Weil et al 2010 PNAS

8/13/2019 Weil et al 2010 PNAS

1/11


2/11


3/11

PNAS proofEmbargoed

ciated with a higher level of excitability and sexual arousal (Fig. 2).High arousal males exhibited more mounts before intromission(Fig. 2 A and E), and then fewer intromissions before ejaculating(Fig. 2 Band F), and they ejaculated more quickly after the rstintromission (Fig. 2 C and G). Additionally, the percentage ofmount attempts that was successful in leading to intromission was

signicantly lower among male mice from the high arousal line(Fig.2D andH). Thepattern of sexualbehaviorindicates that high-arousal males were excitable in an inappropriate manner, as indi-cated by the very low intromission:total mount ratio. Importantly,the temporal structure of the mating bout was similar between thelines as there were no differences in the latency to mount, intromit,or ejaculate between the genetic lines and between offspring highand low arousal groups (Fig. 3 AF). Supplementary videos illus-trate the differences in the patterns of sexual behavior .

Next, we asked the question, do increases in CNS arousaltranslate into increases in anxiety-like/exploratory behaviors?High and low mice of both sexes were tested on the elevated plusmaze and light-dark transition tasks. Interestingly, differences

were signicant according to the parental level of arousal but

were not systematically different among animals that differed inarousal themselves. In the elevated plus maze, mice from thehigh line exhibited an overall increase in anxiety-like behaviors (adecrease in exploration), as indicated by less time spent in theopen arms (Fig. 4A), a longer latency to rst enter an open arm(Fig. 4B), and an overall decrease in exploratory behavior asindicated by total arm entries (Fig. 4D). In all cases, females,independent of breeding type, also spent less time in the openarm and exhibited a longer latency to rst enter an open arm.There was not a consistent relationship between the offspringarousal scores and behavior in the elevated plus maze (Figs. 4EH). In the light-dark test, mice from the high line did notdiffer in the time spent in the light (Fig. 5A) but entered the dark

side of the box after a longer interval (Fig. 5B) and had sig-nicantly fewer transitions between the two sides of the box (Fig.5C). Again, only the parental arousal and not the offspring sarousal were predictive of behavior in the light-dark task (Fig. 5DF). Overall, animals from the high line exhibited moreanxiety-like behavior and a reduction in overall exploration.

Finally, to extract information about the most prominent fea-ture of the data gathered from our generalized arousal assay, weused a mathematical method called principal components anal-

ysis. This method is used here to analyze the relative con-tributions of motor, sensory, and emotional (fear) measures asthey inuence the largest, most elementary dimension of arousal.That is, the most generalized, elementary force operating in ourarousal assay is revealed by a forced one-component solution ofour data set (2). The most interesting comparisons to come out ofthe principal components analysis are illustrated in Fig. 6. Itdemonstrates the separate contributions of Motor Activity,Olfactory Responsivity, and Fear to Principal Component #1, thecomponent that quanties the most generalized, powerful forcegenerating behavior in these arousal assays. In Fig. 6, when a

measure has a (

) sign, that means that it was, indeed, groupedwith the forces on Principal Component #1 but in the reversedirection (lowvalues of that behavior are strongly associated withPrincipal Component #1s contribution to the production ofarousal-related behaviors). Principal Component #1 reects ahigh degree of motor activity. Our analysis raises the question of

whether the structures of arousal functions are the same in malesand females.

Fig. 6 shows that the major differences between HM and LMcome from the large contribution of motor activity of HM toPrincipal Component #1, as well as a difference in the con-tribution of fear. In fact, it is the failure of motor activity driven byPrincipal Component #1 that makes those males LM rather than

Fig. 2. High generalized arousal is associated with high

sexual arousal. All data are presented as mean (SEM). Total

number of mounts before rst intromission broken out by (A)

parental arousal, (E) offspring arousal, and (I) across both

conditions. The number of intromissions before the rst

mount for (B) parental arousal and (F) offspring arousal.

Latency to ejaculate after the rst intromission in animals

divided into (C) parental arousal and (G) and offspring

arousal and intromission: mount ratio (number of successful

intromissions/total number of mounts + intromissions) bro-

ken up by (D) parental arousal and (H) offspring arousal.

Differences are considered statistically signicant ifP< 0.05.

HM,n =6, LM, n = 6.

