nature neuroscience: doi:10.1038/nn...supplementary figure 2 high-frequency optogenetic stimulation...

Supplementary Figure 1

Quantification of spontaneous EPSPs and paired-pulse parameters

(a) Representative voltage responses to current injections (left) and light pulses of varying intensities (right) in a mPFC L2 pyramidal

neuron from a mouse injected with rAAV5-CaMKIIα-ChETATC-eYFP in the ipsilateral BLA. (b) Average light dose-response curves of oEPSPs

evoked in regular-spiking (blue) and fast-spiking (red) neurons by 445 nm light pulses of increasing irradiance. (c) Spontaneous EPSP rates

before and after optical stimulation protocols (control, N = 10 cells; short oHFS, N = 11 cells, long oHFS, N = 19; individual points represent

single recordings. Averages are shown in black, green and blue for control, short and long oHFS, respectively (P = 0.74 t(9) =0.4234, P = 0.85

t(10) =1.9234 and P = 0.42 t(18) = 0.1795), respectively; paired two-tailed t-test). (d) Amplitude of first (left) and second (right) oEPSCs

during the paired-pulse protocol, taken before and after oHFS (left and middle). Grey lines represents average amplitude of 60 successfully

evoked oEPSCs in the individual cells before and after oHFS; red lines depict average over cells (N = 7 cells; oEPSC1: P = 0.0037, oEPSC2: P =

0.035; Paired two-tailed t-test). Paired Pulse Ratio is computed from 60 trials in which both light pulses successfully evoked an oEPSC (right;

P = 0.43, unpaired two-tailed t-test). (e) Failure rates, calculated as the percentage of 60 consecutive traces in which no oEPSC was evoked

within a 10 ms search window after light pulse onset (P = 0.03 and P = 0.12, respectively; unpaired one-tailed t-test). Error bars represent

s.e.m; * P < 0.05.

Nature Neuroscience: doi:10.1038/nn.4523


High-frequency optogenetic stimulation using ChETATC leads to rapid adaptation of synaptic release in BLA–mPFC terminals.

(a) Representative whole cell recordings of a layer 2/3 pyramidal neuron in mPFC acute slices during application of a single test light

pulse (top) and 100 Hz, 3 ms light pulse train for 9 s (long oHFS, bottom). Traces indicate that synaptic release rapidly adapts during

oHFS, indicating that depletion is unlikely to be a major cause of oHFS-induced synaptic depression. (b) Individual recording traces

(gray) and across-cell average (red) showing baseline-corrected Vm responses during oHFS (N = 10 cells), demonstrating similar post-

synaptic response to ChETATC-mediated stimulation of BLA-mPFC terminals using a single 3 ms light pulse stimulation (left) and a long

oHFS protocol (right; 900 3 ms pulses delivered at 100 Hz).



Optrode placement during recording experiments.

(a) Representative slice depicting electrode placement in the mPFC (scale bar: 500 m). (b) Schematic representation of optrode placement for the

fear-conditioning and recording experiment described in figure 3 (scale bar: 100 m). (c) Schematic representation of the final positions of optrode

drives following recording experiments with movable optrode. Electrode locations were estimated based on a dorso-ventral distance of 200-500

m from the tip of the optical fiber.



mPFC unit firing rates during oHFS trains and recovery of evoked responses.

(a) Within-train activity: average FR during a 9-second baseline period, followed by a 9-second oHFS train and a subsequent no-light period. PSTH

(Top) and raster plot (Center) depicting the spiking of all 57 responsive units before, during and after the first of 15 oHFS trains. (Bottom) Average

FR of the responsive units before, during and after the first oHFS train (ANOVA F(2,54)=2.531, P = 0.084; post-hoc tests: P = 0.553 and P = 0.068,

respectively.). Error bars represent s.e.m. (b) Histograms depicting change in evoked FR for each responsive unit, at each of the measured time

points, from T0 (top) to T90 (bottom). Change in evoked FR is measured for each unit against the evoked FR prior to oHFS. Binomial tests: T0,

0.000002; T30, 0.00025; T60, 0.003; T90, 0.124; corrected α=0.0125. (c) Left: Scatter plot depicting the correlation between the dorso-ventral (D/V)

location of the recording electrode (mm ventral from bregma) and the oHFS-induced change in evoked response (N = 57; R = 0.0029; P = 0.9836).

