Supplementary Figure 1 Bidirectional optogenetic modulation of the tonic activity of CEA PKCδ + neurons in vitro. a, Top, Cell-attached voltage recording illustrating the blue light-induced increase in firing rate of a PKC + neuron infected with raav 2/7 EF1a::DIO-ChR2(H134R)-2A-NpHR-2A-Venus. Bottom, Enlarged traces showing firing rates at time points indicated by numbers. b, Rate histogram of the cell shown in the panel a. c, Blue light-stimulation reversibly increases the firing rate of ChR2-expressing PKC + neurons in acute CEA slices (n = 5 cells/ 5 slices/ 2 mice). *P = 0.009, Kruskal-Wallis One-Way ANOVA on Ranks followed by Tukey s multiple comparisons test, H = 9.380, 2 degrees of freedom, light off vs. light on. d, Top, Cell-attached voltage recordings illustrating the yellow light-induced decrease in firing rate of a PKC + neuron infected with raav 2/5 CBA::DIO-ARCH-GFP. Bottom, Enlarged traces showing firing rates at time points indicated by numbers. e, Rate histogram of the cell shown in the panel d. f, Yellow light-stimulation of reversibly decreases the firing rate of ARCH-expressing PKC + neurons in acute CEA slices (n = 6 cells/ 6 slices/ 2 mice). *P = 0.006, Kruskal-Wallis One-Way ANOVA on Ranks followed by Tukey s multiple comparisons test, H = 10.140, 2 degrees of freedom, light off vs. light on. All error bars indicate mean ± s.e.m.
Supplementary Figure 2 Modulation of open field anxiety, but not freezing, by manipulation of PKC + neurons. a, No difference in overall EPM track length during the baseline period (before optogenetic stimulation) between the three experimental groups (ChR2, n = 7 mice; Control, n = 9 mice; ARCH, n = 6 mice; P = 0.766 with one-way ANOVA). b, Optogenetic manipulation of PKC + neurons does not induce freezing in naïve animals in the open field arena (P = 0.656; paired t-test between on/off condition in ChR, t = -0.463, 8 degrees of freedom; P = 0.445; paired t-test between on/off condition in Control, t = -0.790, 12 degrees of freedom; P = 0.464; paired t-test between on/off condition in Arch, t = 0.769, 8 degrees of freedom). c, Blue light stimulation of ChR2-expressing PKC + neurons does not induced freezing behavior in a small arena (ChR2, n = 7 mice; P = 0.124; paired t-test, t = 1.789, 6 degrees of freedom). d, Time spent in the center of the open field arena is bi-directionally modulated by optogenetic manipulations of PKC + neurons (n = 7 mice each group; *P = 0.031, Wilcoxon signed rank test for ARCH, ***P < 0.001 for ChR2, paired t-test, t = 7.039, 6 degrees of freedom, for the control group P = 0.895 between light on and light off condition, t = -0.138, 6 degrees of freedom). e, Overall track length in the open field arena is bi-directionally modulated by optogenetic manipulations of PKC + neurons (n = 7 each group; ***P < 0.001 for ChR2, paired t-test, t = 7.383, 6 degrees of freedom, **P = 0.016, Wilcoxon signed rank test for ARCH, but for the control group P = 0.081 between light on and light off condition, t = 2.100, 6 degrees of freedom). f, Time spent in the center of the open field arena is reduced by the expression of 5 -shrna relative to scrambled control (n = 7 mice each group; **P < 0.001 by unpaired t-test for ChR2, t = 7.039, degrees of freedom; P = 0.895, paired t-test, t = -0.138, 6 degrees of freedom; *P = 0.031, Wilcoxon signed rank paired t-test for ARCH). g, Overall track length in the open field arena is reduced by the expression of 5 -shrna relative to scrambled control (n = 7 each group; *P = 0.011, paired t-test, t = 2.629, 12 degrees of freedom). h, Number of center crossings is reduced by the expression of 5 -shrna relative to scrambled control (n = 7 each group; ***P < 0.001, paired t-test, t = 5.564, 12 degrees of freedom). All error bars indicate mean ± s.e.m.
