP-32+ neurons (Fig. 3). In contrast, in the dorsal parts of the archistriatum mainly
neuropil was labeled, with exception of a small dorsomedial rim of labeled perikarya (Fig.
5C).
Other limbic structures in the telencephalon of the pigeon include the hippocampal system and
the septum. Neuropil labeling in most regions of the hippocampus was comparable to that of the
archistriatum and higher sensory and multimodal areas of the neostriatum. Also, a high number
of DARPP-32+ neurons was observed in the ventromedial area and at the lateral border, which
probably correspond to the V-shaped region and area 7 of Krebs et al. (1991) and Erichsen et
al. (1991) (Fig. 2D). The medial border, however, which is part of the ventromedial and
inferior dorsomedial region as defined by these authors, was found to be completely blank. In
the septum, DARPP-32 immunoreactivity strongly paralleled the distribution of TH+ fibers.
Only two restricted portions of the lateral and ventromedial septum, the latter probably
corresponding to the n. commissuralis of Baylé et al., (1974) contained numbers of DARPP32+
cells, while the whole septum demonstrated very low levels of DARPP-32+ neuropil staining
(Fig. 5D).
Co-occurrence of DARPP-32 and tyrosine hydroxylase baskets
‘Baskets’, which consist of fibers densely coiling around single perikarya and initial dendritic
segments, represent a striking feature of catecholaminergic innervation in avian telencephalon
(Fig. 6A, B). As illustrated in Fig. 6C-E and Fig. 7A, a number of DARPP-32+ neurons receive
input from TH+ baskets. To obtain estimates of the percentage of DARPP-32+ neurons located
in TH-baskets, a total of 3692 neurons were examined. Analysis of several regions were
pooled and classified into one of three categories. Because of the extremely dense meshwork of
TH+ fibers and the very high DARPP-32 abundance in regions of the avian basal ganglia
(paleostriatum augmentatum, paleostriatum primitivum, lobus parolfactorius), reliable countings
were impossible here and these areas were not included in the analysis.
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 165
The highest percentage of co-occurrence was seen in the multimodal lateral and caudal
neostriatum (area temporo-parieto-occipitalis / corticoidea dorsolateralis / neostriatum caudale
/ neostriatum caudolaterale: 15 - 30 %). This value was lower in the intermediate medial and
frontal neostriatum (including ectostriatal belt / neostriatum frontolaterale / neostriatum frontale
trigeminale: 5 - 15 %). The lowest percentage of co-occurrences was observed in the
hyperstriatal and hippocampal regions (hyperstriatum accessorium / area parahippocampalis /
hippocampus: < 5 %), although these areas were rich in DARPP-32+ neurons. Also, many TH-
baskets containing no DARPP-32-labeled neurons were observed throughout the telencephalon.
Forebrain regions differed with respect to the number of fibers constituting single baskets.
Baskets in the caudal neostriatum consisted of a large number of axonal loops surrounding
Figure 6. TH+ fibers and DARPP-32+ neurons in the pigeon caudal neostriatum. (A, B) TH+ baskets consist of axons denselycoiling around single perikarya and initial dendritic segments, so that neurons virtually become visible by the TH+ signal alone. (C-E) VIP-stained DARPP-32+ neurons enwrapped by DAB-stained TH+ fibers. Scale bars represent 30 µm (A), 20 µm (B, D, E),50 µm (C).
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 166
perikarya and initial dendrites, while TH+ fibers in the frontal neostriatum coiled only a few
times around single somata (Waldmann and Güntürkün, 1993).
Co-occurrence of glutamate decarboxylase and tyrosine hydroxylase baskets
To examine whether GAD+ neurons receive TH-basket-type input, 861 GAD+ neurons and 636
TH-baskets throughout the telencephalon were inspected. As demonstrated in Fig. 7B, no co-
occurrence was found. However, in the lobus parolfactorius, paleostriatum augmentatum and
paleostriatum primitivum reliable counting could not be obtained due to the dense net of
catecholaminergic fibers in these regions.
Co-localization of glutamate decarboxylase and DARPP-32
To investigate the possible colocalization of DARPP-32 and GAD antigen, a total of 939
DARPP-32+ and 1149 GAD+ neurons throughout the telencephalon were examined. No
colocalization was observed. Fig. 7C shows examples of neurons in the neostriatum, labeled
for GAD and DARPP-32, respectively. However, in the basal ganglia the possibility of
colocalization cannot be excluded with certainty, since the high DARPP-32 staining density in
these regions hampered the analysis.
DISCUSSION
The present study describes the distribution of DARPP-32 in the avian telencephalon and
provides a classification of telencephalic regions in terms of their neuropil staining intensity
and DARPP-32+ neuron numbers. Additionally, colocalization of GAD-and DARPP-32-
labeled cells and their co-occurrence with TH-labeled axonal baskets were investigated.
Distribution of DARPP-32
The strongest DARPP-32 immunoreactivity was observed in parts of the avian basal ganglia,
namely the paleostriatum augmentatum, lobus parolfactorius and paleostriatum primitivum,
which are regarded as equivalent to the mammalian caudate-putamen and globus pallidus,
respectively (Veenman et al., 1995). In the paleostriatum augmentatum/lobus parolfactorius
both fibers and somata were densely labeled. In these areas, TH+ and DA+ fibers have been
shown to contact predominantely shafts and necks of dendritic spines, (Karle et al., 1996)
suggesting possible sites for dopaminergic receptors. The strong staining in the paleostriatum
primitivum on the other hand resulted almost exclusively from neuropil labeling (see Fig. 5B).
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 167
This clear difference in somatic labeling between the paleostriatum augmentatum/lobus
parolfactorius and paleostriatum primitivum corresponds to results obtained with other markers
of the dopaminergic system in mammals and birds: the abundance of dopaminergic
Figure 7. Fluorescence double-labeling against (A) DARPP-32 and TH, (B) GAD and TH, and (C) DARPP-32 and GAD. Thephotomicrographs of each pair were taken from the same site in caudal neostriatum of the pigeon telencephalon. Two of threeDARPP-32+ neurons (A1) are surrounded by TH+ baskets (A2). GAD+ neurons (B1) do not receive input from TH+ baskets(B2). GAD+ neurons (C2) do not show DARPP-32 immunoreactivity (C1), although GAD+ and DARPP-32+ neurons mayoccur in close vicinity to each other. Arrows mark corresponding positions in each pair. In (C), open and filled arrows indicatepositions of DARPP-32+ and some GAD+ cells, respectively. Scale bars represent 30 µm (A, B), 40 µm (C).
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 168
fibers as well as of D1 and D2 receptors is highest in the caudate-putamen and the
paleostriatum augmentatum/lobus parolfactorius of the mammalian and avian basal ganglia, but
is relatively low in the globus pallidus and the paleostriatum primitivum, respectively (Ball et
al., 1995; Berger et al., 1991; Brock et al., 1992; Joyce et al., 1993; Moons et al., 1994;
Richfield et al., 1987; Schnabel and Braun, 1996; Stewart et al., 1996; Wynne and Güntürkün,
1995; Yung et al., 1995). The dense neuropil labeling in the paleostriatum primitivum may stem
from descending D1-positive axons, as in the globus pallidus of rats where DARPP-32- and
D1- but not D2 receptor-positive axons have been described surrounding unlabeled perikarya
and dendrites (Quimet and Greengard, 1990; Yung et al., 1995). These axons probably belong
to the descending striatonigral and striatoentopeduncular tract (Yung et al., 1995).
Besides the basal ganglia, several other structures of the avian motor and limbic system showed
medium-to-high DARPP-32 immunoreactivity, namely the archistriatum and the area
parahippocampalis/hippocampus. According to Zeier and Karten, (1971; 1973) the anterior two
thirds of the archistriatum (archistriatum pars dorsalis and archistriatum intermedium)
correspond to the mammalian motor cortex, whereas the archistriatum posterior and pars
ventralis have been compared to the mammalian amygdala. In the present study, neurons in the
ventral parts of the motor as well as of the limbic archistriatum were strongly DARPP-32
labeled while in the dorsal parts only neuropil was labeled. This finding stands in contrast to
the pattern described for dopaminergic fibers in the archistriatum by Wynne and Güntürkün,
(1995) who found DA baskets, presumably innervating somata, especially in those archistriatal
portions where no or few DARPP-32 cells were labeled. D2 receptors cannot account for the
observed mismatch, since these are known to have extremely low densities outside the basal
ganglia (Dietl and Palacios, 1988; Richfield et al., 1987; Stewart et al., 1996). However,
differences regarding the dopaminergic innervation and the distribution of dopamine receptors
also exist in motor output areas of various mammalian species. In primates, the motor cortex
receives an exceptionally strong dopaminergic innervation (Berger et al., 1991; Björklund and
Lindvall, 1984; Wiiliams and Glodman-Rakic, 1993) but displays only a sparse distribution of
DARPP-32 labeled cells and contains only medium densities of D1 receptors (Berger et al.,
1990; Cortés et al., 1989; Lidow et al., 1989; Lidow et al., 1991; Quimet et al., 1992; Richfield
et al., 1989).
DARPP-32 soma and neuropil labeling revealed a general pattern of low or no staining in
primary and much stronger labeling in secondary and multimodal association areas (see Fig. 3).
This is in good agreement with studies on the distribution of dopaminergic fibers and D1
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 169
receptors in birds (Dietl and Palacios, 1988; Moons et al., 1994; Wynne and Güntürkün, 1995).
It is also in accordance with data from rodents and primates, where the density of D1-receptors
and dopaminergic fibers is lowest in primary sensory but higher in secondary and association
cortices (Berger et al., 1991; Berger et al., 1988; Joyce et al., 1993; Lewis et al., 1986).
The n. basalis is a notable exception from the rule outlined above. Although showing weak
neuropil labeling, it contains a high percentage of DARPP-32+ neurons (see Fig. 4A). This
observation contrasts with results for other primary sensory regions and could be linked to
other peculiarities of the pigeon's trigeminal system. The n. basalis receives, among other
afferents, direct input from the n. sensorius principalis nervi trigemini and projects through
other forebrain areas to rhombencephalic structures related to pecking (Schall, 1987; Schall et
al., 1986; Wallenberg, 1903; Wild et al., 1985). It is thus the only avian forebrain structure with
sensory afferents bypassing the thalamus. The projection via the n. basalis, neostriatum frontale
trigeminale and archistriatum constitutes a trigeminal circuit for the control of pecking(Schall,
1987; Schall, 1989; Zeigler, 1976). Within this circuit, sensory signals and motor outputs
probably have to be integrated within extremely short time spans. Therefore, dopaminergic
modulation (see general discussion) might start earlier in this system than in other sensorimotor
circuits.
The presence of DARPP-32+ cells within the n. basalis contrasts with a number of previous
anatomical studies. According to Dietl and Palacios (1988) the levels of D1 receptors in the n.
basalis are negligibly low. Kusunoki (1969) could also not reveal monoaminoxidase activity in
the n. basalis, and Wynne and Güntürkün (1995) were unable to detect dopaminergic fibers in
this structure. In contrast to these anatomical results, behavioral data make it likely that
dopaminergic modulation exists within this nucleus. Apomorphine-induced pecking fits are
accompanied by an increase of glucose uptake in the n. basalis (Delius, 1985) and intracranial
apomorphine injections into this structure induce stereotypical pecking bouts (Lindenblatt and
Delius, 1988). Therefore, n. basalis was thought to be a central element of apomorphine
triggered pecking stereotypies and to possess dopamine receptors (Delius, 1985). This last
assumption was strengthened by the finding of Wynne and Delius (1996) that n. basalis-lesions
significantly reduce pecking fits after apomorphine injection. The present finding of a prominent
group of DARPP-32+ cells in the n. basalis does not fit easily with the chemoanatomic results
gathered up to now but may provide an anatomical clue to behavioral data.
According to biochemical, hodological and behavioral results, the neostriatum caudolaterale
has been suggested as an avian equivalent of the prefrontal cortex (Divac and Mogensen, 1985;
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 170
Divac et al., 1985; Divac et al., 1994; Güntürkün, 1997; Mogensen and Divac, 1982; Mogensen
and Divac, 1993; Reiner, 1986; Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995).
