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Regulating anxiety with extrasynaptic inhibition

A Corrigendum to this article was published on 25 November 2015

This article has been updated

Abstract

Aversive experiences can lead to complex behavioral adaptations including increased levels of anxiety and fear generalization. The neuronal mechanisms underlying such maladaptive behavioral changes, however, are poorly understood. Here, using a combination of behavioral, physiological and optogenetic approaches in mouse, we identify a specific subpopulation of central amygdala neurons expressing protein kinase C δ (PKCδ) as key elements of the neuronal circuitry controlling anxiety. Moreover, we show that aversive experiences induce anxiety and fear generalization by regulating the activity of PKCδ+ neurons via extrasynaptic inhibition mediated by α5 subunit-containing GABAA receptors. Our findings reveal that the neuronal circuits that mediate fear and anxiety overlap at the level of defined subpopulations of central amygdala neurons and demonstrate that persistent changes in the excitability of a single cell type can orchestrate complex behavioral changes.

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Figure 1: Fear conditioning enhances anxiety.
Figure 2: Bidirectional regulation of fear generalization and anxiety through optogenetic control of CEA PKCδ+ neurons.
Figure 3: Extrasynaptic inhibition mediated by α5-GABAARs controls the spontaneous firing of PKCδ+ neurons.
Figure 4: Experience-dependent reduction of extrasynaptic inhibition predicts fear generalization.
Figure 5: Brain area–specific conditional genetic deletion of α5-GABAAR expression induces fear generalization and anxiety behavior.
Figure 6: Brain area– and cell type–specific knockdown of α5-GABAAR expression induces anxiety behavior and fear generalization.

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  • 05 October 2015

    In the version of this article initially published, the grant number for U.R. was given as R01MH80006 instead of R01MH080006 and grants to J.M.C. from the National Institute of Neurological Disorders and Stroke (R01NS076517) and the National Institute of Mental Health (R01MH096463), US National Institutes of Health, were missing. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank all members of the Lüthi laboratory for discussions and critical comments on the manuscript. In particularly, we thank P. Tovote, S. Wolff, S. Ciocchi, F. Grenier, J. Gruendemann, C. Müller, K. Bylund and A. Loche for comments and for technical support, and W. Sieghart (Medical University of Vienna) and D. Anderson (California Institute of Technology) for antibodies and mice. This work was supported by the Novartis Research Foundation, by the National Center of Competences in Research program 'SYNAPSY — The Synaptic Bases of Mental Diseases' (financed by the Swiss National Science Foundation (SNSF)) as well as by an SNSF core grant to A.L. J.P.F. was supported by a NARSAD fellowship. C.X. was supported by an EMBO Long-Term Fellowship. P.S. and L.X. were supported by the Australian National Health and Medical Research Council. U.R. was supported by grant R01MH080006 from the US National Institute of Mental Health, National Institutes of Health. Funding for J.M.C. was provided by the National Institute of Neurological Disorders and Stroke (R01NS076517) and the National Institute of Mental Health (R01MH096463), US National Institutes of Health. Y.K. and F.F. were supported by Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung) Sonderforschungsbereich grant F44-17.

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Contributions

The manuscript was prepared by A.L. and P.B. Electrophysiological recordings in vitro, behavioral experiments and viral injections were performed by P.B. Pharmacology combined with single-unit recordings in freely moving animals were designed and performed by L.D. In vivo recordings from optogenetically identified neurons were performed by M.M. and J.P.F. Immunohistochemistry was accomplished by C.X. T.L. cloned the conditional shRNA plasmid. M.M.P. and J.M.C. provided the PWZ-029. L.X. tested the shRNA efficacy on α5GABAAR protein in HEK293T cells and P.S. provided the plasmid expressing shRNA. U.R. provided perfused brains of α5−/− mice and of α5fl/fl mice. All electron microscopy experiments were performed and analyzed by Y.K. and F.F.

Corresponding author

Correspondence to Andreas Lüthi.