Supplementary Figure 3 Extrasynaptic inhibition controls excitability of PKCδ + neurons. a, Representative current trace illustrating the lack of the effect of CGP 52432, a GABA B receptor antagonist, on extrasynaptic inhibition in a PKC + neuron (scale bar: 20 pa, 1 min). b, Bar graph illustrating lack of effect on extrasynaptic inhibition by the application of 1 or 10 M CGP 52432 (n = 4 cells/ 4 slices/ 2 mice). Extrasynaptic inhibition is fully blocked by the application of 100 M PTX (n = 4 cells/ 4 slices/ 2 mice; ***P < 0.001, One-Way ANOVA on Ranks followed by Tukey s pairwise multiple comparison test, F = 22.394, 2 degrees of freedom). c, Top, Representative current trace illustrating the effect of 1 and 50 M SR-95531 and 100 M PTX on
extrasynaptic inhibition in a PKC + neuron (scale bar: 20 pa, 1 min); Bottom, Enlarged traces for during baseline, or during application of 1 or 50 M SR-95531 or 100 M PTX as indicated by the grey rectangles (scale bar: 20 pa, 300 ms). d, Bar graph illustrating effects on extrasynaptic inhibition by the application of 1 or 50 M SR-95531 or 100 M PTX (1 M SR-95531, n = 6 cells/ 6 slices/ 2 mice; 50 M SR-95531, n = 15 cells/ 15 slices/ 6 mice; 100 M PTX, n = 12 cells/ 12 slices/ 6 mice; ***P < 0.011, Mann-Whitney rank sum t-test). Sample t-test vs. control 0: P = 0.259 for 1 M SR-95531, P = 0.130 for 50 M SR-95531, P < 0.001 for each concentration of SR-95531. e, Left, Complete blockade of sipscs by application of 1 or 50 M SR-95531 (1 M: n = 6 cells/ 6 slices/ 2 mice; ***P < 0.001, Wilcoxon signed rank paired t-test; 50 M: n = 15 cells/ 15 slices/ 6 mice; ***P < 0.001, Wilcoxon signed rank paired t-test). f, Representative traces illustrating membrane potential depolarisation and action potentials elicited by current steps of different amplitudes under baseline conditions, or in the presence of SR95531 (1 M; blue traces) or Picrotoxin (100 M; red traces). g, Normalized firing rates plotted against the normalized amplitude of the injected current steps reveals a selective increase in the excitability in the presence of PTX (n = 10), but not SR95531 (n = 10). Scale bar: 20 mv, 400 ms. h, Representative trace illustrating membrane potential changes elicited by a 6 pa depolarizing current step before and after the application of SR95531 (1 M; blue trace) or PTX (100 M; red trace). Scale bar: 10 mv, 500 ms. i, j, Application of PTX (n = 6), but not SR95531 (n = 4) increases input resistance (left panel: R in ; *P = 0.010, Mann-Whitney rank sum unpaired t-test SR vs. PTX; middle: n = 4; P = 0.188, paired t-test for SR, t = -1.171, 16 degrees of freedom; right: n = 5; *P = 0.031, Wilcoxon signed rank paired t-test for PTX). Sample t-test vs. control 0: P = 0.259 for 1 M SR-95531, P = 0.130 for 50 M SR-95531, P < 0.001 for 100 M PTX. k, Example traces illustrating action potential firing elicited by injection of a Gaussian white noise current before and after the application of SR95531 (1 M, blue trace) or PTX (100 M, red trace). Scale bar: 20 mv, 1 s. l, m, Application of PTX (100 M; n = 10), but not SR95531 (1 M; n = 10) induces a depolarising shift in the resting membrane potential (left panel: RMP; *P = 0.044, paired t-test, SR vs. PTX, t = -2.270, 11 degrees of freedom; middle: n = 4; P = 0.428, paired t-test for baseline vs. SR, t = -0.841, 7 degrees of freedom; right: n = 5; *P = 0.008, paired t-test for baseline vs. PTX, t = -3.383, 9 degrees of freedom). Sample t-test vs. control 0: P = 0.214 for 1 M SR-95531, P = 0.008 for 100 M PTX. All error bars indicate mean ± s.e.m.