Fig. 3. Sexual behavior in high generalized arousal mice

retains temporal structure. All data are presented as mean

(SEM). Latency to mount (A, D, andG), latency to intromit

(B, E, and H), and latency to ejaculate (C, F, and I) do not

differ between the lines or based on offspring arousal. Dif-

ferences are considered statistically signicant if P < 0.05.

HM, n = 6, LM, n =6 .

Weil et al. PNAS Early Edition | 3 of 6

NEUROSCIEN

CE


4/11

PNAS proofEmbargoed

HM. HM have high movement rates and are skittish. Females aredifferent. The major difference between HF and LF comes fromthe strong reactivity of the HF to olfactory input. With respect tosex differences, between HM and HF there are large differencesin the contributions of olfactory responsiveness to PrincipalComponent #1, as well as fear. We speculate that an HF femaleready to mate, having spent much time in her burrow, will emergefrom her burrow just before ovulation. She must lack fear andlocomote extensively, spreading the odor of vaginal secretions, a

form of courtship behavior that encourages males to mate just asshe is ovulating. In turn, her powerful olfactory response will helpher choose healthy vigorous males as potential fathers of her litter(23, 24). Between LM and LF, the major difference is due to thefact that the LF had a large motor contribution to PrincipalComponent #1, as well as a smaller difference between LM andLF in fear. From this application of principal components anal-

ysis, we infer that the structure of the primary arousal componentis not the same in males as in females.

Discussion

These data provide evidence that genetically altering generalizedarousal has a profound inuence on sexual and exploratory/anxiety behaviors. The male sexual behavior in this study wascharacterized by HM animals being more excitable, showingmore rapid movements, exhibiting many premature and unsuc-cessful mounts before their rst successful penile insertion, andthen ejaculating rapidly after a minimal number of additionalintromissions. Thus, the ratio of intromissions to excited prein-

tromission mounts was signi

cantly lower in HM compared toLM animals. Interestingly, these results appeared whether ani-mals were sorted by their parentsor by their own arousal scores.

Additionally, selection for high levels of generalized arousalresulted in reduced exploratory behavior in the light-dark andelevated plus maze tasks. This result, surprisingly, was a functionof parental arousal scores and was independent of generalizedarousal scores in the tested animals themselves.

If generalized arousal has implications for specic arousalsubtypes, then it stands to reason that there should be physio-

Fig. 4. Selection for high generalized arousal induces anxiety-like behavior. All data are presented as mean (SEM). Mice from the high Ag lines time spent

in the both the open (A) and closed arms (B) and exhibited longer latency to enter an open arm (C) and exhibited fewer total arm entries (D). There was norelationship between the individual scores on the arousal assay ( EH). Differences are considered statistically signicant ifP< 0.05. HF,n = 22, LF,n = 22, HM,

n = 24, LM, n = 15.

Fig. 5. Selection for high generalized arousal alters light-

dark transition behavior. The lines did not differ in total

time spent in the light (or dark) (A) but high Ag animals

exhibited a longer latency to enter the dark side of the

chamber (B) and fewer overall light-dark transitions ( C).

There was no relationship between the individual scores on

the arousal assay arousal (DF). HF, n = 22, LF, n = 22, HM,

n = 24, LM, n = 15.

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.0914014107 Weil et al.
http://www.pnas.org/cgi/doi/10.1073/pnas.0914014107http://www.pnas.org/cgi/doi/10.1073/pnas.0914014107


5/11

PNAS proofEmbargoed

logical links between the two concepts. In the current work, wefocused on two specic heightened states of arousal: sex andfear. We also have recently delineated a pattern of increases infemale generalized arousal and sexual arousal following estrogenadministration (3, 25). This occurs both directly and also indi-rectly, via the induction of specic and generalized arousal-related neurochemicals. Indeed, a number of neurochemicalsignals that promote generalized arousal throughout the CNSalso promote sexual arousal in limbic structures (1, 26). Forinstance, the arousal-related neurotransmitter histamine is apotent arousal-promoting chemical that increases cortical activ-ity and inhibits the sleep promoting neurons of the ventrolateral

preoptic area. Male mice lacking the histamine synthesis enzymehistidine decarboxylase exhibited reduced mating behavior andprenatal exposure to antihistamines permanently impaired malesexual behavior (27, 28). Additionally, histamine in the ven-tromedial hypothalamus facilitates both electrical activity andlordosis behavior in female rodents (21, 29). Similarly, hypo-cretin peptides increase overall CNS arousal and microinjectionsof hypocretin into the medial preoptic area increase male sexualbehavior (22). The inverse example also provides evidence forthe same point, anesthetics administered before priming doses ofestradiol prevent the expression of lordosis behavior and theinduction of mating-related genes (30, 31) whereas amphetamineadministration facilitates estrogenic induction of mating (32).Taken together, these data forge a link between sexual behavior

and generalized arousal and indicate potential mechanisms bywhich genetic selection for high generalized arousal could impactspecic arousal states such as sex.