The dopaminergic innervation of the neostriatum caudolaterale is among the strongest in the
pigeon telencephalon, (Divac et al., 1994; Waldmann and Güntürkün, 1993; Wynne and
Güntürkün, 1995) just as it is in the mammalian prefrontal cortex (Berger et al., 1988; Joyce et
al., 1993; Lewis et al., 1986; Williams and Goldman-Rakic, 1993). The percentage of DARPP-
32+ neurons in the neostriatum caudolaterale, as revealed in the present study, is lower than in
the secondary sensory areas ectostriatal belt or neostriatum frontale trigeminale, while this
pattern is reversed for the innervation by dopaminergic fibers in these regions (Wynne and
Güntürkün, 1995). Again, this finding parallels data from mammals: The dorsolateral prefrontal
cortex of adult monkeys also displays a low number of DARPP-32+ neurons despite its dense
dopaminergic innervation (Berger et al., 1990).
DARPP-32 as a marker for D1-receptors
In rodents and primates the distribution of DARPP-32 has been found to parallel the
distribution of cells possessing D1 receptors (Berger et al., 1990; Hemmings and Greengard,
1986; Quimet et al., 1992; Quimet et al., 1984; Walaas and Greengard, 1984). Receptors of the
D1-family are positively coupled to adenylyl cyclase activity and can thus stimulate cAMP
synthesis (Kebabian and Calne, 1979). This in turn leads to phosphorylation of DARPP-32 via
cAMP-dependent protein kinase, resulting in inhibition of phosphatase-1 (Hemmings et al.,
1984; Hemmings et al., 1987). DARPP-32 acting as an intracellular “third messenger” is thus
involved in mediating the D1 receptor dependent effects of dopamine.
Key features of the distribution of dopaminergic fibers and D1-receptors in avian, such as
sparing of primary sensory areas or dense innervation of structures of the basal ganglia, (Ball et
al., 1995; Dietl and Palacios, 1988; Moons et al., 1994; Richfield et al., 1987; Stewart et al.,
1996; Waldmann and Güntürkün, 1993; Wynne and Güntürkün, 1995) could also be observed in
the distribution of DARPP-32. There are some exceptions, however Richfield et al. (1987) and
Stewart et al. (1996) demonstrated higher D1-specific binding in hyperstriatum ventrale than in
neostriatum, while this pattern is reversed with DARPP-32. Mismatches in the distributions of
DARPP-32 and D1 receptors have also been noted in mammals, e.g. in primate motor cortex
(Berger et al., 1990). In general, however, these distributions overlap to a high degree, and
DARPP-32 thus seems to represent a useful marker for dopaminoceptive cells expressing D1
receptors within the mammalian as well as within the avian telencephalon.
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 171
It has been noted that glia may also contain DARPP-32 (Berger et al., 1990; Quimet et al.,
1984). Thus, the cell counting analysis presented here may contain overestimations. However,
most DARPP-32+ cells observed were larger than glia cells (Ø > 6 µm) or expressed clear
neuronal properties like dendritic elements (see Fig. 2B-F). Also, in Nissl-counterstained
preparations, very few DARPP-32+ cells were observed resembling glia cells. The estimation
error due to glia staining therefore was probably very low in our preparations.
Co-localization of glutamate decarboxylase with DARPP-32 and co-occurrence with
tyrosine hydroxylase
GAD is relatively homogeneously distributed throughout the pigeon telencephalon (Domenici et
al., 1988; Veenman and Reiner, 1994). Veenman and Reiner (1994) report that the distributions
of GAD+ and GABA+ cells overlap almost completely, and they estimate the fraction of
GABAergic neurons to be about 10-12 % in the neo- and hyperstriatum, showing only slight
regional differences. According to the present results, GAD+ neurons do not express DARPP-
32 and do not receive input from TH-baskets. Therefore, GABAergic cells, at least in the avian
‘neocortex’, do not seem to be the target of the dense type of dopaminergic innervation and
probably do not express D1 receptors. Yet the possibility remains that these cells express
receptors of the D2 receptor family. In mammalian neocortex, there is indeed
electrophysiological evidence that GABAergic neurons may be modulated by dopamine via D2
receptors (Pirot et al., 1992; Rétaux et al., 1991). However, receptor autoradiography studies
in birds have shown that, with exception of the basal ganglia, the abundance of D2 receptors in
the telencephalon is very low (Dietl and Palacios, 1988; Richfield et al., 1987; Schnabel and
Braun, 1996; Stewart et al., 1996). Thus, GABAergic cells in the pigeon telencephalon might
not, or at least not to an important extent, be innervated by dopamine. Therefore presumably
excitatory neurons are the main target of dopaminergic afferents. This interpretation would also
be consistent with data from mammalian frontal cortex, where dopaminergic axons mainly, but
not exclusively, (Smiley and Goldman-Rakic, 1993) synapse on presumably glutamatergic
pyramidal cells, (Berger et al., 1991; Goldman-Rakic et al., 1989; Smiley and Goldman-Rakic,
1993) which also constitute the major cell population expressing DARPP-32 (Berger et al.,
1990).
In the pigeon basal ganglia also no colocalization of GAD and DARPP-32 was detected. In
contrast, GABAergic neurons in mammalian caudate-putamen express D1-receptors and very
likely also DARPP-32 (Joyce et al., 1993; Quimet et al., 1992;Yung et al., 1995). This
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 172
discrepancy may be explained in terms of another difference between avian and mammalian
basal ganglia. The avian basal ganglia contain only 5-10 % GAD+ cells (Veenman and Reiner,
1994) as opposed to the vast majority of medium spiny neurons in mammalian basal ganglia,
which are supposed to utilitize GABA (Groves et al., 1995). Yet, for several reasons we
cannot exclude the possibility that some GAD/DARPP-32 colocalizations went unnoticed in the
present study. First, as noted in the results section, colocalizations were difficult to analyze in
the avian basal ganglia due to the high DARPP-32 neuropil staining. Second, it is conceivable
that some GABAergic cells were indeed colocalized with DARPP-32 but were not labeled by
GAD immunohistochemistry, since Veenman and Reiner (1994) report a subpopulation of small
cells which show GABA but not GAD immunoreactivity. Third, the methods used in the present
study may have been not sensitive enough to detect all GAD+ cells. However, although
Veenman and Reiner (1994) note that pre-treatment of pigeons with colchicine enhances GAD
perikaryal labeling, the percentage of GAD+ cells in the paleostriatum as estimated by these
authors (5-10 %) seems to be in the same range as in the present study.
In conclusion, the present results make it likely that dopamine in the pigeon's forebrain exert its
effects primarily by changing physiological parameters of non-GABAergic and thus presumably
excitatory units, although this conclusion has to be treated with caution with respect to the avian
basal ganglia.
Co-occurrence of DARPP-32 and tyrosine hydoxylase
In the present study, a large number of DARPP-32+ neurons localized in TH-baskets have been
observed, although the majority of labeled perikarya did not receive this type of dense
catecholaminergic innervation. The highest percentage of DARPP-32/TH co-occurences was
found in the lateral and caudal parts of the neostriatum and thus in parts of the neostriatal
association areas. In contrast to primary and secondary sensory areas, these association areas
are also known to have a direct link to the avian basal ganglia (Veenman et al., 1995). The
finding that there are many ‘empty’ TH-baskets could be due to noradrenergic fibers, and TH-
baskets of non-dopaminergic origin have indeed been demonstrated (Wynne and Güntürkün,
1995). Alternatively, neurons expressing other than D1 dopamine receptor subtypes could also
be the target of the basket-type input.
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 173
General discussion
In birds, dopaminergic innervation of the telencephalon with clear regional differences arises
from the midbrain nuclei tegmenti pedunculopontinus pars lateralis and area ventralis
tegmentalis, comparable to the projections from the mammalian substantia nigra pars compacta
and area ventralis tegmentalis, respectively (Kitt and Brauth, 1986; Waldmann and Güntürkün,
1993). Our study shows that dopaminergic innervation of the pigeon telencephalon is
comparable to that of mammals in three important respects. First, DARPP-32, and therefore
presumably the D1 receptor, is most abundant in parts of the avian basal ganglia and is present
in medium to high concentrations in most other telencephalic secondary sensory, associative,
limbic and motor regions. Second, the primary auditory and visual areas show no or only weak
DARPP-32 labeling. Third, GAD+ neurons are not colocalized with DARPP-32 and do not
receive TH-basket input. Thus, dopamine presumably mainly innervates excitatory forebrain
units in pigeons, a situation at least partly comparable to mammalian neocortex (Berger et al.,
1991; Gaspar et al., 1995; Goldman-Rakic et al., 1989; Smiley and Goldman-Rakic, 1993).
What are the possible functional implications of the present findings and the fact that a similar
organization of the dopaminergic innervation exists in mammals? The sparing of most primary
sensory regions by markers of dopaminergic innervation and the medium to high levels in most
other telencephalic structures imply that dopamine has its most prominent role in higher level
sensory and motor processes. Based on the findings by Schultz and coworkers, (Apicella et al.,
1992; Ljungberg et al., 1992; Schultz and Romo, 1990; Schultz et al., 1993; Schultz et al., 1995)
who showed that dopaminergic midbrain neurons are activated in new and unexpected
situations, and computational studies, (Durstewitz and Güntürkün, 1996) one may infer that
dopamine allows for rapid strengthening of couplings between higher sensory representations,
motor outputs, and the reward value of the behavior. By stabilizing such new configurations, the
dopaminergic system may enable the organism to develop predictions of behavioral
consequences (Montague et al., 1996; Schultz et al., 1995). This type of fast and highly flexible
learning may not be that important in primary sensory regions for two reasons. First, most
structural modifications in these areas occur during pre- and early postnatal development. In
contrast, in adult primary sensory structures there may be need for only slight incremental
modifications of, e.g., receptive field properties. In line with this assumption, considerable
amounts of DARPP-32 are present in the visual cortex of infant monkeys but not in that of adults
(Berger et al., 1990). Second, only higher elaborated sensory representations may participate in
those associations reinforced by the dopaminergic system because only these representations
Chapter 5: Distribution of DARPP-32 in pigeon telencephalon 174
may stand in a unique relationship to relevant aspects of the situation. In contrast, there may be
too many interpretational possibilities for activity patterns in primary sensory areas. Therefore,
these patterns possibly cannot become directly coupled to certain motor functions but must first
be resolved by higher processes.
In cortex, dopamine has been shown to inhibit spontaneous and evoked activity in vivo,
(Bernardi et al., 1982; Ferron et al., 1984; Godbout et al., 1991; Sesack and Bunney, 1989;
Verma and Moghaddam, 1996) to reduce excitatory synaptic transmission, (Law-Tho et al.,
1994; Pralong and Jones, 1993) and to shift the probability of long term modifications in favor
of long term depression as opposed to long term potentiation (Chen et al., 1995; Law-Tho et al.,
1995). Computational modeling suggests that dopamine, by these physiological mechanisms,
suppresses the recall of previously learned associations during learning and thereby enables the
fast formation of new couplings (Durstewitz and Güntürkün, 1996). This hypothesis implies that
dopaminergic fibers mainly innervate excitatory neurons and that these cells are the biggest
subpopulation expressing dopaminergic receptors. This prediction is consistent with the present
findings that GABAergic units do not express DARPP-32 and are not targets of a strong
dopaminergic input.
A remaining question concerns why only a certain fraction of DARPP-32+ neurons receives the
dense basket-type catecholaminergic input. It is conceivable that these neurons serve special
functions in the innervated neural networks. One hypothesis derives from the proposed role of
dopamine in fast associative learning (Durstewitz and Güntürkün, 1996). Fast learning
presumably requires the respective neurons or cell populations to be synchronously active for
some time to allow for long term synaptic modifications (Singer, 1993). The dopaminergic
system may be involved in keeping new patterns active by inducing oscillatory processes
(Montaron et al., 1982). In the avian telencephalon, the strong basket-type dopaminergic input
may terminate on those excitatory cells which can acquire pacemaker-like characteristics in
learning situations. Thereby, dopamine in the avian brain may excert some control over the long
term formation of associations between certain situations and profitable behaviors. Of course,
these notions are highly speculative and clearly more evidence is needed. However, our
assumptions may guide further research on the possible functions of dopamine in learning in
different species.