Supplementary Figure 4 Activation mechanisms of extrasynaptic GABA A Rs. a, Example current trace (left), and bar graphs (right) illustrating the effect of the sequential co-application of bicuculline (20 M; BIC), SR95531 (20 M; SR) and picrotoxin (100 M; PTX)(n = 6 cells/ 6 slices/ 2 mice). Application of BIC blocks both sipscs and, due to its inverse agonistic effect, induces a shift in the holding current. This shift is antagonised by subsequent application of SR95531 indicating that ligand-independent activation of extrasynaptic GABA A Rs contributes to the total extrasynaptic inhibition in PKC + neurons. Finally, application of PTX, which acts on a different site, completely abolishes extrasynaptic inhibition. Scale bar: 5 pa, 2 min. F (5,10) = 21.17, *P < 0.001, One-Way repeated measures ANOVA followed by Dunnett s multiple comparisons test. b, Top, Example trace showing sipscs recorded from a PKC + neuron during application of tetrodotoxin (1 M; TTX). Scale bar: 20 pa, 1 s. Bottom, TTX significantly decreases sipsc frequency in PKC + neurons (n = 7 cells/ 7 slices/ 3 mice; *P = 0.010, paired t-test, t = 3.698, 6 degrees of freedom). c, Top, Example trace showing averaged sipsc waveforms recorded from a PKC + neuron before (baseline) and during application of TTX (1 M; scale bar: 8 pa, 12 ms). Bottom, TTX has no effect on sipsc amplitude (n = 7 cells/ 7 slices/ 3 mice; P = 0.904 paired t- test, t = 0.201, 6 degrees of freedom). d, Bar graph illustrating the effect of TTX and PTX on extrasynaptic inhibition in PKC + neurons (*P = 0.016, ***P < 0.001, sample t-test). e, Regression plot reveals significant correlation between TTX-sensitive extrasynaptic inhibition (ps/pf) and TTX-induced reduction in sipsc frequency (n = 7 cells/ 7 slices/ 3 mice; P = 0.028; linear regression: R = 0.85). All error bars indicate mean ± s.e.m.
Supplementary Figure 5 Immunohistochemical staining of 5 -GABA A Rs in CEA. a, Example anti 5 -GABA A R staining in CEA of a wild type mouse (Gabra5 (+/+) ). b, Anti- 5 staining is completely absent in slices from 5 deficient knock-out animals (Gabra5 (-/-) ). c e, Double-staining against 5 -GABA A Rs and CFP expressed in PKC neurons of PKC - Cre animals (PKC + (CFP)) reveals co-localisation. Scale bar: 20 µm. f, Pie chart illustrating the percentage of PKC + neurons expressing 5 -GABA A Rs (n = 5 sections from 5 animals). All error bars indicate mean ± s.e.m.
Supplementary Figure 6 Pharmacology of extrasynaptic inhibition in PKC + neurons. a, Representative current traces recorded from PKC + neurons showing the effect of the 5 -GABA A R inverse agonist ethyl (13aS)-7- methoxy-9-oxo-11,12,13,13a-tetrahydro-9h-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate (L-655,708, 50 nm, left trace), of the neurosteroid 3,5 - tetrahydrodeoxycorticosterone (THDOC; 10 100 nm, middle trace), a modulator of -containing GABA A Rs, and of the agonist of α 1 -containing GABA A Rs benzodiazepine, N,N-dimethyl-2-(6-methyl-2-p-tolylimidazo[1,2-a]pyridin-3- yl)acetamide (Zolpidem, 20-300 nm, right trace) on extrasynaptic inhibition. b, Extrasynaptic inhibition (expressed in ps/pf) is reduced by the application of the 5 -GABA A R inverse agonist (L-655,708; 50 nm, 5 and 50 M; n = 6 9 cells/ 6 9 slices/ 4 5 mice). THDOC, 10 100 nm, and Zolpidem, 20 to 300 nm, have no significant effect (n = 8 cells/ 8 slices/ 3 mice). The total extrasynaptic inhibition is revealed by the application of picrotoxin (PTX, 100 M; n = 14 cells/ 14 slices/ 6 mice). *P = 0.019 for PWZ (100 µm), P = 0.024 for L- 655,708 (50 nm), **P = 0.004 0.009, ***P < 0.001, sample t-test versus the control baseline. P = 0.012, for 50 M L-655,708 vs. 100 nm THDOC. F (5,48) = 12.90, P < 0.001 for 100 M PTX vs. all other conditions, Kruskal-Wallis One-Way ANOVA on Ranks followed by Tukey s multiple comparisons test, H = 53.167, 10 degrees of freedom. c, Top, Representative traces showing sipscs recorded from PKC + neuron before (baseline) and during application of PWZ-029 (1 M). Scale bar: 20 pa, 400 ms. Bottom, Two different inverse agonists at 5 -GABA A Rs (L-655,708 and PWZ-029) have no effect on sipsc frequency in PKC + neurons (L-655,708; 50 nm, 5 and 50 M: n = 6 9 cells/ 6 9 slices/ 4 5 mice, P = 0.325 0.593, sample t-test; PWZ-029, 1 nm and 1 µm: n = 4 5 cells/ 4 5 slices/ 2 mice, P = 0.325 0.902, sample t-test). d, Top, Representative trace of averaged sipsc waveforms recorded from PKC + a neuron before (baseline) and during application of PWZ-029. Scale bar: 8 pa, 8 ms. Bottom, Two different inverse agonists at 5 -GABA A Rs (L- 655,708 and PWZ-029) have no effect on sipsc amplitude in PKC + neurons (L-655,708; 50 nm, 5 and 50 M, n = 6 9 cells/ 6 9 slices/ 4 5 mice, P = 0.144 0.208, sample t-test; PWZ-029, 1 nm and 1 M, n = 4 5 cells/ 4 5 slices/ 2 mice; P = 0.240 and P = 0.532, sample t-test). All error bars indicate mean ± s.e.m.