Genetic selection for high levels of generalized arousal resultedin reduced exploratory behavior in the light-dark and elevatedplus maze assays. A reduction in exploratory behavior in thesetasks could be conceptualized as indicative of anxiety-likebehavior (33). That is, high levels of generalized CNS arousalproduced greater home-cage locomotor activity but reducedexploratory activity in a novel environment (light-dark box andelevated plus maze). We infer that the increased locomotor drivein High arousal animals is more than offset by arousal-inducedincreases in anxiety-like states that would serve to suppressexploration. Importantly, although sexual behavior was affectedby both parental and offspring arousal, exploratory/anxiety-like

behavior was only altered by the arousal state of the parents.Exactly how this parental strain effect occurred remains tobe determined.

A linkage of the generalized arousal concept to specicbehavioral states is also implied by results with other systems.For instance, a circadian function that has received intensescrutiny is the regulation of sleep. Currently, more than 15% ofadults in the United States have some kind of sleep disorder.

Although the biological functions of sleep remain controversial,the locations and some of the properties of neurons turned offduring sleep have been charted (34). Ultimately, the regulationof sharp transitions between sleeping and waking may depend on

negative feedback loops among neurons in the hypothalamusand basal forebrain (4). Problems in the controls over arousalmechanisms manifest as sleep problems are extremely common(35), as are arousal problems related to depression (36) andstress (37). Further, Aston-Jones et al. (38) have been able toimplicate the activation of arousal-related hypocretin neurons inthe lateral hypothalamus in reward-seeking behaviors. For eachof these specic behavioral statese.g., sleep, stress, mood andreward-seekinggeneralized arousal provides the most ele-mentary, primitive neuronal force for the activation of behavior,and its exact behavioral impact is shaped by the specicenvironmental circumstance.

Alternative Interpretations. Our measures of arousal are all

dependent on locomotor activity as the principal readout.Therefore, it could be argued that articial selection for a genethat would tend to increase the overall level of locomotor activity

would be interpreted (or misinterpreted) as an increase in CNSarousal. However, the specic details of our arousal assay do notsupport such a simplistic explanation. Indeed, for the sensoryand fear components of the assay, we subtract backgroundactivity from the poststimulation responses to determinebehavioral reactivity. As an additional issue of interpretation, itshould be pointed out that differential sleep states are a poten-tial random confound for the sensory component of the assay.However, that confound is minimized by the presentation of therotational (vestibular) stimuli that is likely to awaken animalsbefore the olfactory stimulation that is the particular subtestupon which selection was based. In any case, in novel contexts

Fig. 6. Mathematical structure of generalized arousal isdifferent between males and females selected for divergent

levels of generalized arousal. Differential contributions of

motor, sensory (olfactory), and emotional (fear) measure-

ments to the most generalized force driving behavior in the

arousal assay, namely, Principal Component 1. The patterns

of these contributions differed between HM and LM,

between HF and LF, between HM and HF, and between LM

and LF. For example, LMs were low because motor meas-

urements did not drive their Principal Component 1. Fur-

thermore, HM had a high, positive contribution of fear to

Principal Component 1 (HF did not), but HM lacked HFs

strong contribution of olfactory responsiveness. Controls

using these same large sets of data scrambled and controls

using random numbers failed to yield similar patterns and

sharply reduced the percentage of the data explained by

Principal Component 1.

Weil et al. PNAS Early Edition | 5 of 6

NEUROSCIEN

CE


6/11

PNAS proofEmbargoed

like the elevated plus maze and light-dark box, the selection forhigher generalized arousal produces less overall exploratorybehavior as compared to Low-selected mice. Rather than simplyselecting for greater locomotor activity, the High mice appear tobe exhibiting greater generalized CNS arousal in concert withincreases in behaviors related to both fear and sex drives.