Chapter 6: Actions of dopamine on neurons in the chick NCL 175
Effects of dopamine on the excitability of neurons in the chick NCL
INTRODUCTION
In the previous chapters the importance of the NCL for sensorimotor integration and „frontal“
excecutive functions has been outlined repeatedly. Most of the evidence that supports the
involvement of the NCL in working memory (Mogensen and Divac, 1982, 1993, Gagliardo et
al., 1996, 1997, Güntürkün, 1997; Kalt et al., 1999), reversal learning (Hartmann and
Güntürkün, 1998), response inhibition (Güntürkün, 1997, Aldavert-Vera et al., 1999), and
spatial orientation (Gagliardo and Divac, 1993) stems from lesion studies. More recent studies
have begun to unravel the cellular mechanisms that underlie these functions of the NCL.
Electrophysiological single-unit recordings in awake pigeons which performed a delayed
Go/NoGo task, have elucidated the contribution of neurons in the NCL to response inhibition
and working memory (Kalt et al., 1999). In this study single neurons showed an increase in their
firing frequency relative to baseline conditions during the delay period of the task. Neuronal
activity changes also signalled the onset of the stimulus or the delivery of the reward. Some
neurons furthermore seemed to code the behavioral significance of the stimulus, such that the
neuronal activity, either an increase or a decrease in spike frequency, was statistically different
between Go and NoGo trials (Kalt et al., 1999).
In the prefrontal cortex (PFC) of mammals, task-related electrical activity is modulated by
dopamine (DA), mainly via the D1 receptor (Sawaguchi et al., 1988, 1990a,b; Williams and
Goldman-Rakic, 1995). Dopaminergic midbrain neurons are activated at the onset of working
memory tasks (Schultz et al., 1993) and DA levels in the PFC increase during delay-task
performance (Watanabe et al., 1997). Blockade of the dopaminergic input to the PFC or of
dopaminergic D1 receptors in the PFC disrupt delay-task performance (Brozoski et al., 1979;
Simon et al., 1980; Sawaguchi and Goldman-Rakic, 1994; Seamans et al., 1998). Similarly, in a
behavioural study in the pigeon, blockade of D1 receptors in the NCL by a competitive
antagonist (SCH-23390), strongly impaired performance in a working memory task (Güntürkün
and Durstewitz, in press).
One concept regarding the functions DA might play during working memory, is to increase the
signal-to-noise ratio and to stabilize neural representations (Sawaguchi et al., 1990a,b;
Durstewitz et al., 1999, 2000). This stabilizing effect on the network could enable sustained
Chapter 6: Actions of dopamine on neurons in the chick NCL 176
task-related activity, even in the presence of interfering input (Durstewitz et al., 1999), which in
mammals appears to be a unique feature of the PFC (Miller et al., 1996).
In this chapter I will present preliminary data on the effects of DA on the firing behaviour of
chick NCL neurons recorded in-vitro, using both whole-cell and perforated patch-clamp
techniques. The electrophysiological findings are complemented with morphological data from
double-labeling experiments, in which neurons that were intracellularly filled during whole-
cell recordings were immunohistochemically stained and colocalized with labeling of tyrosine-
hydroxylase, as a marker of the dopaminergic innervation. It can be assumed that the basket-like
innervation provides a means by which the dopaminergic system could selectively modulate a
specific class of neurons, and/or that the strong somatic innervation renders these neurons
particularly susceptible for the effects of DA. The results show that DA can increase the
excitability of NCL neurons and profoundly alter their firing pattern, which in turn might sustain
neuronal delay activity during delay-task performance. However, the responses of neurons,
which were anatomically shown to receive basket-like dopaminergic innervation, to DA
application did not differ from those that received only „en-passant“ innervation, thus
suggesting that the different modes of dopaminergic innervation exert no qualitatively different
influence over their target neurons.
MATERIALS AND METHODS
Preparation of slices and electrophysiological recording: For preparation of brain slices a total of 32 chicks
(5 - 12 days post-hatch, median 7 days) were decapitated and the brains were rapidly transferred to iced
extracellular solution. Coronal slices (350 µm thick) of the caudal telencephalon were cut on a vibratome.
Slices were incubated in extracellular solution consisting of (in mM): 119 NaCl, 2.5 KCl, 1 NaH2PO4, 26.2
NaHCO3 ,10 D-glucose, 4.5 CaCl2, and 1.3 MgCl2, saturated with 95% O2 /5% CO2, pH 7.3. Slices were
allowed to recover for at least 1 hour, before being transferred to the recording chamber. All recordings were
made at room temperature (21 - 23°C) in a submerged slice chamber perfused with extracellular solution.
Whole-cell and perforated-patch recordings were made from neurons in the caudolateral subventricular region
of the chick forebrain, using the blind-patch technique (Blanton et al., 1989). Electrodes (6 – 8 MΩ resistance)
were filled with an intracellular solution consisting of (in mM): 135 K-gluconate, 20 KCl, 2 MgCl2 , 10 N-(2-
hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 0.1 ethylene glycolbis (β-aminoethyl ether)-
N,N,N´,N´,-tertraacetic acid (EGTA), 4 Na2-ATP, and 0.5 Na2 -GTP, and adjusted to pH 7.3 with KOH. In some
recordings 3 - 3.5 mg/ml of the dipotassium salt Lucifer Yellow (LY, Sigma, Deisenhofen, Germany) were
added to the intracellular solution and neurons were filled by diffusion. Perforated-patch recordings were made
by adding nystatin to the pipette solution (final concentration was 500 µg/ml, 0.3 % DMSO). The membrane of
a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity
Chapter 6: Actions of dopamine on neurons in the chick NCL 177
between the pipette and the cytoplasm of the cell without the loss or alteration of cytoplasmic compounds
necessary for the maintenance of the effects under study.
Recordings were made using a HEKA (Lambrecht, Pfalz, Germany) EPC-7 patch-clamp amplifier, filtered at 3
kHz, and sampled at 10 kHz. The voltage drop across the pipette could not be bridge-balanced. Sampling was
done with a Digidata 1200 interface using pClamp 6.0 software (Axon Instruments, Foster City, CA), and data
were stored on computer for off-line analysis with the CLAMPFIT module of pClamp.
Experimentally produced changes in membrane potential (Vm) during current-clamp recordings were
compensated with a bias current to maintain Vm at baseline levels.
The apparent input resistance (Ri) was monitored continuously by applying brief (50 ms) hyperpolarizing
current pulses before each depolarizing current step. The amplitude of these hyperpolarizing current pulses (10
- 50 pA) was adjusted for each cell to be in the linear range of the current–voltage relationship, as estimated
from the cells response to a series of depolarizing and hyperpolarizing current injections.
Experimental paradigm: To study cell excitability, action potentials were evoked by 1.1 sec-long depolarizing
current injections with various amplitudes (100 - 800 pA). The amplitude of the current used for statistical
comparisons of drug effects was adjusted to be in the mid-range between the current that depolarized the
neuron just above spike-threshold and the close-to-maximal stimulation. In the majority of cells the time
course of drug action and wash-out of the effect was measured continuously by applying 5 current steps (at 0.2
Hz) every 30 or 45 seconds. After baseline measurements of action-potential number and Ri, dopamine (1 - 50
µM) was bath applied for approximately 1 minute. Two gravity-fed perfusion lines were used to change
solutions. Both control and drug ACSF were continuously oxygenated throughout experiments.
The dopamine D1-receptor agonist SKF-38393, a D1 antagonist (SCH-23390), and the D2-receptor agonist
(quinpirole) were prepared as stock solutions and stored frozen until diluted to the final concentration. In a
number of experiments DA stock solutions were prepared in a similar way and ascorbate (3 mM) was added to
the solution to reduce oxidation. In all other experiments DA solutions were prepared at the desired final
concentration immediately before application. Dopaminergic agonists and antagonists were obtained from
Research Biochemicals (Natick, USA). Only one neuron experiencing drug treatment was used from each brain
slice. In several instances different agonists were applied successively to one cell to test its differential
response to various pharmaka. Usually, either a D1 or D2 receptor agonist (SKF-38393, or quinpirol) were
followed by a single application of DA. In these cases only the response to the agonist that was first applied
was used for statistical analysis. In all cases, only cells with a resting potential of at least -60 mV and stable
baseline responses were given drug treatments.
Analysis and statistics. For each cell, the number of action potentials evoked by depolarizing current steps was
quantified by averaging the number of spikes produced in 5 consecutive traces before, at the end of, and
approximately 5 minutes after drug application. The input resistance (Ri) served as a measure for the stability
of a given recording. Thus, cells that showed a non-reversible change of input resistance larger than 15 % were
excluded from analysis to control for possible unspecific changes in recording conditions. Furthermore, for
cells that showed a change in the number of evoked spikes during a given drug treatment, only those that
exhibited a reversible effect (a washout of at least 50% of the effect) were used for analysis. The effects of DA
Chapter 6: Actions of dopamine on neurons in the chick NCL 178
or its agonists on the number of evoked action potentials were expressed as the percentage change relative to
baseline conditions to account for the variation in the absolute number of spikes which could be evoked in
neurons of different cell types (c.f. chapter 3).
Comparisons of drug effects across concentrations relative to control recordings were made using univariate
analysises of variance (ANOVA). Subsequent tests for statistical significance of effects were done using
Student´s t test for unpaired samples. For multiple comparisons the alpha-niveau was Bonferroni-adjusted.
„Within-group“ comparisons used a Student’s t test for paired samples where noted.
Histological procedures
Following recording, slices were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in 0.12 M phosphate
buffer (4°C, pH 7.4) for about 15 hours. They were then transferred to a solution of 30% sucrose in phosphate
buffer containing 0.9% NaCl (PBS; pH 7.4) and 0.01% NaN3 as a preservative. Slices were resectioned at 70
µm on a freezing microtome and collected in PBS. For double-labeling immunohistochemistry of LY and
tyrosine-hydroxylase (TH), endogeneous peroxidases were first blocked by preincubating slices in 0.5% H2O2
for 30 minutes. Slices were then washed in PBS and floating sections were incubated overnight at 4°C in a
monoclonar anti-TH antibody from mouse (Boehringer; working dilution 1: 200) in PBS containing 0.3 %
Triton-X (Sigma, Deisenhofen). The following steps were carried out at room temperature, separated by three
washes in PBS of 10 min each. After washing, slices were incubated for 2 hours in the secondary antibody
directed against mouse from horse (Vector Labs, Burlingame, CA), diluted at 1:200 in PBS containing 0.3 %
Triton-X. After washing, slices were incubated for 2 hours in the avidin-biotin complex (ABC Elite, Vector
Labs; 1:100 in PBS with 0.3% Triton X-100). Washes in PBS were followed by additional washes in 0.12 M
acetate buffer (pH 6). Staining was achieved by the 3,3'-diaminobenzidine (DAB) technique with heavy-metal
amplification (modified from Adams, 1981) by adding H8N2NiO8S2 (2.5 g/ 100 ml), NH4Cl and CoCl2 (both
40 mg/ 100 ml), which yields a black signal. After 20 minutes of preincubation the reaction was catalyzed using
0.3% H2O2. The reaction was stopped by rinsing the tissue in acetate buffer and PBS. The same staining
procedure was then repeated for labeling of LY. Therefore, slices were incubated overnight at 4°C in a
monoclonar biotinylated anti-LY antibody from rabbit (Molecular Probes, Leiden, The Netherlands; 1:200
working dilution) in PBS containing 0.3% Triton X-100 (Sigma). After washing, slices were incubated for 2
hours in the avidin-biotin complex (1:100 in Triton-PBS). Washes in PBS were followed by additional washes
in acetate buffer. Labeling of LY was again achieved by incubating slices in DAB, but without addification of
heavy-metal compounds, thus resulting in a brown staining product. The reaction was stopped by rinsing the
tissue in acetate buffer and PBS. Slices were then mounted, dehydrated and coverslipped. Camera lucida
reconstructions of filled cells were drawn using an Olympus BH-2 with a drawing tube at x 125 magnification.
RESULTS
A total of 84 neurons that met the criteria specified in Materials and Methods were analyzed.