Supplementary Figure 7 5 -GABA A R-mediated extrasynaptic inhibition in PKC + versus PKC neurons. a, Top, Representative trace of sipscs recorded from a PKC - neuron before (baseline) and during application of 100 nm or 1 M PWZ-029. Scale bar: 30 pa, 300 ms. Bottom, 5 -GABA A R-mediated extrasynaptic inhibition (expressed in ps/pf) in PKC - neuron was significantly greater compared to PKC + neurons (n = 5 6 cells/ 5 6 slices/ 2 mice; *P = 0.021, paired t-test, t = 3.406, 6 degrees of freedom). b, Pre-embedding double-labeling electron microscopy detected 5 -GABA A R expression in both PKC + and PKC - neurons of the CEl. PKC + neurons were revealed by HRP-DAB immunoreactivity whereas immunometal (gold/silver) particles identified 5 - GABA A R. Immunometal particles present at the plasma membrane (arrows) were in extrasynaptic locations. In both PKC + and PKC - neurons, immunometal particles were more frequently observed within the cytoplasmic area, and in particular around mitochondria. Synapses are indicated by arrowheads. Scale bars: upper left panel 1 μm; all others 500 nm. c, Bar graphs show quantification for plasma membrane-associated (PKC + : 0.17 ± 0.02, n = 70; PKC - : 0.48 ± 0.08, n = 58; ***P < 0.001 Mann-Whitney rank sum unpaired t-test) and intracellular (PKC + : 3.4 ± 0.3, n = 70; PKC - : 8.4 ± 0.8, n = 58; ***P < 0.001 Mann-Whitney rank sum unpaired t-test) immunometal particles in PKC + and PKC - dendrites collected from at least 2 tissue blocks/mouse (n = 2). All error bars indicate mean ± s.e.m.
Supplementary Figure 8 5 -GABA A R-mediated extrasynaptic inhibition controls the firing of CEA neurons in vivo. a, Schematic representation of the technical procedure. Electrodes and glass pipette or a cannula were implanted in CEA allowing the recording of single units and application of PWZ-029 (PWZ) or vehicle in freely moving animals. b, Example time course histogram of the tonic firing of a CEA single unit (expressed in Hz) before, during and after vehicle application. Light grey bar marks the 10 min application of vehicle. Time bin: 1 min. c, Same as b but for the application of PWZ-029 application (light pink bar). d, Bar graph illustrating the change in firing rate as compared to pre-drug baseline (expressed in Hz) for application of vehicle (n = 8 units from 2 animals) or PWZ-029 (n = 6 units from 2 animals). ***P < 0.001, Mann-Whitney rank sum unpaired t-test. All error bars indicate mean ± s.e.m.
