Two-photon photostimulation and imaging of neural circuits

Volodymyr Nikolenko, Kira E Poskanzer & Rafael Yuste

Supplementary figures and text:

Supplementary Figure 1. Optical design of microscope

Supplementary Figure 2. Loading cortical slices with mag-Indo 1AM

Supplementary Figure 3. mag-Indo-1-AM loads mostly neurons

Supplementary Figure 4. Simultaneously acquired input maps from four neurons

Supplementary Figure 5. Analysis of all-optical stimulation experiments

Supplementary Note. Complex target uncaging

Supplementary Methods Note: In the version of this supplementary file originally posted online, several references were incorrect. The errors have been corrected in this file.

Nikolenko et al., Suppl. Figure 1, p.1

Supplementary Figure 1: Optical design of microscope.

Our system is a modification from a previously described custom two-photon

fluorescence (2PF) and second harmonic generation (SHG) microscope1, 2. Specifically,

the intensity of a beam from a NIR pulsed mode-locked Ti:Sapphire laser (1) is

controlled by a Pockels cell (2); the collimated beam is then focused by a plano-convex

lens (3a) onto a pinhole (3b) that serves as a spatial filter, and later re-collimated by a

second lens (3c); a diffractive optical element (DOE, 4) splits the laser beam onto five

individual beamlets (see also Figure 2 B) that spread at 0.23˚ inter-beamlet angle; this

angle is decreased by a second telescope (two plano-convex lenses 5a and 5c) that also

images the DOE onto the optical plane of scan mirrors while keeping individual beamlets

collimated (7b); an iris (5b) is placed in between the lenses 5a and 5c to switch between

single-beam raster-mode imaging (when it is closed it allows only for the central beamlet

to pass) and five-beamlet DOE vector mode imaging/photostimulation (when the iris is

completely opened); a 700nm long-pass filter (6a) removes residual visible-light radiation

Nikolenko et al., Suppl. Figure 1, p.2

from the laser and periscope mirrors (6) deliver the beam from the optical table plane to

the scanning unit (7) that consists of a holder for IR-reflecting mirror or dichroic (7a) and

galvanometer scan mirrors (7b); a scan lens (or “pupil-transfer” lens, 7c) mechanically

couples the scanning unit to the upright microscope and forms a telescope with a tube

lens of the microscope (8b) to deliver the collimated beam to the back aperture of the

objective (8c); it also and optically conjugates the scan mirrors and the objective. The

two-photon fluorescence (2PF) signal is collected by the same objective and is separated

from the excitation light by a short-pass dichroic (8a) and is detected by a PMT (9) after

being filtered through additional color filter(s) (9a) and focused onto a small active area

of the PMT by an additional lens (9b); a fast shutter (9c) protects the PMT from

overloading during uncaging pulses; alternatively 2PF or second-harmonic generation

(SHG) signals can be collected via the microscope condenser (8d) and detected by a

second PMT assembly (12). The electrical signal from the PMTs is amplified by pre-

amplifier(s) (10) and digitized by a data acquisition board (11).

1. Majewska, A., Yiu, G. & Yuste, R. A custom-made two-photon microscope and deconvolution system. Pflugers Arch 441, 398-408 (2000). 2. Nikolenko, V., Nemet, B. & Yuste, R. A two-photon and second-harmonic microscope. Methods 30, 3-15 (2003).

Nikolenko et al., Suppl. Figure 2, p.1

Supplementary Figure 2: Loading of cortical slices with mag-Indo 1AM.

Example of two-photon fluorescence image from a P13) somatosensory (S1) neocortical

slice, loaded with mag-Indo-1AM. The image was acquired in raster mode (725nm

excitation) with a 10x 0.3NA objective, and a single optical section in axial (Z) direction

is shown, with no additional scanning zoom. Scale 200μm. The pial surface is on the top,

and all cortical layers are visible.

Nikolenko et al., Suppl. Figure 3, p.1

Supplementary Figure 3: mag-Indo-1- AM loads mostly neurons.