An additional alternative explanation is that we have produceddifferences in a single gene involved in the regulation of arousal-related neurotransmission. Because the arousal neurochemical

systems are reciprocally regulated and overlapping the alterationin a gene for one of these systems could induce increases ingeneralized CNS arousal and behavior. Further, we recognizethat we have presented results only from the most recent gen-eration; consequently, the one that showed the greatest quanti-tative separation between High Arousal and Low Arousal lines.Data from additional generations will become available in thecoming year. These lines may be useful to investigate the ana-tomical and genetic mechanisms that environmental pressureshave engaged to adjust generalized arousal across developmentalstages between sexes and over the 24 h light/dark time period.Future, studies will address the neurochemical, anatomical andgenomic differences between the High and Low lines.

In summary, mice genetically selected for high levels of gen-eralized arousal are producing a clear phenotype characterizedby greater home cage activity, sensory responsivity, and emo-tional lability. Interestingly, a High Arousal phenotype is asso-

ciated with exaggerated sexual arousal and reductions inexploratory behavior, when compared to mice selected for lowlevels of arousal. Taken together, these data provide support fora potential role for generalized arousal as a driver of specicmotivated behaviors.

Methods

Methods. All experiments were carried out in accordance with National

Institutes of Health Guidelines and followed procedures approved by The

Rockefeller University Institutional Animal Care and Use Committee.Thestrain used in this study wasderivedfroman extensively outbred stock,

Het-8, that resulted from an extensive intercross of more than eight outbred

strains followed by more than 60 generations of structured outbreeding (39).

Mice were housed in groups of four to ve same-sex siblings in standard

laboratory conditions with ad libitum access to food and ltered tap water.

All animals were housed in 12:12 light/dark cycles (lights on at 0600).

Breeding Procedure. The animals in this manuscript represent our sixth

generation of selectionfor both high andlow levelsof generalized arousal. In

each of the subsequent generations, mice were tested in the generalized

arousal assay and an overall arousal score was generated (see SI Textfor

descriptions of the behavioral tests). Briey, the total distance traveled over

the 24 h day in the home cage assay, the horizontal activity from the

olfactory stimulus, and the relativized change in vertical activity on the fear

training session were used as selection variables. Animals were rank-ordered

for their scores on each of these variables, the scores were added and the

animals with the most extreme scores (six highest and lowest) were selected

as the founders of the next generation.

1. Pfaff DW (2006) Brain Arousal and Information Theory: Neural and Genetic

Mechanisms (Harvard University Press, Cambridge, MA).

2. Gorsuch RL (1983)Factor Analysis(Lawrence Erlbaum Associates) .

3. Garey J, et al. (2003) Genetic contributions to generalized arousal of brain and

behavior.Proc Natl Acad Sci USA 100:1101911022.

4. Saper CB, Scammell TE, Lu J (2005) Hypothalamic regulation of sleep and circadian

rhythms.Nature437:12571263.

5. Fox MD, et al. (2005) The human brain is intrinsically organized into dynamic,

anticorrelated functional networks.Proc Natl Acad Sci USA 102:96739678.

6. Fox MD, Raichle ME (2007) Spontaneousuctuations in brain activity observed with

functional magnetic resonance imaging. Nat Rev Neurosci8:700711.

7. Vincent JL, et al. (2007) Intrinsic functional architecture in the anaesthetized monkey

brain.Nature 447:8386.

8. Saper CB, Chou TC, Scammell TE (2001) The sleep switch: Hypothalamic control ofsleep and wakefulness. Trends Neurosci24:726731.

9. Cruikshank SJ, Connors BW (2008) Neuroscience: State-sanctioned synchrony.Nature

454:839840.

10. Poulet JFA, Petersen CCH (2008) Internal brain state regulates membrane potential

synchrony in barrel cortex of behaving mice. Nature454:881885.

11. Martin EM, Pavlides C, Pfaff DW (2009) Multi-modal sensory responses of nucleus

reticularis gigantocellularis and the responses relation to cortical and motor

activation.J Neurophysiol, in press0 .

12. Leung CG, Mason P (1999) Physiological properties of raphe magnus neurons during

sleep and waking. J Neurophysiol81:584595.

13. Leung CG, Mason P (1998) Physiological survey of medullary raphe and magnocellular

reticular neurons in the anesthetized rat. J Neurophysiol80:16301646.

14. Lu J, Sherman D, Devor M, Saper CB (2006) A putativeip-op switch for control of

REM sleep. Nature441:589594.