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Competing interests

In the last 3 years, U.R. has received compensation for professional services from Concert Pharmaceuticals.

Integrated supplementary information

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 PKCδ+ 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 PKCδ+ 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, lightoff vs. lighton. d, Top, Cell-attached voltage recordings illustrating the yellow light-induced decrease in firing rate of a PKCδ+ 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 PKCδ+ 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, lightoff vs. lighton. All error bars indicate mean ± s.e.m.

Supplementary Figure 2 Modulation of open field anxiety, but not freezing, by manipulation of PKCδ+ 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 PKCδ+ 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 PKCδ+ 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 PKCδ+ 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 lighton and lightoff 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 PKCδ+ 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 lighton and lightoff 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 α5-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 α5-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 α5-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 GABAB receptor antagonist, on extrasynaptic inhibition in a PKCδ+ neuron (scale bar: 20 pA, 1 min). b, Bar graph illustrating lack of effect on extrasynaptic inhibition by the application of 1 or 10 μM CGP 52432 (n = 4 cells/ 4 slices/ 2 mice). Extrasynaptic inhibition is fully blocked by the application of 100 μM 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 μM SR-95531 and 100 μM PTX on extrasynaptic inhibition in a PKCδ+ neuron (scale bar: 20 pA, 1 min); Bottom, Enlarged traces for during baseline, or during application of 1 or 50 μM SR-95531 or 100 μM 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 μM SR-95531 or 100 μM PTX (1 μM SR-95531, n = 6 cells/ 6 slices/ 2 mice; 50 μM SR-95531, n = 15 cells/ 15 slices/ 6 mice; 100 μM 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 μM SR-95531, P = 0.130 for 50 μM SR-95531, P < 0.001 for each concentration of SR-95531. e, Left, Complete blockade of sIPSCs by application of 1 or 50 μM SR-95531 (1 μM: n = 6 cells/ 6 slices/ 2 mice; ***P < 0.001, Wilcoxon signed rank paired t-test; 50 μM: 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 μM; blue traces) or Picrotoxin (100 μM; 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 μM; blue trace) or PTX (100 μM; 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: Rin; *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 μM SR-95531, P = 0.130 for 50 μM SR-95531, P < 0.001 for 100 μM 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 μM, blue trace) or PTX (100 μM, red trace). Scale bar: 20 mV, 1 s. l, m, Application of PTX (100 μM; n = 10), but not SR95531 (1 μM; 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 μM SR-95531, P = 0.008 for 100 μM PTX. All error bars indicate mean ± s.e.m.

Supplementary Figure 4 Activation mechanisms of extrasynaptic GABAARs.

a, Example current trace (left), and bar graphs (right) illustrating the effect of the sequential co-application of bicuculline (20 μM; BIC), SR95531 (20 μM; SR) and picrotoxin (100 μM; 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 GABAARs contributes to the total extrasynaptic inhibition in PKCδ+ 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 PKCδ+ neuron during application of tetrodotoxin (1 μM; TTX). Scale bar: 20 pA, 1 s. Bottom, TTX significantly decreases sIPSC frequency in PKCδ+ 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 PKCδ+ neuron before (baseline) and during application of TTX (1 μM; 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 PKCδ+ 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 α5-GABAARs in CEA.

a, Example anti α5-GABAAR staining in CEA of a wild type mouse (Gabra5(+/+)). b, Anti- α5 staining is completely absent in slices from α5 deficient knock-out animals (Gabra5(-/-)). c–e, Double-staining against α5-GABAARs and CFP expressed in PKCδ neurons of PKCδ-Cre animals (PKCδ+ (CFP)) reveals co-localisation. Scale bar: 20 µm. f, Pie chart illustrating the percentage of PKCδ+ neurons expressing α5-GABAARs (n = 5 sections from 5 animals). All error bars indicate mean ± s.e.m.