Slices were simultaneously loaded with mag-Indo1-AM and Sulforhodamine 101

(SR101, 20 μM) and imaged at their respective wavelengths. (A) and (B) show images

of the same slice taken at 10x (0.3 NA) of mag-Indo1-AM (A; 725nm excitation,

480/540nm filter) and SR101 (B; 850 nm excitation, 595/615nm filter) across all cortical

layers. Scale 100μm. (C) and (D) show cells from the slices above at higher

magnification (60x, 0.9 NA). Note the higher density of mag-Indo1-AM-positive cells

(C) than SR101-positive cells (D) and overall low degree of overlap. Arrow in (D)

Nikolenko et al., Suppl. Figure 3, p.2

indicates a SR101 labeled cell (presumably an astrocyte) also loaded with mag-Indo1-

AM. Scale 10μm.

Nikolenko et al., Suppl. Figure 4, p.1

Supplementary Figure 4. Simultaneously acquired input maps from four neurons.

Example of mapping synaptic inputs by using quadruple recording from adjacent

neurons. Same experiments as Figure 3A. Four layer 5 pyramidal cells were patched

Nikolenko et al., Suppl. Figure 4, p.2

(white arrows point to locations of corresponding cell bodies) and four corresponding

input maps were acquired during a single run of the stimulation protocol. The

morphological reconstructions of dendritic trees for neurons A, B and D are

superimposed. Scale bar 100μm. (a-d) Input color-coded maps for all four neurons (color

scheme proportional to amplitude of EPSP, but reversed relative to figure 3a, in order to

enhance visibility on the dark background: lighter shades of orange correspond to larger

peak amplitude). (e, f) Representative maps of 2nd and 3rd order overlap between neurons

A&B and A&B&C respectively.

Nikolenko et al., Suppl. Figure 5, p.1

Supplementary Figure 5. Analysis of all-optical stimulation experiments.

High resolution plot of bottom panel from figure 6C.

Supplementary Note: Complex target uncaging

Glutamate uncaging in complex targets triggers action potentials

In previous uncaging experiments on dendritic spines, the effective radius of

glutamate photorelease triggered by two-photon excitation was very small (~2-3 µm in all

dimensions1, 2), so it became difficult to activate enough glutamate receptors to bring a

neuron to action potential threshold. Indeed, upon two-photon uncaging of individual

spines, the average size of recorded somatic events was less than 2mV (for 2-10ms pulses).

The size of these events depends on many factors: the duration of the uncaging pulse, the

concentration of the MNI-glutamate, the laser intensity and the local density of glutamate

receptors. The laser intensity is usually limited by a photodamage threshold (no more than

25-35mW on the sample per diffraction-limited spot with 0.8-0.95NA objectives, in our

experiments), and the concentration of caged neurotransmitter is limited by the solubility of

the compound in the water (10-50 mM of MNI-glutamate in case of local application

(“puffing”) as well as by the chemical purity (caged glutamate always has trace amounts of

free glutamate, and it is not practical to use more than 5mM of MNI-glutamate in the case

of bath application). Thus, the rate and amplitude of depolarization is effectively controlled

by diffusion of the photoreleased free glutamate towards glutamate receptors, and, since the

chemical reaction of uncaging itself is very fast3, possibly by the diffusion of MNI-

glutamate into the illuminated area.

By targeting neuronal somata instead of spines, we previously reported that it is

possible to use two-photon uncaging of MNI-caged glutamate to trigger action potentials in

cortical neurons4. Using long laser pulses (10-50ms) in positions close to the cell soma, we

were able to depolarize cells by 5-10mV, but it was difficult to reach the action potential

threshold. In addition, such long uncaging durations were capable of inducing

photodamage and also compromised the spatial resolution, by generating spill-over effects

at these durations.

To make neurons fire more reliably, a logical solution was to use several uncaging

locations and photorelease a larger amount of glutamate over a larger area. We thus

designed “complex targets” (Figure 2A) for simultaneous photostimulation and calcium-

imaging. A complex target consists of several uncaging “stimulation sub-targets” and one

“imaging sub-target” centered on the detected center of mass of the neuron. The beam is

sequentially placed at each stimulation location, at high laser intensity, then, for imaging, the

laser intensity is lowered to a lower level and the laser beam is positioned onto the imaging

target. For efficient uncaging with high NA objectives, laser power levels of approximately

30mW were used on the sample, whereas ~5mW on the sample were sufficient for point-

measurements of fluorescence signals, with a good signal-to-noise ratio and without any

detectable uncaging.