All neurons could be assigned unequivocally to one of the 4 cell classes described in chapter 3
Chapter 6: Actions of dopamine on neurons in the chick NCL 179
by their responses to depolarizing and hyperpolarizing current pulses. Of these 84 neurons, 37
were characterized as type I neurons by their burst firing pattern of action potentials, and 33
showed a regular or phasic-tonic firing, characteristic of type II neurons. Five cells displayed
the high-frequency firing typical of type III interneurons, and the remaining 9 cells were
classified as type IV neurons by their burst firing of action-potentials and the absence of
prominent inward rectification.
10 µM DA washbaseline
200 ms
20 mVA
10 µM DA washbaseline
200 ms
20 mVB
200 ms
20 mV
10 µM DA washbaseline
C
200 ms
20 mV
10 µM DA washbaseline
D
Type I
Type II
Type III
Type IV
-30pA
180pA
-30pA
180pA
-100pA
550pA
-100pA
600pA
Figure 1: The effects of dopamine application in the 4 major celltypes of the chick NCL. Dopamine at 10 µM could largelyincrease the number of spikes evoked by a constant current injection in the three spiny cell types (types I, II and IV, shown inA, B, and D, respectively), but not in type III interneurons (C). The enhancement of neuronal excitability by dopamine appearedto be independent from changes in the passive properties of the cells.
Chapter 6: Actions of dopamine on neurons in the chick NCL 180
Effect of dopamine application on neuron excitability
The effect of DA on the excitability of NCL neurons was studied by measuring changes in spike
number in response to 1.1-sec-long depolarizing current injections. The effect of DA did not
vary with the recording technique used, therefore, for comparison of dopaminergic effects
across concentrations data obtained during whole-cell and perforated-patch recordings were
pooled. In control recordings (n = 9), neurons that were recorded under the same conditions
and over comparably long periods as experimentally-treated cells (at least 20 minutes), showed
only a slight decrease of 4.5 ± 4% in the number of evoked spikes. This change from baseline
was neither reversible nor significant (df = 8; t = 1.05; p > 0.05; Student’s t test for paired
samples).
Application of DA reversibly increased the number of spikes in 32 of 43 neurons tested (74%).
However, the effect of DA differed with the cell-type studied. Dopamine increased the number
of evoked action potentials in neurons of all spiny cell-classes (i.e. types I, II, and IV), but not
in type III interneurons (figure 1). Thus, application of dopamine increased the number of spikes
in 13 of 16 type I neurons, in 14 of 17 type II neurons, and in 4 of 5 type IV neurons. In contrast,
in 4 of 5 type III neurons tested, application of DA resulted in no change in the number of
evoked spikes, or in a small decrease, similar to that observed in controls. In one neuron there
was a small increase of 11% (9 vs. 8 spikes). To account for these differences in responsivity,
type III neurons were excluded from further analysis of drug effects.
(n=7)(n=5)
**(n=17)
*(n=5)
(n=4)
(n=9)
control 1 µM 5 µM 10 µM 20 µM 50 µM
concentration of dopamine
% c
hang
e in
num
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kes
140
120
100
80
60
40
20
0
-20
Figure 2: The effect of dopamine at various concentrations. In contrast to control recordings, in which a small decrease in thenumber of evoked spikes occured during prolonged recording, application of dopamine consistently increased the firing of spinyNCL neurons. The change in the number of action potentials was significantly different (p < 0.01) from changes in controls at 5µM and 10 µM dopamine. At higher concentrations the effect of dopamine did not saturate, but rather diminished, resulting in aninverted U-shape of the dose-response curve.
Chapter 6: Actions of dopamine on neurons in the chick NCL 181
The increase in spike number induced by DA across the range of concentrations studied (i.e. 1,
5, 10, 20, and 50 µM; N = 38) was significantly different from changes observed in controls (df
= 46; F = 3.7; p < 0.01; ANOVA). When grouped according to the concentration of DA applied,
significant increases in the spike number compared to control recordings occured only at
concentrations between 5 and 10 µM DA, indicating a dose dependence of the effect of DA
(figure 2). In 5 µM DA the mean number of spikes increased by 74.5 ± 9.7 % (df = 12; t = -8.9;
p < 0.01, α-adjusted; Student´s t test for unpaired samples) and in 10 µM DA by 112.5 ± 22.6
% (df = 24; t = -5.1; p < 0.01, α-adjusted; Student´s t test for unpaired samples). The effect of
DA did not saturate, but rather decreased with higher concentrations, resulting in almost no
change in the number of spikes at 50 µM DA (n = 4; c.f. figure 2). This finding is in agreement
with an inverted U-curve that has been used to describe the range of optimal dopaminergic
activation (Williams and Goldman-Rakic, 1995; Murphy et al., 1996; Zahrt et al., 1997). The
effect of DA usually developed within 3 minutes and peaked about 5 - 10 minutes after the end
of bath-application (figure 3). The effect was long lasting, sometimes persisting up to about an
hour, depending on the concentration of DA that was applied.
0 2 40
2
4
6
8
10
12
14
16
18
20
22
24
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
10 µM DA 10 µM DA
Time (min)
Num
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Figure 3: Example for the time course of dopamine-induced enhancement of cell excitability in a type IV neuron. The dopamineeffect usually developed within a few minutes after dopamine application in the bath. In most neurons the strongest effect onthe number of evoked action potentials was seen approximately 5 - 10 minutes after cessation of dopamine application, and itcould last for more than an hour. The short application-time of dopamine (∼1 minute) served to minimize rapid D1 receptordesenzitation.
Chapter 6: Actions of dopamine on neurons in the chick NCL 182
To examine possible mechanisms by which DA might regulate the excitability of NCL neurons,
the near-threshold firing behaviour of 20 neurons that had received DA applications at doses
shown to effectively increase the number of spikes (i.e. 5 and 10 µM) was compared with
changes in controls. The effects of DA on the latency and the threshold of the first spike were
studied, and in addition the change in the duration of the first inter-spike-interval was analyzed.
The results show that application of DA led to a negligible reduction in the latency of the first
spike (mean changes -6.5 ± 3.8 % for DA, and +4.3 ± 6.1 % for controls; df = 27; t = 1.50; p >
0.05; Student´s t test for unpaired samples). In current-clamp recordings the threshold for
generation of an action-potential varied considerably over the duration of the experiment (a
change of +1.69 ± 1.4 mV in controls); application of 5 or 10 µM DA, however, resulted in a
small shift of the spike-threshold towards hyperpolarized potentials (-1.14 ± 0.72 mV), and this
change showed a statistical trend when compared to controls (df = 27; t = 1.98; p = 0.058;
Student´s t test for unpaired samples). Dopamine had its strongest impact on the duration of the
first inter-spike-interval, which usually occured at more depolarized potentials than the first
spikes. Application of DA significantly shortened the first inter-spike-interval when compared
to changes in control recordings (mean changes -23.5 ± 4.4 % for DA, and +32.6 ± 14.4 % for
controls; df = 27; t = 4.89; p < 0.01; Student´s t test for unpaired samples).
Application of DA had little effect on the passive membrane properties of NCL neurons. There
was only a negligible overall reduction in input resistance (-2.66 ± 1.4 %) after DA application
compared to controls (df = 46; F = 0.36; p > 0.05; ANOVA).
In addition to its effects on cortical pyramidal neurons in mammals, DA has been shown to
modulate the cell excitability of GABAergic interneurons (Penit-Soria et al., 1987; Retaux et
al., 1991; Pirot et al., 1992; Zhou and Hablitz, 1999). Because spontaneous synaptic input can
strongly modulate the passive properties (i.e. input resistance) of neurons (Paré et al., 1998), I
tested whether DA might increase the excitability of NCL neurons by decreasing spontaneous
inhibitory synaptic transmission. Therefore, in 6 principal neurons the GABAA receptor blocker
picrotoxin (100 µM) was added to the extracellular solution before the application of 10 µM
DA. In the presence of picrotoxin, DA still produced a significant increase in the number of
spikes of 82.6 ± 26.5 % (df = 13; t = -3.24; p < 0.05; Student´s t test for unpaired samples).
This result indicates that the effect of DA on evoked spikes is not mediated indirectly by a
decrease in spontaneous inhibitory synaptic transmission.
Chapter 6: Actions of dopamine on neurons in the chick NCL 183
Pharmacology of dopaminergic enhancement of excitability
To determine which DA receptor subtype might mediate the observed changes in spike number,
the effects of selective agonists of either the D1 or D2 family of DA receptors were studied. In
the same line of evidence the effect of DA was challenged by a specific antagonist to
dopaminergic D1 receptors (figure 4).
Application of the D1 receptor agonist SKF-38393 mimicked the effects of DA in that it
significantly increased the number of spikes, both at 10 and 25 µM (df = 23; F = 5.75; p < 0.01;
ANOVA; figure 4A, D). The mean increase in the relative number of action potentials was 79.3
± 19.1 % compared to control recordings. At 10 µM (n = 7), SKF-38393 led to an increase in
the number of spikes of 98.9 ± 33.9 % (df = 14; t = -3.02; p < 0.05, α-adjusted; Student´s t test
for unpaired samples), while at 25 µM (n = 8) the increase was 67.4 ± 24.3 % (df = 15; t = -
3.04; p < 0.05, α-adjusted; Student´s t test for unpaired samples).
Conversely, when 10 µM DA was applied approximately 5 min after bath application of the D1
receptor antagonist SCH-23390 (45 µM), the change in the number of action potentials was not
significantly different from control recordings. In the presence of SCH-23390, DA (n = 5) had
only a small (mean increase 16.0 ± 21.3%), non-significant effect on the number of evoked
spikes (df = 12; t = -0.94; p > 0.05). Bath application of SCH-23390 for 5 - 10 minutes alone,
following 5 minutes of baseline measurements, had no effect on the number of evoked spikes (df
= 4; t = 0.87; p > 0.05; Student´s t test for paired samples; figure 4B, D).
The D2 receptor agonist quinpirole (n = 6) had no effect on the excitability of NCL neurons.
Bath application of 25 or 50 µM quinpirol resulted only in a small, non-significant decrease in
the number of action-potentials by 1.53 ± 1.5 % (df = 13; t = -0.57; p > 0.05; Student´s t test for
unpaired samples), similar to that observed in controls (figure 4C, D). In 3 of the 6 neurons 10
µM DA was applied subsequently to the establishment of the quinpirole effect (at least 20
minutes after application of quinpirole). In all 3 cells DA application led to an increase in the
number of spikes (mean 74.8 ± 41%), showing that these cells were not generally unresponsive
to dopaminergic transmission (figure 4 C). This result supports the assumption drawn from the
anatomical data reviewed in chapter 4, that D2 receptor mediated mechanisms have only
relatively little, if any, influence on neurons in the caudal forebrain of birds. Taken together,
these data suggest that DA modulates cell excitability in the NCL via activation of D1 type
receptors by a mechanism that appears to be independent of input resistance.
Chapter 6: Actions of dopamine on neurons in the chick NCL 184
(n=9)
10 µM 25 µM
SKF 38393
% c
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140
120
100
80
60
40
20
0
-20 quinpiroleSCH 23390(+ 10 µM DA)
*(n=8)
*(n=7)
(n=6)
(n=5)
control
baseline
20 mV
200 ms
25 µM SKF 38393
wash
baseline
20 mV
200 ms
45 µM SCH 23390+ 10 µM DA
wash
20 mV
200 ms
baseline
50 µM quinpirole
wash
1 µM DA
A B C
D
400pA 600pA 550pA
-100pA-100pA -50pA
Figure 4: Pharmacology of the enhancement of cell excitability. Activation of the D1, but not the D2 receptor, mimicked theeffects of DA in that it led to a significant increase in evoked action potentials. A: Example of a regular spiking neuron in whichthe selctive D1 agonist SKF-38393 was bath applied. B: Dopamine D1 receptor blockade by application of the competitive D1receptor antagonist SCH-23390 before and during application of dopamine results in no change in the number of spikes in thisneuron, and only in a small non-significant increase across all neurons tested (c.f. D). C: Application of the selective D2receptor agonist quinpirole has no effect on cell excitability in this type I neuron, and in all neurons tested (c.f. D). Application ofDA in the same neuron, however, could largely increase the number of spikes in a burst, demonstrating that this neuron was notgenerally irresponsive to dopaminergic stimulation. D: Overview of the effects of different DA receptor agonists andantagonists on cell excitability compared to control recordings.