15. Sutcliffe JG, de Lecea L (2002) The hypocretins: Setting the arousal threshold.Nat Rev

Neurosci3:339349.

16. Sherin JE, Elmquist JK, Torrealba F, Saper CB (1998) Innervation of histaminergic

tuberomammillary neurons by GABAergic and galaninergic neurons in theventrolateral preoptic nucleus of the rat. J Neurosci18:47054721.

17. Adamantidis AR, Zhang F, Aravanis AM, Deisseroth K, de Lecea L (2007) Neural

substrates of awakening probed with optogenetic control of hypocretin neurons.

Nature450:420424.

18. Rossato JI, Bevilaqua LRM, Izquierdo I, Medina JH, Cammarota M (2009) Dopamine

controls persistence of long-term memory storage. Science 325:10171020.

19. Harris GC, Aston-Jones G (2006) Arousal and reward: a dichotomy in orexin function.

Trends Neurosci29:571577.

20. Edwards CM, et al. (1999) The effect of the orexins on food intake: comparison with

neuropeptide Y, melanin-concentrating hormone and galanin. J Endocrinol 160:

R7R12.

21. Donoso AO, Broitman ST (1979) Effects of a histamine synthesis inhibitor and

antihistamines on the sexual behavior of female rats. Psychopharmacology (Berl)66:

251255.

22. Gulia KK, Mallick HN, Kumar VM (2003) Orexin A (hypocretin-1) application at the

medial preoptic area potentiates male sexual behavior in rats. Neuroscience 116:

921923.

23. Kavaliers M, Choleris E, Pfaff DW (2005) Recognition and avoidance of the odors of

parasitized conspecics and predators: Differential genomic correlates. Neurosci

Biobehav Rev29:13471359.

24. Kavaliers M, Choleris E, Pfaff DW (2005) Genes, odours and the recognition of

parasitized individuals by rodents.Trends Parasitol21:423429.

25. Ribeiro AC, Pfaff DW, Devidze N (2009) Estradiol modulates behavioral arousal and

induces changes in gene expression proles in brain regions involved in the control of

vigilance. Eur J Neurosci29:795801.

26. Lee AW, et al. (2006) Functional genomics of sex hormone-dependent

neuroendocrine systems: Specic and generalized actions in the CNS.Prog Brain Res

158:243272.

27. Chiavegatto S, Bernardi MM, de-Souza-Spinosa H (1989) Effects of prenatal

diphenhydramine administration on sexual behavior in rats. Braz J Med Biol Res 22:

729732.

28. Pr G, Szekeres-Barth J, Buzs E, Pap E, Falus A (2003) Impaired reproduction Q:11of

histamine decient (histidine-decarboxylase knockout) mice is caused predominantly

by a decreased male mating behavior. Am J Reprod Immunol50:152158.

29. Zhou J, et al. (2007) Histamine-induced excitatory responses in mouse ventromedial

hypothalamic neurons: ionic mechanisms and estrogenic regulation. J Neurophysiol

98:31433152.

30. Roy EJ, Lynn DM, Clark AS (1985) Inhibition of sexual receptivity by anesthesia during

estrogen priming. Brain Res 337:163166.

31. Quiones-Jenab V, Zhang C, Jenab S, Brown HE, Pfaff DW (1996) Anesthesia during

hormone administration abolishes the estrogen induction of preproenkephalin

mRNA in ventromedial hypothalamus of female rats. Brain Res Mol Brain Res 35:

297303.

32. Holder MK, et al. (2009) Methamphetamine Facilitates Female Sexual Behavior and

Enhances Neuronal Activation in the Medial Amygdala and Ventromedial Nucleus of

the Hypothalamus(Psychoneuroendocrinology) Q:12.

33. Rodgers RJ, Cao BJ, Dalvi A, Holmes A (1997) Animal models of anxiety: Anethological perspective.Braz J Med Biol Res 30:289304.

34. Siegel JM (2005) Clues to the functions of mammalian sleep. Nature437:12641271.

35. Mahowald MW, Schenck CH (2005) Insights from studying human sleep disorders.

Nature437:12791285.

36. Krishnan V, Nestler EJ (2008) The molecular neurobiology of depression.Nature455:

894902.

37. Zhou Z, et al. (2008) Genetic variation in human NPY expression affects stress

response and emotion.Nature 452:9971001.