Spatial resolution of complex target uncaging

In the case of a circular arrangement of stimulation targets, as shown on Figure 2A,

the diameter of the stimulation targets pattern corresponded approximately to the size of a

typical neuronal soma. From our experience, this arrangement guaranteed repeatable

triggering of action potentials in practically all stimulated neurons with 30-50ms uncaging

pulses (the total duration of all stimulation sub-targets) and provided good spatial resolution

of photostimulation (Figure 2A, B). The actual parameters of the complex targets (number

of stimulation sub-targets, circular pattern diameter, duration for each sub-target and

uncaging power) were adjusted individually for different types of experiments, objective

lens magnification and NA.

References:

1. Matsuzaki, M., et al. Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat Neurosci 4, 1086-1092. (2001). 2. Araya, R., Jiang, J., Eisenthal, K.B. & Yuste, R. The spine neck filters membrane potentials. Proc. Natl. Acad. Sci. USA 103, 17961-17966 (2006). 3. Canepari, M., Nelson, L., Papageorgiou, G., Corrie, J.E. & Ogden, D. Photochemical and pharmacological evaluation of 7-nitroindolinyl-and 4-methoxy-7-nitroindolinyl-amino acids as novel, fast caged neurotransmitters. J Neurosci Methods 112, 29-42 (2001). 4. Nikolenko, V., Ellis-Davies, G.C.R. & Yuste, R. Simultaneous optical stimulation and two - photon imaging of neocortical circuits. in Society for Neuroscience Annual Meeting (Society for Neuroscience, New Orleans, 2003).

Nikolenko et al., Suppl. Methods, p.1

Supplementary Methods

Input mapping protocols

After developing a basic two-photon uncaging technique capable of stimulating

individual neurons, we used this method for mapping input connections onto a neuron, as

has been done with to one-photon glutamate uncaging photostimulation1-7. One limitation

of one-photon uncaging is the lack of spatial resolution in the lateral, and particularly in

the axial, direction. Although a certain level of spatial resolution is achievable with high-

NA objective lenses4, researchers often use the cylindrical profile of uncaging UV beam3

to generate two-dimensional maps. Thus, the maps generated do not have single cell

resolution, but rather reflect the activation of small territories.

The nonlinear nature of two-photon uncaging allowed us to overcome these

limitations and perform mapping with single-cell precision.

Photostimulation experiments proceeded as follows:

1. Acute slices were loaded with AM-dye (usually mag-Indo1AM); the area of interest

was identified.

2. One or several neurons were patch-clamped in whole-cell mode (in current clamp).

3. The objective lens was switched to a lower magnification one, to visualize all cortical

layers in a single field of view; a 2D raster-mode image was acquired.

4. The image was analyzed, individual neurons were detected, and center of mass

coordinates were loaded into a custom software program for sequential photostimulation

in pseudo-random order.

5. The photostimulation protocol was started. Each neuron was stimulated by 3-6 laser

pulses with ~0.5s interval between pulses. The total duration of the protocol was usually

approximately 25 min when stimulating 500 neurons.

Nikolenko et al., Suppl. Methods, p.2

6. Electrophysiological recordings were analyzed and events resembling synaptic EPSP

from a photostimulated cell (i.e. time-locked to uncaging pulses) were identified and

maps of input connections were generated.

Electrophysiological methods.

Neurons were patch clamped and the intracellular potential was monitored

simultaneously during imaging/uncaging. Whole-cell current-clamp recordings were

made using a BVC-700 (Dagan Corp., Minneapolis MN) or Axoclamp 700B (Axon

Instruments, Foster City CA) amplifiers. 6-10MΩ micropipettes were filled with (in

mM): 130K-methylsulfate, 10 KCl, 10 Na-HEPES, 2.5ATP-Mg, and 0.3GTP-Na and

0.35% biocytin, pH 7.4 (294-6mOsm), and 50µM fluorescent dyes Indo-1-K

pentapotassium salt (for calibration experiments) or Alexa-594 (for mapping experiments

to visualize the dendritic tree by using an additional 600 nm long-pass filter in front of

the PMT; both dyes are from Molecular Probes, OR). Neurons filled with biocytin during

recording were processed for morphological identification.