Chapter 6: Actions of dopamine on neurons in the chick NCL 185
Cell type specifity
One aim of the present experiments was to determine whether the response to DA in cells that
receive the basket-type dopaminergic innervation differ from those that receive only „en-
passant“ innervation of their dendrites. Type I and type II neurons are the most plausible
candidates to receive basket-type innervation, as baskets preferentially innervate neurons with
large soma sizes (c.f. chapter 4) and the anatomical data presented in chapter 5 also had shown
that GABAergic interneurons do not receive dopaminergic basket input.
In addition, spikes are unitary events and I had expressed the effects of DA as the percentage
change occuring between baseline and drug condition. Thus, although the spike number was
averaged across 5 consecutive sweeps, this procedure might have led to a significantly larger
statistical increase in type I neurons, as they have a low basal firing rate and might therefore
„profit“ more from small variations in the number of spikes. However, a comparison of the
effect of 5 - 10 µM DA on type I and type II neurons, respectively, showed no significant effect
of cell type. Dopamine increased the mean number of spikes in type I cells (n = 10) by 120 ±
20.7 %, and in type II neurons (n = 11) by 73.5 ± 23.1 % (df = 19; t = 1.77; p > 0.05; Student´s
t test for unpaired samples). This result shows that DA appears to modulate the large principal
cell types in the NCL in the same manner.
Morphology
It was attempted to identify the class of neurons that receives basket-type dopaminergic input by
colocalizing Lucifer Yellow-intracellularly filled neurons with tyrosine-hydroxylase containing
fibers by means of double-label immunohistochemistry.
In 15 slices in which neurons were filled with Lucifer Yellow, TH-labeling was sufficiently
good enough to examine the catecholaminergic innervation of the soma in detail. Of these 15
neurons 12 showed only few TH immunopositive fibers in the vicinity of their soma or initial
dendrites, making no, or seemingly only „random“ contact. The remaining 3 neurons received
catecholaminergic input from TH-labeled fibers showing numerous varicose-like swellings in
close apposition to the soma and the initial segments of dendrites, resembling the basket-type
innervation (figure 5). All of these 3 neurons were characterized as type I neurons by their burst
firing pattern of action potentials. In each of these neurons application of DA (n = 2) or SKF-
38393 (n = 1) resulted in a marked increase in the number of spikes. This finding shows that
burst-firing neurons in the NCL appear to be a main target of the dense basket-like somatic
innervation, however, due to the small sample size, it would
Chapter 6: Actions of dopamine on neurons in the chick NCL 186
Figure 5: Anatomy of the dopaminergic innervation of electrophysiologically characterized neurons that were shown to beresponsive to dopamine application. Neurons were intracellularly filled with Lucifer Yellow (LY) during recording, and double-labeled immunohistochemically for LY and tyrosine-hydroxylase (TH) as a marker of dopaminergic fibers. A: Camera lucidareconstruction of the basket-type innervation of a type I neuron by TH-positive fibers (red). For reason of clarity only theproximal innervation by dopaminergic fibers is shown. The axon of the cell is shown in light green. B, C: Photomicrographs ofthe cell shown in A. D: Another example of a neuron that received numerous TH-positive varicosities in close apposition to thesoma and initial dendrites (arrows). E: Photomicrograph of a cell that did not receive basket-like innervation. Although thenumber of labeled fibers is generally lower than in C (due to differences in staining quality), several basket-like structures can stillbe seen (arrows; c.f. also F). Furthermore, there is a large number of varicose-like swellings in close apposition to the shafts andspines of the cells dendrites. F: Detail of the TH-labeling shown in E. The arrow points to a basket-like structure that coilsarround the soma of an unlabeled neuron. Scale bars are 25 µM in A, B, D, F, and 50 µM in C, E.
Chapter 6: Actions of dopamine on neurons in the chick NCL 187
be premature to conclude that they constitute the only cell class receiving basket-innervation.
Moreover, the observed changes in the spike-pattern following DA application in neurons
innervated by basket-forming dopaminergic fibers did not differ qualitatively nor quantitatively
from cells that received diffuse „en-passant“ innervation to their dendrites and somata. Thus, it
appears that the different modes of dopaminergic innervation exert no qualitatively different
influence over their target neurons.
DISCUSSION:
The data presented here, suggest that DA modulates cell excitability in the NCL via activation
of D1-type receptors. The enhancement of membrane excitability appears to be independent of
changes in input resistance, and an indirect effect on GABAergic transmission. The latter was
inferred from direct recordings of putative GABAergic fast-spiking interneurons, and by the
absence of an effect of the Cl- channel blocker picrotoxin on the DA-induced increase in
projection neurons.
The effect of DA appeared to be dose-dependent, being maximally in a narrow range at
concentrations of 5 - 10 µM DA in the sample of neurons recorded here. This finding is in
agreement with numerous studies which have demonstrated that an optimal degree of activation
of the dopaminergic system is necessary for the active retention of information, or the planning
of motor sequences in working memory (Sawaguchi and Goldman-Rakic, 1991; Williams and
Goldman-Rakic, 1995; Murphy et al., 1996; Sawaguchi, 1997; Zahrt et al., 1997; Arnsten and
Goldman-Rakic, 1998).
Similarly to the present results in birds, in mammals an enhancement of cell excitability in
vitro, or an increase in task-related activity in vivo, have been shown to depend on activation
of D1-like receptors (Sawaguchi et al., 1988, 1990,b; Sawaguchi and Goldman-Rakic, 1991,
1994; Williams and Goldman-Rakic, 1995; Yang and Seamans, 1996; Shi et al., 1997;
Sawaguchi, 1997; Kita et al., 1999). It should be noted, however, that several other studies
have observed an inhibitory action of DA on pyramidal cells (Geijo-Barrientos and Pastore,
1995; Gulledge and Jaffe, 1998; Zhou and Hablitz, 1999; Geijo-Barrientos, 2000), and a
possible involvement of D2 receptors in this depressant effect has been reported (Gulledge and
Jaffe, 1998).
One possible explanation for these conflicting results is a differential, and possibly also time-
dependent activation of D1- and D2-type receptors by DA, which might result in exactly
Chapter 6: Actions of dopamine on neurons in the chick NCL 188
opposite effects (Zheng et al., 1999). Thus, activation of D2-like receptors may require higher
concentrations of DA than those needed to activate D1-like receptors, as DA has a higher
affinity for D1-like receptors than for D2-like receptors when measured at the high affinity state
of the receptors (Seeman and Van-Thol, 1993). Furthermore, in the neocortex there are much
more D1 -like receptors than D2-like receptors (e.g. Lidow et al., 1991; Joyce et al., 1993), a
situation also seen in the dorsal forebrain of birds (Schnabel and Braun, 1996; see chapter 4).
In addition, the depressant effects of DA may be time-dependent and/or related to receptor
desensitization. Thus, studies in PFC neurons that utilized long (5 min) DA or D2-like agonist
applications, noted a decrease of input resistance and a concurrent reduction in the number of
evoked action-potentials (Geijo-Barrientos and Pastore, 1995; Gulledge and Jaffe, 1998; Zhou
and Hablitz, 1999). In contrast, the short duration application time (about 1 min.) used in the
experiments presented here, served to minimize rapid D1 receptor desensitization (Jarvie et al.
1993; Ng et al., 1995). Thus, for a given depolarizing input, time-dependence (desensitization)
or concentration-dependence of the DA effect might in principle account for the different results
obtained in various studies. In this view, the D2-like receptor may induce an „early“ transient
suppression of neuronal excitability, which in-vitro becomes only evident after desensitization
of the D1 receptor (Geijo-Barrientos and Pastore, 1995; Gulledge and Jaffe, 1998), whereas
D1/5 receptor activation may induce a „late“, prolonged (tens of minutes) enhancement of
neuronal excitability in response to depolarizing inputs (Yang and Seamans, 1996; Shi et al.,
1997; present study). However, the cellular basis for a possible D2 receptor mediated effect in
the cortex is not known. More importantly, in the present experiments no depressant action of
DA, or an involvement of D2 receptors in the mediation of the dopaminergic effects was found.
The ionic mechanisms by which DA exerts its effects in the frontal cortex following stimulation
of the D1 receptor are much better studied. By reviewing some of these findings I hope to
reveal some parallels and differences regarding the possible effects of DA in mammalian
cortical neurons and cells of the NCL. However, voltage-clamp studies into the effect of DA on
isolated currents in NCL neurons are necessary to directly study the ionic mechanisms by which
DA appears to modulate cell excitability.
In the cortex DA, via its action at the D1 receptor, appears to shift the threshold of the persistent
Na+ current (INaP) towards more hyperpolarized potentials (Yang and Seamans 1996; Gorelova
and Yang, 1997; but see Geijo-Barrientos and Pastore, 1995; Geijo-Barrientos, 2000). This
current is responsible for inward rectification in the depolarized voltage range and serves
important functions in setting the firing threshold for repetitive spike firing (Schwindt, 1992)
and signal amplification (Stuart and Sakmann, 1995). The existence of such a current in neurons
Chapter 6: Actions of dopamine on neurons in the chick NCL 189
of the bird´s caudal forebrain was shown in the zebrafinch HVC (Kubota and Saito, 1991), and
indicated by a TTX-sensitive inward rectification in NCL neurons (chapter 3). The tendency of
DA to lower the spike threshold and to prolong repetitive firing in NCL neurons, might reflect
an action of DA on the persistent Na+ current.
In contrast, the observed reduction in the duration of the first inter-spike-interval in the present
study might result from an action of DA on K+ currents, e.g. the Ca++ activated K+ current
termed IAHP, which is involved in spike-frequency adaptation (Madison and Nicoll, 1982;
Storm, 1990), or the slowly inactivating K+ current (IKS) that is responsible for membrane
outward rectification in the depolarized voltage range (Schwindt et al. 1988; Kitai and
Surmeier, 1993; Yang and Seamans, 1996). The latter will functionally counteract sustained
membrane depolarization and suppress repetitive spike firing. A strong outwardly rectifying
conductance was also seen in the spiny projection neurons of the NCL (see chapter 3). Thus in
the NCL, DA might shorten inter-spike-intervals and reduce accomodation by inactivating an
outwardly rectifying K+ conductance, as has been shown in the PFC and striatum (Yang and
Seamans, 1996; Nisenbaum et al., 1998).
The concurrent actions of DA on both, INaP and IKS, have been proposed to reduce spike treshold
and to contribute to the increased firing rate observed in vivo (Sawaguchi et al., 1988; 1990a,b)
and in vitro (Cepeda et al., 1992; Yang and Seamans, 1996; Shi et al., 1997; Yang et al., 1999).
In the present study, the facilitatory effects of DA could be seen most clearly at membrane
potentials considerably above threshold, i.e. in response to large depolarizing current pulses.
This might indicate a dopaminergic modulation of ionic currents that are maximally active at
depolarized potentials, such as high-voltage activated (HVA) Ca++ channels of the L, P, and N
type (Sayer et al., 1990; Seamans et al., 1997; but see Durstewitz et al., 2000). High-threshold
Ca++ channels are activated by suprathreshold synaptic inputs that elicit spike firing in the soma
and which then backpropagates into the dendritic arbor (Stuart and Sakmann, 1994; Spruston et
al., 1995). Dendritic high-voltage-activated Ca++ currents are likely to participate in multiple
functions that are important for burst firing mechanisms, sustaining repetitive firing, and
enduring forms of changes in synaptic plasticity (Yang et al., 1999). The existence of Ca++
currents that are capable of initiating slow Ca++ spikes has also been shown in neurons of the
NCL (c.f. chapter 3), but the voltage-dependence of these currents was not studied.