38. Harris GC, Wimmer M, Aston-Jones G (2005) A role for lateral hypothalamic orexin

neurons in reward seeking. Nature437:556559.

39. Mclearn GE, Wilson JR, Meredith W (1970) The use of isogenic and hetergenic mouse

stocks in behavioral research. Contributions to Behavior-Genetic Analysis: The Mouse

as a Prototype, eds Lindzey G, Thiessen DD (Appleton-Century Crofts, New York), pp

122.

6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.0914014107 Weil et al.
http://www.pnas.org/cgi/data/0914014107/DCSupplemental/Supplemental_PDF#nameddest=STXThttp://www.pnas.org/cgi/doi/10.1073/pnas.0914014107http://www.pnas.org/cgi/doi/10.1073/pnas.0914014107http://www.pnas.org/cgi/data/0914014107/DCSupplemental/Supplemental_PDF#nameddest=STXT


7/11

Q: 1_Please contact [email protected] you have questions about the editorial

changes, this list of queries, or the figures in your article. Please include your manuscript number in

the subject line of all e-mail correspondence; your manuscript number is 200914014. Please (i)

review the author affiliation and footnote symbols carefully, (ii) check the order of the author

names, and (iii) check the spelling of all author names and affiliations. Please indicate that the

author and affiliation lines are correct by adding the comment OK next to the author line. Please

note that this is your opportunity to correct errors in your article prior to publication. Corrections

requested after online publication will be considered and processed as errata.

Q: 2_PNAS allows up to five key terms that (i) do not repeat terms present in the title (which is searchable

online) and (ii) do not include nonstandard abbreviations. You may add one term. Also, please check

the order of your key terms and approve or reorder them as necessary.

Q: 3_Please review the information in the author contribution footnote carefully. Please make sure that the

information is correct and that the correct author initials are listed. Note that the order of author

initials matches the order of the author line per journal style. You may add contributions to the list in

the footnote; however, funding should not be an authors only contribution to the work.

Q: 4_You will receive a notification from the PNAS eBill system in 1-2 days. Each corresponding author is

required to log in to the system and provide payment information for applicable publication charges

(purchase order number or credit card information) upon receipt of the notification. You will have theopportunity to order reprints through the eBill system if desired, as well. Failure to log in and provide

the required information may result in publication delays.

Q: 5_Reminder: You have chosen not to pay an additional $1275 (or $950 if your institution has a site

license) for the PNAS Open Access option.

Q: 6_The contributed by author must be the or one of the corresponding authors. Please edit

correspondence footnote as appropriate.

Q: 7_Please verify that all supporting information (SI) citations are correct. Note, however, that the

hyperlinks for SI citations will not work until the article is published online. In addition, SI that is notcomposed in the main SI PDF (appendices, datasets, movies, and "Other Supporting Information

Files") have not been changed from your originally submitted file and so are not included in this set

of proofs. The proofs for any composed portion of your SI are included in this proof as subsequent

pages following the last page of the main text. If you did not receive the proofs for your SI, please

contact [email protected].

Q: 8_Please provide these SI materials or delete this statement.

Q: 9_Please provide a publisher for ref. 2

AUTHOR QUERIES

AUTHOR PLEASE ANSWER ALL QUERIES 1
http://-/?-http://-/?-


8/11

Q: 10_Is ref. 11 in press or has it been published? Please clarify. If article has been published pleaseprovide volume and page numbers.

Q: 11_Please verify author name in ref. 28.

Q: 12_Please provide a place of publication for ref. 32.

Q: 13_Please verify figure legend against figure. Need to add labels?

AUTHOR QUERIES

AUTHOR PLEASE ANSWER ALL QUERIES 2


9/11

PNAS proofEmbargoed

Supporting Information

Weil et al. 10.1073/pnas.0914014107

Generalized Arousal Assay

Home Cage Motor Activity. Mice were housed in a home cagebehavior monitoring system (Accuscan Instruments) under

standard laboratory conditions. Each animal

s cage was sur-rounded with a set of infrared photobeams. Disruption of a beamwas recorded as an activity count. Data were collected with a PCusing Versamax software (Accuscan Instruments). We allowedmice to acclimate to this environment for up to 5 days and datafor analyses was taken from the last 4 days of behavioral re-cordings. During motor activity testing, total distance traveled(TD), horizontal activity (HA) and vertical activity (VA) werecollected in 60-min bins throughout the 24-h clock.