Supplementary Figure 9 Specificity of fear conditioning induced changes in extrasynaptic inhibition. a, Representative current traces recorded from PKC - neurons before (baseline) and during application of 100 M PTX in slices obtained from animals exposed to the CS only or from fear conditioned animals (CS-US). Scale bar: 20 pa, 5 s. b, Fear conditioning increased extrasynaptic inhibition in PKC - neurons relative to control animals (CS only: n = 8 cells/ 8 slices/ 5 mice; CS-US: n = 12 cells/ 12 slices/ 7 mice, **P = 0.008 Mann-Whitney rank sum unpaired t-test). c, There was no significant correlation between extrasynaptic inhibition in PKC - neurons and behavioral fear generalization in fear conditioned animals (n = 7; P = 0.188, R = 0.1). d, Representative current traces recorded from PKC + neurons before (baseline) and during the application of 10 and 100 nm THDOC and 100 M PTX in CS only and fear conditioned (CS-US) animals. e, THDOC had no effect on extrasynaptic inhibition in PKC + neurons (n = 5 cells / 5 slices/ 2 mice for each group, **P = 0.001, ***P < 0.001, repeated measures One-Way ANOVA, PTX vs. THDOC). f, Fear conditioning does not change the frequency or amplitude spontaneous inhibitory postsynaptic currents (sipscs) in PKC + neurons (n = 23 cells/ 8 mice each group; sipscs amplitude: P = 0.495, t = 0.689, 44 degrees of freedom, unpaired t-test between CS only and CS-US groups; sipscs frequency: P = 0.762, Mann-Whitney rank sum unpaired t-test between CS only and CS- US groups). g, Contextual fear conditioning increases EPM anxiety behaviour. Mice were subjected to five unsignaled USs (n = 11 mice) on day 1 and tested on the EPM on day 2. Similar to animals subjected to cued fear conditioning (CS-US), animals subjected to
contextual fear conditioning spent less time on the EPM open arms. h, Following the EPM test, US-only animals were tested for context discrimination (5 min in each context). Contextual fear generalization was quantified as the freezing ratio in a novel context (CTX - ) vs. the conditioning context (CTX + ). Animals exhibited an inverse correlation between the time spent in the open arms (expressed in %) and contextual fear generalisation (CTX - /CTX + ) (n = 11 mice; P > 0.001, R = 0.8, linear regression). i, Similar to animals subjected to cued fear conditioning (CS-US, n = 26 cells/ 26 slices/ 8 mice, Fig. 4), PKC + neurons recorded in slices obtained from animals subjected to contextual fear conditioning (US only, n = 6 cells/ 6 slices/ 3 mice) exhibited a reduction in the total PTX-sensitive extrasynaptic inhibition compared to PKC + neurons recorded in control animals (P < 0.001, Kruskal-Wallis One-Way ANOVA on Ranks followed by Dunn multiple comparison post hoc test, H = 23.935, 2 degrees of freedom, CS only vs. CS-US or vs. US only). j, Similar to animals subjected to cued fear conditioning (CS-US, 28 cells/ 28 slices/ 8 mice, Fig. 4), PKC + neurons recorded in slices obtained from animals subjected to contextual fear conditioning (US only, n = 6 cells/ 6 slices/ 3 mice) exhibited a reduction in 5 -GABA A R- mediated L-655,708-sensitive extrasynaptic inhibition compared to PKC + neurons recorded from control animals (CS only, n = 14 cells/ 14 slices/ 8 mice; F (2,42) = 10.66, **P = 0.004, Kruskal-Wallis One-Way ANOVA on Ranks followed by Dunn multiple comparison post hoc test, H = 11.597, 2 degrees of freedom, CS only vs. CS-US or vs. US only). All error bars indicate mean ± s.e.m.
Supplementary Figure 10 Cre-regulated knockdown of 5 subunits. a, structure of the plasmid expressing shrna in a Cre-dependent manner packed into a paav virus. The arrow indicates the direction of expression. The right expression of shrna is observed with Tdtomato expression. b, Bottom, Picture of transiently transfected P19 cells in cell culture with a plasmid expressing GFP and Cre (CMV-CRE-eGFP, green), TdTomato and shrna (EF1-DIO-U6-TdtomatoshRNA-KD, red) and the merge picture (yellow). c, Western blots from HEK293T cells transfected with rat 5 subunit and four different RNAi oligonucleotides (RNAi 5.1 to RNAi 5.4; lanes 2-5). dsred was co-transfected to mark transfected cells. Lane1 show cells transfected with α 5 alone, lane6 shows cells transfected with dsred alone and lane7 are non-transfected cells. Alpha tubulin was used as the loading control. Blots were probed with a 5 antibody. d, RNAi5.2 or a scrambled oligo (Scr) was cloned into a floxed lentilox and HEK 293 cells with transfected with or without Cre-recombinase. Transfected HEK293T cells were harvested 3 days after transfection and western blots probed with an anti- 5 antibody (Novus).