In neurons of the PFC and the striatum DA has been shown to activate D1/5 receptors on
pyramidal dendrites to directly attenuate the amplitude of high-voltage-activated Ca++ spikes, or
to augment the duration of a dendritic HVA Ca++ plateau, respectively (Surmeier et al., 1995;
Yang and Seamans, 1996; Hernández-López et al., 1997; Yang et al., 1998). At moderate
Chapter 6: Actions of dopamine on neurons in the chick NCL 190
concentrations, DA application results in a weak suppression of N-type Ca++ channels, that
cluster in the distal dendrites (Westenbroek et al., 1992). This action of DA may serve to
„sharpen” incoming depolarizing synaptic signals (from layer I–II inputs) arriving at the distal
dendrite, such that only sufficiently strong depolarizing inputs are propagated to the soma. Large
suppression of N-type Ca++ channels, however, may „uncouple” the distal dendrites, preventing
distal signals from reaching the soma (Yang and Seamans, 1996; Yang et al., 1999). This latter
action may help to explain in part why dopamine has often been attributed as having an
“inhibitory” or “suppressive” effect on spontaneous firing or activities evoked by specific
inputs in the PFC1. At the same time DA augments the duration of a Ca++ plateau, which is
mediated by L-type Ca++ channels that cluster in the soma and proximal dendrites of pyramidal
neurons (Westenbroek et al., 1990; Yang et al., 1998). Functionally, activation of L-type Ca++
channels may enhance the amplitude and duration of subthreshold excitatory synaptic inputs
(Seamans et al., 1997), and the dopaminergic modulation of L-type Ca++ currents has been
shown to increase the excitability of striatal neurons to NMDA application and depolarizing
current injections (Hernández-López et al. 1997; Cepeda et al. 1998). Through its action on L-
type Ca++ channels, DA will thus complement the increased cell excitability caused by the
enhancement of INaP, eventually leading cortical neurons to sustained firing (Yang et al., 1999).
Both, L-type Ca++ channels (Westenbroek et al., 1990; Seamans et al., 1997), and the persistent
Na+ conductance (Westenbroek et al., 1989; Stuart and Sakmann, 1995) are found
predominantly in the soma and basal dendrites of cortical neurons, thus enabling a differential
regulation of distal vs. proximal inputs by DA, as originally proposed by Yang and Seamans
(1996). While distal signals may be inhibited through the actions of DA on N- and P-type Ca++
channels, proximal inputs may be facilitated by dopamine´s enhancement of INaP and L-type
currents.
In the mammalian PFC, the network-effects of DA also appear to involve a modulation of
GABAergic interneurons. Dopamine directly depolarizes interneurons in the PFC and enhances
spontaneous and evoked IPSPs recorded in pyramidal neurons (Penit-Soria et al., 1987;
Godbout et al., 1991; Rétaux et al., 1991; Pirot et al., 1992; Zhou and Hablitz, 1999; Durstewitz
et al., 2000; but see Law-Tho et al., 1994). Accordingly, the suppressive action of DA on
spontaneous firing of PFC neurons in vivo is blocked by previous application of a GABA
antagonist (Pirot et al., 1992), suggesting that DA acts through interneurons in the PFC to reduce
spontaneous activity of pyramidal neurons. In the NCL, however, a significant contribution of
1 This effect of DA might also in part explain the downward slope of the dose-response curve at highconcentrations of DA (inverted U-shape).
Chapter 6: Actions of dopamine on neurons in the chick NCL 191
GABAergic interneurons is highly unlikely, as in the present study DA did not modulate the
firing-pattern of presumed GABAergic fast-spiking interneurons, and its actions on spiny
principal neurons were unaffected by depressing spontaneous inhibitory transmission.
It is important to note that in vivo the effect of DA within the PFC is determined by the activity
level of PFC neurons and the specific inputs that are driving this activity. Although DA inhibits
spontaneous activity within the PFC of the anesthetized rat (Ferron et al., 1984; Mantz et al.,
1988; Pirot et al., 1992), it enhances task-related single-unit activity more than background
activity in the behaving primate (Sawaguchi et al., 1988, 1990a). DA (but not VTA stimulation)
also enhances responses evoked by hippocampal stimulation in the anesthetized rat and use-
dependent changes in synaptic efficacy in the hippocampal-PFC pathway that may be associated
with learning (Doyère et al., 1993; Jay et al., 1995). Similarly, in vitro the effects of DA are
voltage-dependent, favouring excitatory actions (e.g. via INaP, HVA-Ca++, or NMDA currents),
when the membrane already is in a depolarized state (Cepeda et al., 1998; Durstewitz et al.,
2000). Thus, DA may enhance task- or learning-related activity relative to spontaneous or
background activity within the PFC, and very likely also in the NCL. In the context of a delay-
task, the DA-induced increase in action-potential firing might augment the reliability of
transmission and thus provide the stability needed to hold online goal-related information in a
recurrent network.
The cellular basis by which DA in the NCL could achieve such a selective enhancement of
task-related inputs or reverbarating activity in a local network, remains unclear. One working
model assumed that basket- and non-basket-type innervation are dichotomic modes of
dopaminergic innervation which control different postsynaptic targets, or which strongly differ
in the degree of modulation of cell excitability. From the present results, burst firing neurons of
type I appear to be a main target of the dopaminergic basket-type innervation. Cells in this class
display a high threshold for action-potential generation and may thus require strong spatially or
temporarily summated inputs to fire. On the other hand, their burst of action-potentials results in
a faithfull transduction onto their target cells (Snider et al., 1998). An increase in the phasic
response of type I neurons will effectively drive more postsynaptic cells and „bind“ them in a
network. Enhanced firing of bursting neurons by DA could therefore establish a „pacemaker“-
activity in a network.
However, neurons from the other cell types in the NCL were modulated in the same way by
DA, and there was no obvious difference in the size of the response between cells receiving
basket and non-basket innervation. In addition, due to the small sample size of neurons that
Chapter 6: Actions of dopamine on neurons in the chick NCL 192
could morphologically be shown to receive basket-type innervation, it should not be excluded
that members of other cell-types also receive basket-type dopaminergic innervation. In any
way, the observed enhancement of firing in regular-spiking neurons of the type II, will also
strongly support sustained firing in a reverberating network, and thus functionally might enable
active retention of information in the NCL.
General discussion 193
General discussion
The experiments presented in this thesis provide an attempt to delineate an associative region in
the forebrain of birds, which has been shown to be involved in executive functions, such as
working memory and response control. The input- and output relations of the neostriatum
caudolaterale (NCL) with telencephalic and thalamic structures were studied with pathway
tracing techniques (chapters 1 and 2), and the direct sensory projection from the dorsal
thalamus was tested in-vivo (chapter 2). The principal celltypes of the NCL were described
physiologically and anatomically in-vitro (chapter 3), and the results give hints about their
possible roles in a proposed canonical circuit. Furthermore, the relationship of the
dopaminergic innervation with immunohistochemically or electrophysiologically identified
elements of this circuit was analyzed at the light-microscopic level (chapters 5 and 6). Finally,
the effect of dopamine or its receptor-specific agonists on the excitability of NCL neurons were
studied in-vitro (chapter 6).
Elements of a proposed circuit involved in working memory and executive functions
Fuster (1989) has proposed that the temporal organization of behaviour is at the heart of
prefrontal cortex functions. The cognitive operations that underly this „memory for action“ are
essentially targeted by the logic of delay tasks: „If earlier that, then now this“. This logic
requires a dynamic process of internal transferring of information across time and of cross-
temporal matching, the contents of which must be constantly changing as behaviour evolves,
especially in situations in which the outcome of the behaviour is yet not fully predictable, e.g.
when learning a new task (Fuster, 1989).
The PFC seems essential for three cognitive functions necessary to mediate contingencies
across time, (i) a temporally retrospective function of short-term memory (working memory),
(ii) a temporally prospective function of anticipatory set or preparation (planning and
foresight), and (iii) a protective function of interference control (inhibition of false response
tendencies).
Behavioural studies have shown that the NCL serves at least two of these „prefrontal“
functions. Lesions of the NCL show behavioural impairments in a variety of working memory
tasks (Mogensen and Divac, 1982, 1993, Gagliardo et al., 1996, 1997, Güntürkün, 1997).
General discussion 194
Similarly, the role of the NCL in interference control has been tested with Go/NoGo tasks and
reversal-learning (Hartmann and Güntürkün, 1998; Güntürkün, 1997, Kalt et al., 1999).
The prospective coding functions of the NCL have not been studied yet, but Kalt and coworkers
(1999) observed neurons that increased their firing towards the end of the delay, which, in
accordance to the findings in primates, has been interpreted as „reward expectancy“ or a
preparatory motor function of these cells. Therefore, it is highly likely that the anticipation of
events and the preparation for them, also takes part in the NCL.
As briefly outlined in chapter 1, the temporal organization of behaviour requires to bridge
temporally separate elements of action and sensorium. The anatomical data presented in
chapters 1 - 3 strongly suggest that the NCL may serve such a synthetic function in the
perception-action cycle. Similar to the PFC of mammals, the NCL receives converging afferents
from various telencephalic and thalamic areas that relay input from the major sensory
modalities of somesthesis, vision, and audition (chapters 1 and 2). At least the
intratelencephalic projections are reciprocal which might allow the NCL to control its own
input. The pattern of subtelencephalic projections is not known and need to be studied with
more sensitive anterograde tracers. In addition, the NCL is reciprocally connected with the
avian homologue of the amygdala (chapter 1), a connection which is also characteristic for the
PFC (Condé et al., 1995; Reep et al., 1996; Sesack, et al., 1989; Barbas and DeOlmos, 1990),
and projects to most parts of the somatic and limbic striatum, as well as the sensorimotor
archistriatum (chapters 1 and 3). Via the projections through the basal ganglia and the
archistriatum, the NCL does not only exert indirect influence over motor structures in the
brainstem (Wild, 1993; chapter 1), but may also control its own dopaminergic innervation from
the substantia nigra and AVT.
In the context of working memory tasks it is generally assumed that items from long-term
memory relevant to the task at hand are retrieved from stores outside the PFC. Functional
imaging studies in humans, and electrophysiological recordings in animals performing delay
tasks indeed show time- and domain-dependent activation within distributed networks related
to the PFC (Cohen et al., 1997, Belger et al., 1998; Chafee and Goldman-Rakic, 1998).
It is reasonable to believe that such a gross anatomical arrangement of different sensory-
specific networks also exists between the NCL and other associational or sencondary sensory
forebrain areas in the avian brain. This is inferred from the sheer necessity to save memory
„storage“, and the fact that both, at the areal level (chapter 1), and at the single cell level
General discussion 195
(chapter 3) a large number of cells were seen to project back to sensory areas in the anterior
forebrain. The reciprocal connection of the NCL with virtually all of its sources of afferents
suggest the existence of multiple distributed networks within the avian forebrain. One such
functional network probably inolves the connection between the NCL and the parasensory area
in the neostriatum intermedium (NIM, or MNH in chicks). This area sends a massive projection
to large parts of the NCL and in turn receives considerable feedback from the NCL (chapter 1).
Both forebrain areas receive a distinct innervation from the polymodal DLP (chapter 2) and the
termination field of the DLP within NCL completely overlaps with the termination area of the
NIM (c.f. figs. 10, 11 in chapter 1, and fig 1f in chapter 2).
The exact homologue of the DLP remains unclear (c.f. detailed discussion in chapter 1),
functionally, however, the DLP-NCL projection has been implicated in the proper execution of
working memory tasks (Güntürkün, 1997), similar to the MD-PFC projection in mammals
(Sakurai and Sugimoto, 1985; Fuster, 1989 for review). It is therefore not unreasonable to
speculate that the DLP-NIM projection also conveys information pertinent to these tasks. The
NIM receives afferents from areas in the Wulst and archistriatum, and its projection onto NCL
could complement the parallel input the NCL receives from these areas. Via the projections of
the NCL and NIM to the basal ganglia and the archistriatum, this loop through the forebrain
could also influence activity in the dorsal thalamus (including DIP and DLP) and lower
mesencephalic areas (e.g. the optic tectum, or AVT; c.f. fig. 13 in chapter 1).