The change is calculated as (FR(T0)-FR(Tpre))/(FR(T0)+FR(Tpre)). Right: Mean change in light-evoked spiking in units within the PL (D/V < 2.6 mm) and

IL (D/V > 2.6 mm) regions (N(PL) = 24; N(IL) = 33; T(52) = -0.8433; P = 0.4029). Error bars in all panels indicate s.e.m.



oHFS effects do not back-propagate from mPFC to the BLA.

(a) Schematic representation of experimental setup. Mice were injected unilaterally in the BLA with rAAV5-CaMKIIα-ChETATC-eYFP and implanted

above the ipsilateral PL with an optical fiber. Eight weeks later, mice were anesthetized and an electrode array was lowered into the BLA. (b) Raster

plots overlaid with PSTHs showing the spiking of representative light-responsive BLA units (n = 48 light responsive units, out of 112 BLA units

recorded), before and immediately after oHFS. Test pulses were delivered to the mPFC at 5 Hz. Top: Representative short-latency response,

showing low jitter and minimal oHFS-induced attenuation. Bottom: Representative unit with longer latency response, showing larger jitter and

stronger oHFS-induced attenuation. (c) Within-train activity: average PSTH of FR of all light-responsive units (n = 48) during the first 9-second oHFS

train (bin size: 10 ms). (d) Bar plot depicting the mean FR of all responsive units before, during and after the first of the three oHFS trains. (e)

Average PSTHs for all 48 BLA units that showed responses to PFC light stimulation. Top: Average PSTH for responses to 5 Hz light stimuli (triggered

on light pulse onset, 300 individual light pulses); Bottom: Average PSTH for responses of the same 48 units to 100 Hz oHFS light stimuli (triggered

on light pulse onset, 300 first light pulses out of 900 during the first oHFS train). (f) A histogram presenting response latencies of all responses. (g)

oHFS-induced attenuation in evoked response plotted against the response latency for individual BLA units (N = 48). Population data (gray circles)

overlaid with mean and s.e.m. (black). Shading or error bars in all panels indicate s.e.m.



mPFC-projecting BLA cells predominantly target the mPFC.

(a) Representative CLARITY image from a mouse injected bilaterally with rAAV5-CaMKIIα-eYFP in the BLA. Scale bar, 1 mm. (b) Top: Representative

confocal images of coronal slices from a mouse injected in the BLA with rAAV5- CaMKIIα-eYFP. Slices are taken from anteroposterior positions -1.8,

+2.0, +1.5 and -1.2 (from left to right; distance in mm from bregma). Overlays indicate anatomical landmarks based on the mouse stereotaxic

atlas58

. Bottom: Representative confocal images of coronal slices from a mouse injected with HSV-EF1α-mCherry-IRES-Cre into the mPFC and

rAAV5-EF1α-DIO-eYFP into the BLA. Anatomical locations of slices are as indicated above. Scale bars: mPFC, 100 μm; all others 500 μm. (c)

Quantification of eYFP-labeled BLA afferents in mice injected with CaMKIIα-eYFP (blue, N = 5 mice) and HSV-Cre/DIO-eYFP (red, N = 6 mice).

Fluorescence in each region was normalized to the average fluorescence of BLA axons in all measured slices. Two-way ANOVA revealed a significant

experiment X region interaction (F(8,112)=2.47 p<0.05). A post hoc test showed that in HSV-Cre/DIO-eYFP mice the only significant differences

were between the mPFC and all other measured regions (all p<0.01), while in the CaMKIIα-eYFP controls, mPFC fluorescence differed significantly

only from three regions. Abbreviations: amPFC: anterior mPFC; pmPFC: posterior mPFC; LO: lateral orbitofrontal cortex; AcbSh: nucleus accumbens

shell; MDL: mediodorsal thalamus. LSI: Lateral Septal Nucleus. Hbn: Habenula. SNC: Substantia Nigra. PV: Paraventricular thalamus. Error bars in all

panels indicate s.e.m; * p < 0.05.



Unilateral BLA–PL synaptic depression has no behavioral effect during extinction of cued fear.