Supplementary Figure 6 Pharmacology of extrasynaptic inhibition in PKCδ+ neurons.

a, Representative current traces recorded from PKCδ+ neurons showing the effect of the α5-GABAAR 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 3α,5α- tetrahydrodeoxycorticosterone (THDOC; 10 – 100 nM, middle trace), a modulator of δ-containing GABAARs, and of the agonist of α1-containing GABAARs 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 α5-GABAAR inverse agonist (L-655,708; 50 nM, 5 and 50 μM; 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 μM; 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 μM L-655,708 vs. 100 nM THDOC. F(5,48) = 12.90, P < 0.001 for 100 μM 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 PKCδ+ neuron before (baseline) and during application of PWZ-029 (1 μM). Scale bar: 20 pA, 400 ms. Bottom, Two different inverse agonists at α5-GABAARs (L-655,708 and PWZ-029) have no effect on sIPSC frequency in PKCδ+ neurons (L-655,708; 50 nM, 5 and 50 μM: 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 PKCδ+ a neuron before (baseline) and during application of PWZ-029. Scale bar: 8 pA, 8 ms. Bottom, Two different inverse agonists at α5-GABAARs (L-655,708 and PWZ-029) have no effect on sIPSC amplitude in PKCδ+ neurons (L-655,708; 50 nM, 5 and 50 μM, n = 6 – 9 cells/ 6 – 9 slices/ 4 – 5 mice, P = 0.144 – 0.208, sample t-test; PWZ-029, 1 nM and 1 μM, 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 α5-GABAAR-mediated extrasynaptic inhibition in PKCδ+ versus PKCδ neurons.

a, Top, Representative trace of sIPSCs recorded from a PKCδ neuron before (baseline) and during application of 100 nM or 1 μM PWZ-029. Scale bar: 30 pA, 300 ms. Bottom, α5-GABAAR-mediated extrasynaptic inhibition (expressed in pS/pF) in PKCδ neuron was significantly greater compared to PKCδ+ 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 α5-GABAAR expression in both PKCδ+ and PKCδ neurons of the CEl. PKCδ+ neurons were revealed by HRP-DAB immunoreactivity whereas immunometal (gold/silver) particles identified α5-GABAAR. Immunometal particles present at the plasma membrane (arrows) were in extrasynaptic locations. In both PKCδ+ and PKCδ 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 (PKCδ+: 0.17 ± 0.02, n = 70; PKCδ: 0.48 ± 0.08, n = 58; ***P < 0.001 Mann-Whitney rank sum unpaired t-test) and intracellular (PKCδ+: 3.4 ± 0.3, n = 70; PKCδ: 8.4 ± 0.8, n = 58; ***P < 0.001 Mann-Whitney rank sum unpaired t-test) immunometal particles in PKCδ+ and PKCδ dendrites collected from at least 2 tissue blocks/mouse (n = 2). All error bars indicate mean ± s.e.m.

Supplementary Figure 8 α5-GABAAR-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 PKCδ neurons before (baseline) and during application of 100 μM 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 PKCδ 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 PKCδ neurons and behavioral fear generalization in fear conditioned animals (n = 7; P = 0.188, R = 0.1). d, Representative current traces recorded from PKCδ+ neurons before (baseline) and during the application of 10 and 100 nM THDOC and 100 μM PTX in CS only and fear conditioned (CS-US) animals. e, THDOC had no effect on extrasynaptic inhibition in PKCδ+ 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 PKCδ+ 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), PKCδ+ 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 PKCδ+ 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), PKCδ+ 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 α5-GABAAR-mediated L-655,708-sensitive extrasynaptic inhibition compared to PKCδ+ 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 α5 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-Tdtomato-shRNA-KD, red) and the merge picture (yellow). c, Western blots from HEK293T cells transfected with rat α5 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 α5 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 α5 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-α5 antibody (Novus).

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Botta, P., Demmou, L., Kasugai, Y. et al. Regulating anxiety with extrasynaptic inhibition. Nat Neurosci 18, 1493–1500 (2015). https://doi.org/10.1038/nn.4102

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