In the PFC of mammals, working memory is manifest in the activity of delay-related neurons
that are activated after offset of the cue and continue firing throughout the duration of the delay
(e.g. Fuster, 1973; Sawaguchi and Yamane, 1999). Most cellular models of working memory
assume that the activity of these neurons reflects reverberating activity in a cell assembly that
codes for the given memorandum (Amit, 1995; Goldman-Rakic, 1996; Durstewitz et al., 1999;
2000). In the PFC, reciprocal horizontal projections between neurons in layers III, and V-VI,
respectively, connect discrete „columns“ within the same cortical region, which are assumed to
form functional modules (e.g. Goldman-Rakic, 1996; Melchitzky et al., 1998; González-Burgos
et al., 2000). The local interaction between these cells provides a neural basis for positive
feedback circuits in the PFC that may play a role in sustaining neural activity (Amit 1995;
Goldman-Rakic, 1996; Durstewitz et al., 1999, 2000).
In the pigeon, Kalt and coworkers (1999) have recently shown that neurons in the NCL can
similarly enhance their firing throughout the delay period of a delayed response task to „hold“
information after the offset of the sensory cue. It can be assumed that at least part of the
General discussion 196
sustained activity results from reverbarating activity within the NCL. In accordance with this
view, simultaneous recordings from two NCL neurons during a working memory task show a
significant correlation of task-related activity (Thomas Kalt, personal communication).
The electrophysiological analysis of cell types in-vitro, allows the description of certain
distinct features (e.g. firing pattern, or membrane time constant) that determine their integrative
properties and the impact on other cells (chapter 3). However, the conclusions that can be
drawn from this analysis about a cell´s role within a functional circuit are rather limited. This is
due to the fact that in-vivo the cell´s function might be determined much more by the properties
of the network, than by the properties of the cell itself.
In the NCL, spiny principal neurons of the type II, and smooth type III interneurons showed
sustained firing over a wide range of frequencies and throughout long depolarizing current
pulses. Thus, in principle, the firing patterns of these cells could represent a neural substrate for
active memory retention on the single cell level. Furthermore, Durstewitz and coworkers
(2000) could show, that in a network of the PFC stimulus-specific recurrent activity can be
maintained at frequencies below 20 Hz, which is in the range of the steady-state firing
frequency of type II neurons (c.f. chapters 3 and 6).
However, in contrast, the firing frequency of single NCL neurons in-vivo is not only highly
irregular (as might also be the case in cortical neurons), but furthermore it appears that a large
portion of neurons which show delay-related activity, respond with bursts of action potentials
(Thomas Kalt, personal communication). Thus, reverberating activity in an ensemble of burst-
firing neurons (i.e. cell types I and IV1) also seems to make a major contribution to delay
activity. As bursting neurons showed strong accomodation of firing and were not able to
generate repetitive bursts in response to continued depolarization, phases of refractory
inhibition, most likely provided by feedback from the type III interneurons, are also essentially
to produce the pattern observed in-vivo.
Functionally, burst firing increases the probability to drive the postsynaptic cell beyond spike
threshold (Snider et al., 1998). The high frequency of action potentials during a burst will thus
amplify the neural signal and synchronize the activity in a population of postsynaptic cells
(Hablitz, 1986; Snider et al., 1998). Therefore, burst firing pattern of type I cells might provide
for reliable transmission within an active cell assembly, which is needed for the stability of the
mnemonic representation.
1 As discussed in detail in chapter 3, neurons of type IV are likely to represent immature stages of other spinycell types, and therefore they are not considered here further.
General discussion 197
Modulation by the dopaminergic system
As discussed in detail in chapters 4 and 5, the organization of the mesotelencephalic DA
projections and the regional distribution of dopaminoceptive elements is highly similar in
mammals and birds, despite approximately 250 million years of divergent evolution.
As also shown in chapter 4, the structural similarities extend to functional similarities with
regard to the control of sensorimotor and associative processes, that are controlled by the
dopaminergic system.
In mammals, the integrity of the active short-term memory trace appears to be critically
dependant on an optimal level of DA receptor stimulation. Both, prefrontal DA depletion
(Brozoski et al., 1979; Simon et al., 1980) and excessive stimulation of DA receptors (Murphy
et al., 1996; Sawaguchi, 1997; Zahrt et al., 1997; Arnsten and Goldman-Rakic, 1998), disrupt
performance on delay tasks. Similarly, the administration of high doses of D1 antagonists into
the PFC also impair performance on delay tasks and decrease delay-period activity of PFC
neurons (Sawaguchi et al., 1990b; Sawaguchi and Goldman-Rakic, 1994; Williams and
Goldman-Rakic, 1995; Zahrt et al., 1997), however, application of low doses of D1 antagonists
or DA into the PFC increases delay-correlated activity of PFC neurons relative to background
activity (Sawaguchi et al., 1988; 1990a; Williams and Goldman-Rakic, 1995; Müller et al.,
1998). Thus, too much or too little DA may be detrimental to cognition.
Thus, one major finding of the present studies is that DA also profoundly alters the excitability
of NCL neurons, and that the observed increase in the number of spikes was strongly dose
dependent. Moreover, this dose dependency was best described with an inverted U-curve,
demonstrating also that under physiological conditions in birds, as is the case in mammals, an
optimal level of DA might be required for the changes in neuronal excitability to take place
(chapter 6).
Together with the data by Kalt and coworkers (1999) and the voltage-dependence of the DA
effect (discussed in chapter 6), one can speculate that in-vivo the mesencephalic DA signal to
the NCL enhances the excitability of active cell assemblies, which might result in recurrent
activity that is strong enough to carry the memory trace over the delay.
So far, there exists only one study that has examined the contribution of the DA system on
working memory in birds (Güntürkün and Durstewitz, in press). In support for the hypothesis
outlined above, the results show a severe impairment in working memory functions, following
the blockade of D1 receptors by injections of high doses of SCH-23390 into the NCL.
Therefore, D1 receptor mediated enhancement of neuronal firing might be a requirement for the
General discussion 198
active retention of memory contents both in mammals and birds. To test the validity of this
assumption, it could be attempted to pharmacologically alter the delay-related activity of NCL
neurons through the iontophoretic application of D1 receptor antagonists, or supranormal doses
of DA (c.f. Sawaguchi and Goldman-Rakic, 1991; Williams and Goldman-Rakic, 1995).
However, a general, non-specific increase in activity will not produce a stable representation
in short-term memory. As indicated above, the voltage- (i.e. activity-) dependance of the DA
effects (c.f. discussion in chapter 6) might serve to enhance only those cell assemblies that are
currently activated. Yet, the question remains, how the representation of memory items within
the recurrent activity might be protected from other interfering stimuli.
In mammals, the bilaminar pattern of prefrontal cortical DA innervation might be an inherent
aspect of the dopaminergic modulation of working memory. In the PFC, the innervation by
dopaminergic fibers is densest in the upper layers I-II and V-VI (c.f. figure 4 in the
introduction). The arrangement of long-range associational and local input, respectively, shows
a somewhat parallel pattern. Synaptic inputs from other association cortical regions mainly
arrive in layers I-II, where the apical tuft of the layer III, and V-VI pyramidal neurons extends
(Sesack et al., 1989; Berendse et al., 1992; Condé et al. 1995), and, as outlined above, local
synaptic connections that contact the soma and proximal dendrites arise from neurons from
within the same lamina.
As discussed in detail in chapter 6, DA might modulate distal signals in a manner different from
its effect on proximal inputs. In sum, DA acting on pyramidal neurons of the PFC (i) restricts the
effects of inputs to the apical dendrites of these neurons by attenuating the dendritic HVA Ca++-
mediated amplification of such inputs (Westenbroek et al., 1992; Surmeier et al., 1995; Yang
and Seamans, 1996); and (ii) increases cell excitability through its actions on conductances that
are located mainly in the soma and proximal dendrites (Yang and Seamans, 1996; Yang et al.,
1998). Thus, one effect of DA might be to selectively attenuate distal EPSPs relative to
proximally arriving EPSPs, resulting in a differential „selection“ of synaptic inputs (Yang et al.,
1996b; see also Law-Tho et al., 1994).
In the absence of a layered structure that enables the spatial separation of functionally distinct
inputs, another mechanisms is needed to „switch“ between inputs from long range associational
fibers and from intrinsic connections. In the avian brain, the dichotomic modes of DA
innervation might serve this function.
General discussion 199
Figure 1: Summary of the anatomical data presented here, and the modulatory actions of dopamine on identified neuronalelements in the NCL. The input-output relations of the NCL have been described in chapter 1 - 3. Note that for reason of claritynot all possible afferent and efferent projections are depicted. However, neurons from all spiny cell types (i.e. types I, II, IV [notshown]), but not smooth interneurons (type III), project to the same targets outside NCL. In addition, it can be assumed, thatneurons of all cell types receive the same afferent input. The assignment of DARPP-32 immunoreactivity to certain cell types isinferred from a comparison of the morphology of DARPP-32 positive neurons described in chapter 5 with those identified inchapter 3, and this is complemented by the reaction of these cell types to the application of DA and the D1 receptor agonistSKF-38393. Part of the intrinsic connections of the NCL can be inferred from the pattern of local axonal collateralizations ofdifferent cell types (c.f. chapter 3). However, a detailed description of the intra-NCL connections is still lacking. The insertshows a hypothetical arrangement through which the dopaminergic system could achieve a spatial selection of functionallydistinct synaptic inputs. Local synaptic connections that arrive at the soma of neighbouring cells might be enhanced relative tosynaptic inputs from sensory areas arriving at the distal dendrites. In accordance with the data presented in chapter 6, a type Ineuron is shown to receive basket-type innervation. For the model proposed, however, it is not relevant whether a specific celltype receives basket-innervation, and there is evidence that projection neurons of the other celltypes also receive basket-innervation.
There are several working hypotheses regarding the role of the dopaminergic basket-type
innervation that have been tackled by the studies presented here. One assumption might have
been that the basket-innervation provides a means for the dopaminergic system to selectively
target a certain class of neurons, e.g. GABAergic interneurons, cells with distinct firing
patterns, or distinct axonal connections. Alternatively, it might have been thought that the strong
somatic basket innervation exerts a qualitatively or quantitatively different influence on the
feedback projectionsto secondary sensory areas
thalamic input(DLP, SRt, VIP)
archistriatum (limbic and motor) (limbic and motor)
basal ganglia
archistriatum (limbic and motor)
(limbic and motor)basal ganglia
basal gangliaand archistriatum
dopaminergic fibersfrom SN and VTA
telencephalic input fromsensory and associational areas
intra NCLconnections
type IIDARPP-32+DA and D1 agonistenhance firing
type IDARPP-32+DA and D1 agonistenhance firing
type IIIDARPP-32-no effect of DAon firing pattern
General discussion 200
target cell, in the sense that it provides a higher concentration of DA, which in turn might result
in opposite or quantitatively different effects than comparatively lower concentrations of DA
(e.g. inverted U shape of effects).
Clearly, our understanding about the functional subtypes of NCL neurons is still too limited to
unequivocally rule out any of these hypothesis. However, from the data provided in chapter 6
no obvious qualitative or quantitative difference in the response to DA was evident for neurons
that received strong somatic basket input.
So what is the functional importance of the two modes of dopaminergic innervation then?
As indicated above, I would like to propose that baskets provide a means for spatial selection
of functionally distinct synaptic inputs. A differential effect of DA on distinct inputs might be
achieved if a specific class of connections, e.g. local connections between NCL neurons, make
synapses preferentially on the somata and initial dendrites of neighbouring cells. Such a
hypothetical arrangement is shown in the insert in figure 1. For this model it is not neccessary
that all intrinsic connections of NCL neurons selectively make contacts only with the somata of
other NCL neurons. Dopamine might still enhance these inputs relatively more if only a fraction
of intra-NCL connections synapses on the somata of neighbouring cells. For the differential
effects of DA to take place it is merely critical to assume that a larger number of proximal
inputs originates from intra-NCL projections relative to long-range sensory inputs. Also, the
large number of baskets found in the NCL is not an indicator for a larger number of intrinsic
connections. Other areas in the avian forebrain might possess a similarly large number of local
interconnections, but the functions of these circuits may not depend as much on dopaminergic
modulation as it appears to be the case for the NCL.
In the NCL, however, the proposed „input-selection“ effect of DA could account for
reverbarating activity between NCL neurons, and a concurrent protection of this memory trace
against interfering stimuli. This effect of DA could be brought about by several mechanisms.