All experiments that require simultaneous photostimulation and calcium imaging

were performed in physiological temperature in order to study network activity in a

system that is as close to physiological state as possible. However, to improve the

duration of the whole-cell recordings, we carried out mapping experiments at room

temperature.

Histological methods.

Slices were fixed using a 4% paraformaldehyde solution in 0.12 M phosphate

buffer (PB). Following imaging/uncaging experiments, slices were submerged in room

Nikolenko et al., Suppl. Methods, p.3

temperature fixative and allowed to fix overnight at 4˚C. Slices were then rinsed in

0.12M PB 3 times, cryoprotected in 20% sucrose in 0.12M PB for 12–168 h and frozen

on dry ice in tissue freezing medium (H-TFM, Triangle Biomedical Sciences, Durham,

NC). Upon defrosting, slices were rinsed in 0.12M PB three times and pretreated with 1%

hydrogen peroxide in 0.12M PB for 30 min under agitation at room temperature. The

tissue was then rinsed in 0.02M potassium phosphate saline (KPBS) and incubated in

avidin-biotin-peroxidase complex (catalog number PK-6100, Vector Laboratories, Inc.,

Burlingame, CA) overnight under agitation at room temperature (10µl solution A and

10µl solution B per 1 ml of 0.02M KPBS and 0.3% Triton-X). Slices were rinsed in

0.02M KPBS 3 times and incubated in 0.7mg/ml 3,3'-diaminobenzidine, 0.2 mg/ml urea

hydrogen peroxide, 0.06M Tris buffer (D-4293, Sigma-Aldrich, St. Louis MO) in 0.02M

KPBS for 5–15 min. Upon completed 3,3'-diaminobenzidine reaction, slices were rinsed

in 0.02M KPBS and mounted in Vectashield mounting medium (H-1000, Vector

Laboratories, Inc.). Stained cells were visualized with DIC optics using an Olympus

upright microscope (BX51), and 71 neurons were reconstructed in 3 dimensions using the

Neurolucida workstation (MicroBrightField Inc., Williston, VT). All reconstructions

were done under a 100x, 1.40 NA objective.

References:

1. Callaway, E.M. & Katz, L.C. Photostimulation using caged glutamate reveals functional circuitry in living brain slices. Proc Natl Acad Sci U S A 90, 7661-7665 (1993). 2. Dalva, M.B. & Katz, L.C. Rearrangements of synaptic connections in visual cortex revealed by laser photostimulation. Science (New York, N.Y 265, 255-258 (1994). 3. Shepherd, G.M., Pologruto, T.A. & Svoboda, K. Circuit analysis of experience-dependent plasticity in the developing rat barrel cortex. Neuron 38, 277-289 (2003).

Nikolenko et al., Suppl. Methods, p.4

4. Kotter, R., Schubert, D., Dyhrfjeld-Johnsen, J., Luhmann, H.J. & Staiger, J.F. Optical release of caged glutamate for stimulation of neurons in the in vitro slice preparation. J Biomed Opt 10, 11003 (2005). 5. Dodt, H.U., Schierloh, A., Eder, M. & Zieglgansberger, W. Circuitry of rat barrel cortex investigated by infrared-guided laser stimulation. Neuroreport 14, 623-627 (2003). 6. Boucsein, C., Nawrot, M., Rotter, S., Aertsen, A. & Heck, D. Controlling synaptic input patterns in vitro by dynamic photo stimulation. Journal of neurophysiology 94, 2948-2958 (2005). 7. Shoham, S., O'Connor, D.H., Sarkisov, D.V. & Wang, S.S. Rapid neurotransmitter uncaging in spatially defined patterns. Nature methods 2, 837-843 (2005).