Behavioral data are collected from mice in which electrophysiological recordings were performed for Figure 3. Bottom: Schematic representation of

experimental setup. Recordings were performed 24 hours following cued fear acquisition. The baseline period consisted of 5 CS presentations.

oHFS trains were interleaved with 15 subsequent CS presentations. Left: Both groups no oHFS (N = 5) and oHFS (N = 5) pooled to show freezing

extinction main effect. Center: Unilateral BLA-PL synaptic depression during fear extinction (binned to four 5-tone bins). Right: Bar plot showing

freezing (%) to the tone in both groups during extinction. Shading or error bars in all panels indicate s.e.m.



Effects of BLA–PL short oHFS on conditioning and extinction of cued fear.

Bottom: Behavioral paradigm. Short-oHFS was immediately followed by cued fear conditioning. Extinction training was performed the next day in

context B and an extinction-retrieval test in context B was performed the next day. Far-Left: Freezing (%) of control (black; N = 10) and ChETATC

(blue; N = 8) mice measured during acquisition. Left: Freezing (%) of the two groups during extinction training shows extinction of freezing to the

tone. Center: Freezing (%) of the two groups during early (first 10 trials) and late (last 10 trials) extinction training (Two-way ANOVA group X time

bin interaction F(1,16)=6.6238 p<0.05, post hocs p=0.35, p<0.001 respectively). Right: Freezing (%) of the two groups during an extinction-retrieval

test, 24 hours following extinction training. Far-right: Freezing (%) of the two groups during early (first 8 trials) and late (last 7 trials) extinction-

retrieval (Two-way ANOVA main effect for group F(1,16)=2.9748, p=0.1038; group X time bin interaction F(1,16)=0.2748 p=0.6073 both N.S).

Shading or error bars in all panels indicate s.e.m; * p < 0.05.



Contextual fear memories acquired with cue and general anxiety are not affected by oHFS of BLA–mPFC projections.

(a) Freezing to the context after CS extinction - Bottom: Behavioral paradigm. oHFS was immediately followed by cued fear conditioning. Extinction

training in context B was conducted on the next day. Seven days later, mice were reintroduced to the fear conditioning context (context A). Left:

Freezing (%) during acquisition shows that mice in both groups increased freezing from first to last trials. Right: Following extinction of cued fear,

freezing (%) of the two groups to the fear-conditioned context shows no difference between the groups. (b) No effect of BLA-PL synaptic

depression on contextual fear response. Left: Schematic representation of surgical setup. Mice were injected bilaterally in the BLA with rAAV5-

CaMKIIα-ChETATC-eYFP and implanted above the PL with an optic fiber. Bottom: Behavioral paradigm; oHFS of BLA-PL axons was immediately

followed by contextual fear conditioning. Freezing was measured on the following day in the same context. Center: Freezing (%) of ChETATC (blue, N

= 10) and control mice (black, N = 9) during acquisition (ANOVA revealed main effect of trial F(5,85)=20.13 P < 0.0001, both group effect and group

× trial interaction N.S.). Right: Freezing responses of both groups to the fear-conditioning context were similar on the following day (t(17) = 1.34,

p=0.199 N.S). (c) Bottom: Behavioral paradigm; oHFS was followed by an open field test. Left: Locomotion velocity of mice that underwent oHFS of

BLA-PL projections (N = 10) and control mice (N = 10; t(18) = 0.064; P = 0.949). Right: Mean % time in center is not altered in the same two groups

of mice (t(18) = 0.718; P = 0.4918). Error bars represent s.e.m. (d) Bottom: Behavioral paradigm. oHFS was followed by elevated plus maze test.


Left: Control (N = 9) and ChETATC (N = 10) mice showed similar preference to the closed arms. A two-way ANOVA with stimulation and zone as

factors yielded a main effect for zone (F(2,34)=12.92 P < 0.0001). Post hoc analysis showed no effect of stimulation (F(1,17) = 0.038 P = 0.16). Right:

Mean velocity in the elevated plus maze was similar in control and ChETATC mice (t(17) = -1.581; P = 0.132). Error bars in all panels indicate s.e.m.



Fiber-placement schemes for mice in all behavioral experiments.

Attenuation of blue light from the average location of the fiber (top right, based on Stujenske et al. 2015, Cell Rep. Jul 21;12(3):525-34).

Line plot depicts the light irradiance profile through a vertical projection below the fiber center. Anatomical diagrams depict the fiber-

placement schemes for each of the behavioral experiments conducted in this study.


nature neuroscience: doi:10.1038/nn...supplementary figure 2 high-frequency optogenetic stimulation...

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