Sensory Testing. On the last day of home cage motor activitymonitoring,micewereexposed to a series of three typesof sensorystimuli in the following order: tactile, vestibular, and olfactory. Allstimuli were presented when mice were in a resting state,which

was when there was no home cage activity detected by the com-

puter (i.e., zero TD, HA, or VA) for at least 5 min. During thehome cage activity assay, disruption of a beam was recorded anddata collected for TD, HA, and VA. The stimuli were ad-ministered sequentially and were designed to use different sen-sorymodalities. First, the tactile stimulus consisted of a 2-s air puffdelivered from the cage lid. Second, an olfactory stimulus of theodor from 100% benzaldehyde (Sigma) was applied for a 20-sduration. The change in the mouses home cage activity wasmeasured until the animal reached a resting state again. Third, the

vestibular stimulus consisted of moving the cage in a circularmotion about its vertical axis on an orbital shaker (BarnsteadInternational) for an 8-s period at 90 rpm. Sensory testing oc-curred during the inactive period and was delivered by an auto-mated computer program so as not to confound the results withthe presence of human investigators. Data from total TD, HA,

and VA of the rst 10 min after the stimuli were collected.

Contextual Fear Conditioning. Automated cued fear conditioningwas carried out during the dark cycle. The test consists of onetraining session and one testing session spaced 24 h apart inside asound-attenuated chamber using an arousal assay apparatus. APlexiglas mouse shoebox cage tted with a metal shock grid ooris used instead of our standard home cages. The cage and gridoor are cleaned with 100% alcohol and dried before eachsession. This system trains and tests eight animals in parallel. Thetraining session lasts 10.5 min during which time, activity ismeasured continuously. After the initial 5-min baseline activity, a

30-s tone, accompanied during the last second by a 0.5-mA shock,is administered three times separated by a 1-min interstimulusinterval (ISI). Finally, the mouse was left in the apparatus for anadditional 2 min. Four hours later, the mouse was returned to theisolation box with grid oor cage and activity was measuredcontinuously. During this 13.33-min testing session, animals aregiven a 150-s acclimation period followed byve 30-s tones with100-s ISI and 100 s after the last tone. Comparisons of behaviordue to conditioning were made using the rst 150 s of the trainingday baseline (preshocks) to the total 150 s of the tone pre-sentations during testing day. Fear related activity is reported aspercent change from baseline.

Sexual Behavior Tests. Experimental male mice were removed fromtheir cages and placed in a clean cage with a free cycling femalemouse derived from the same original stock. Mating tests wereperformed for 90 min daily until the female exhibited behavioralsigns of sexual receptivity (the lordosis reex). Tests were per-

formed during the active period, recorded under photographicred light and analyzed ofine by an investigator unaware of thebreeding history of the animals. Latency and frequency of mounts,intromissions, and ejaculations were recorded.

Elevated Plus Maze.All experimental animals (males and females)were tested in the following tasks. The elevated plus maze waselevated 30 cm from the ground with a 5-cm2 center platform andfour radial arms (5 cm wide 30 cm long) with two closed armsencased by 5-cm2 high opaque walls (Rockefeller University In-strument Shop, New York, NY). Mice were placed in thecenter ofthe maze facing an open arm, allowed to explore undisturbed, and

videotaped for 5 min. Video tapes were scored ofine by an in-vestigator unaware of the breeding history of the animals.

Light-Dark Preference Test.The test apparatus consisted of a clearplastic box (50 50 35 cm) with a black (light opaque) covered-plastic box (50 25 25 cm)in one side (the dark side). The blackbox hadan open doorway (2 5 cm) thatled tothe light sideof theapparatus, which was illuminated by a 40-W white bulb (about 420lx Q:1on the oor). At the beginning of the tests, mice were placedinto the light side of the apparatus. For each mouse, the followingmeasurements were recorded for 5 min: total time spent in thedark compartment, total time in the light compartment, numberof transitions between the dark and light compartments, and thelatency to enter the dark compartment. Between the tests, theapparatus was thoroughly wiped clean.

Weil et al.www.pnas.org/cgi/content/short/0914014107 1 of 2
http://www.pnas.org/cgi/content/short/0914014107http://www.pnas.org/cgi/content/short/0914014107


10/11


11/11

Q: 1_Please define lx.

AUTHOR QUERIES

AUTHOR PLEASE ANSWER ALL QUERIES

Documents

Weil et al 2010 PNAS