The easiest model to assume is that the distribution of ionic conductances across the cell
membrane of NCL neurons varies in a manner similar to that found in mammalian neurons, so
that DA will attenuate distal („sensory“) inputs through dendritic HVA Ca++ channels, and will
enhance proximal inputs via its actions on INaP and L-type currents. Indeed it appears, that such
ionic „compartments“ exist not only in neocortical neurons that extend long apical dendrites.
For example, striatal (Surmeier et al, 1995) and cerebellar (Usowicz et al., 1992) neurons
show a similar distribution of N- and P-type currents in the dendritic segments, and a
predominance of L-type currents at the soma and proximal dendrites. Alternatively, DA could
General discussion 201
have dose-dependent effects on these neurons, as DA released from baskets will probably
faster reach significant concentrations at a large number of receptors. However, such an effect
would have went undetected in the experiments presented in chapter 6, in which DA was bath
applied, and the recordings were made (most likely) only from the somata of NCL neurons.
Support for a differential dendritic vs. somatic action of DA may come from the observation by
Schnabel and coworkers (1997), who found that the majority of D1 receptor immunoreactivity
was localized to synaptic and extrasynaptic sites in spines and dendrites, but that in addition
some neurons also showed dense labeling of the somatic cytoplasm.
To test the ideas outlined above, anatomical and electrophysiological studies may be devised.
Clearly, an electronmicroscopic analysis of the synaptic types and the subcellular distribution
of intra-NCL connections is required. To this end, either small injections of an anterograde
tracer confined to the NCL might be utilized, or the axonal arborizations of single, intracellulary
labeled neurons might be studied. Functionally, these connections could also be analyzed by
paired recordings from neighbouring cells in NCL, and the effect of DA on the synaptic weight
may be compared relative to DA-induced changes in synaptic inputs from areas outside the
NCL. Furthermore, direct recordings from dendrites of NCL neurons and pharmacological
isolation of single ionic currents could adress the issue whether DA exerts its effects via a
differential modulation of specific conductances.
In sum, the data presented here describe the organisation of a polymodal associative structure in
the avian forebrain, and they provide a structural and physiological framework in which
sensorimotor integration and working memory may take place. Furthermore, based on a
comparison with the prefrontal cortex of mammals, it appears that functional similarities also
neccessitate some structural similarities. Thus, the localization of a cognitive function within a
neuronal circuit, and a further understanding of the circuit´s structural and physiological
requirements may ultimately help to identify the building blocks of mental computation in
various species.
List of abbreviations 202
Abbreviations used in the text and figures
6-OHDA 6-hydroxydopamine
AMPA α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
AC or Acc nucleus accumbens
Ai archistriatum intermedium, pars centrale
Aid archistriatum intermedium, pars dorsale
Aidd archistriatum intermedium, parsdorsodorsale
Aidv archistriatum intermedium, parsdorsoventrale
AL ansa lenticularis
Am archistriatum mediale
Ap archistriatum posterior
APH area parahippocampalis
Av archistriatum intermedium, pars ventrale
AVT area ventralis tegmentalis; also see AVT
Bas nucleus basalis
BNST bed nucleus of the stria terminalis
Ca++ calcium
CDL corticoidea dorsolateralis
Cl- chloride
CPi cortex piriformis
DA dopamine
DARPP-32 dopamine- and cAMP-regulatedphosphoprotein, Mr = 32,000
DBH dopamine-beta-hydroxylase
DIP nucleus dorsointermedius posterior thalami
DIVA nucleus dorsalis intermedius ventralisanterior
DLL nucleus dorsolateralis anterior thalami,pars lateralis
DLM nucleus dorsolateralis anterior thalami,pars medialis
DLP nucleus dorsolateralis posterior
DMA nucleus dorsomedialis anterior thalami
DMP nucleus dorsomedialis posterior thalami
E ectostriatum
Ep ectostriatal belt
FPL fasciculus prosencephali lateralis
GABA γ-aminobutyric acid
GAD glutamate decarboxylase
GLd dorsolateral geniculate nuclei
HA hyperstriatum accessorium
HD hyperstriatum dorsale
HIS hyperstriatum intercalatus superior
HOM tractus occipitomesencephalicus, parshypothalami
Hp hippocampus
HV hyperstriatum ventrale
HVA high-voltage-activated (Ca++ currents)
HVdv hyperstriatum ventrale dorso-ventrale
HVvv hyperstriatum ventrale ventroventrale
ICo nucleus intercollicularis
IHA nucleus intercalatus of the hyperstriatumaccessorium
IMHV intermediate and medial part of thehyperstriatum ventrale
IKS slowly inactivating potassium current
INaP persistent sodium current
INP nucleus intrapeduncularis
ir immunoreactive
K+ potassium
L1 field L1
L2 field L2
L3 field L3
LFS lamina frontalis superior
LH lamina hyperstriatica
LHy nucleus lateralis hypothalami
LPO lobus parolfactorius
MNH mediorostral neostriatum/hyperstriatumventrale
n. nucleus
List of abbreviations 203
NA noradrenaline
Na++ sodium
NC neostriatum caudale
NCL neostriatum caudolaterale
NCm neostriatum caudomediale
Nd neostriatum dorsale
NF neostriatum frontale
NFL neostriatum frontolaterale
NFT neostriatum fronto-trigeminale
NI neostriatum intermedium
NIL neostriatum intermedium laterale
NIM neostriatum intermedium medialis
NIMl neostriatum intermedium medialis, parslaterale
NIMm neostriatum intermedium medialis, parsmediale
NMDA N-methyl-D-aspartate
OM tractus occipitomesencephalicus
Ov nucleus ovoidalis
PA paleostriatum augmentatum
PFC prefrontal cortex
PNMT phenylethanolamine-N-methyl transferase
PP paleostriatum primitivum
PT nucleus pretectalis
Rt nucleus rotundus
SAC stratum album centrale
SCI stratum cellulare internum
SGC stratum griseum centrale
SGP substantia grisea et fibrosaperiventricularis
SNC substantia nigra, pars compacta
SNR substantia nigra, pars reticulata
SPC nucleus tractus septomesencephalici
SPL nucleus spiriformis lateralis
SRt nucleus subrotundus
T nucleus triangularis
TH tyrosine hydroxylase
Tn nucleus taeniae
TO (chapter 1) optic tectum
TO (chapter 4) olfactory tubercle
TPc nucleus tegmenti pedunculopontinus, parscompacta
TPO area temporo-parieto-occcipitalis
TSM tractus septomesencephalicus
Va vallecula
VIA ventrointermediate area of the thalamus
VTA ventral tegmental area (A10); also seeAVT
VIP ventrointermediate area of the posteriornuclei
VP ventral pallidum
Literature 204
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236
Danksagung
Zuallererst möchte ich meinen Eltern und meiner Frau Ingelore danken, die mir immer alles
ermöglicht haben, und die mir nicht nur während der Zeit dieser Doktorarbeit den Rücken
freigehalten haben.
Meinem Betreuer und Chef Prof. Onur Güntürkün möchte ich dafür danken, dass er mehr Freund
als Chef war, und dass auch er mir fast immer alles ermöglicht hat. Ich glaube, dass ich - und
damit auch diese Arbeit - sehr von seiner offenen und kompetenten Art profitiert habe. Sowohl
die persönlichen und wissenschaftlichen Freiheiten die er mir eingeräumt hat, als auch die
gemeinsamen Diskussionen haben meine Arbeit immer wieder beflügelt.
Grossen Anteil am Gelingen dieser Arbeit haben natürlich auch alle Mitarbeiter und
Mitarbeiterinnen der AE Biopsychologie. Von vielen von ihnen habe ich immer wieder
fachliche oder persönliche Unterstützung erhalten. Das gilt vor allem für Markus „Fast Blue“
Hausmann, der mich jeden Tag ertragen musste; Thomas „Mauve Masher“ Kalt, der mich oft zur
Arbeit hin und meistens auch wieder zurück gebracht hat, wobei er auf beiden Wegen so
manchen Unmut abfangen konnte (und in der Zeit dazwischen auch); und Burkhard „Horst“
Hellmann, der mir mit dem einen oder anderen Tip und Trick ausgeholfen hat („aber dafür hat
man ja Post-docs“). Alle anderen von euch haben die Zeit nicht minder geprägt. Vielen Dank
deshalb auch an Bettina Diekamp, Martina Skiba, Martina Manns, Ariane Schwarz, Sabine
Kesch, und Christel Lehnert. Besonderen Dank auch an Daniel Durstewitz, mit dem ich nicht nur
viel Laborarbeit geteilt habe, sondern mit dem ich auch so manch´ anstrengende Diskussion
geschlagen habe. Jede einzelne davon war es wert!
Bei Prof. Hanns Hatt und meinem zweiten Betreuer PD Kurt Gottmann möchte ich mich dafür
bedanken, dass sie mir in ihrer Arbeitsgruppe am Lehrstuhl für Zellphysiologie eine „zweite
Heimat“ gegeben haben. Auch dort habe ich das Glück gehabt, nicht nur etwas lernen zu können,
sondern vor allem es im Umgang mit freundlichen und hilfsbereiten Menschen zu tun. Besonders
hervorgehoben seien dabei Gunnar, Markus, Ralf und Simon!
Dank auch an Prof. Toru Shimizu, dem ich die anatomischen „basics“ und eine tolle Zeit in
Florida zu verdanken habe, sowie Dr. Ulli Schall, der mir ebenfalls sowohl wissenschaftlich
als auch persönlich eine schöne Zeit in Australien verschafft hat.
Dank euch allen!
237
Lebenslauf
Sven Kröner
persönliche Daten und Schulausbildung
04.03.1971 geboren in Bochum (Nordrhein-Westfalen)
1977 - 1981 Besuch der Grundschule Castroper-Hellweg, Bochum
1981 - 1990: Besuch des Heinrich-von-Kleist Gymnasiums, Bochum:
Abitur im Mai 1990, ebenda
Juli 1998 Heirat mit Ehefrau Ingeborg Kröner, Las Vegas, Nevada
Universitäre Ausbildung
1990 - 1996 Studium der Psychologie an der Ruhr-Universität Bochum
Diplomarbeit: Der "präfrontale Cortex" von Tauben - Modell fürdie Chemoarchitektur kognitiver Prozesse
seit Sept. 1996 Wissenschaftlicher Mitarbeiter an der Fakultät für Psychologie,AE Biopsychologie, bei Prof. Onur Güntürkün
Sept. 1996 - Mai 2000 Promotion: „The caudolateral neostriatum of the avian forebrainand its modulation by dopaminergic afferents“Betreuer der Promotion:Prof. Dr. Onur Güntürkün, AE Biospychologie, RUBPD Dr. Kurt Gottmann, Lehrstuhl für Zellphysiologie, RUB
Tag der mündlichen Prüfung 29. 05. 2000
Forschungsvorhaben im Ausland
Juli - Dez. 1994 Forschungspraktikum an der University of South Florida, Tampa,FL, im Department of Experimental Psychology bei Prof. ToruShimizu: Neuroanatomische Untersuchungen zum caudalenVorderhirn von Vögeln
Dez. 1996 - Apr. 1997 Forschungsaufenthalt am Prince of Wales Hospital, Sydney,Australien, in der Biological Schizophrenia Research Unit bei Dr.Phillip Ward: Untersuchungen zum Einfluß von Amphetamin aufPrepulse-Inhibition und auditorische „Gating“-Mechanismen
Stipendien
Okt. 1996 Stipendium der G.A. Lienert Stiftung:
238
Erklärung
Die hier vorgelegte Dissertation wurde von mir selbst und ohne unerlaubte fremde Hilfe
angefertigt. Außer den in den Anmerkungen im Text und im Literaturverzeichnis genannten
Hilfsmitteln wurden keine weiteren benutzt. Bereits im Druck erschienene oder als Manuskripte
eingereichte Teile der Arbeit sind als solche gekennzeichnet.
Neben der „mit Auszeichnung“ bestandenen Diplom-Hauptprüfung im Fach Psychologie an der
Ruhr-Universität Bochum, habe ich bislang keine weiteren Prüfungen abgelegt.
Ich habe diese Dissertation weder in dieser, noch in irgendeiner anderen Form bereits
vorgelegt. Ich habe darüberhinaus bislang auch keine andere Dissertation vorgelegt.
Bochum, den 27. 03. 2000
Sven Kröner