Hippocampal Somatostatin Interneurons Control the Size of Neuronal Memory Ensembles

Article

Reference

Hippocampal Somatostatin Interneurons Control the Size of Neuronal Memory Ensembles

STEFANELLI, Thomas, et al.

Abstract

Hippocampal neurons activated during encoding drive the recall of contextual fear memory.

Little is known about how such ensembles emerge during acquisition and eventually form the cellular engram. Manipulating the activity of granule cells (GCs) of the dentate gyrus (DG), we reveal a mechanism of lateral inhibition that modulates the size of the cellular engram. GCs engage somatostatin-positive interneurons that inhibit the dendrites of surrounding GCs. Our findings reveal a microcircuit within the DG that controls the size of the cellular engram and the stability of contextual fear memory.

STEFANELLI, Thomas, et al . Hippocampal Somatostatin Interneurons Control the Size of Neuronal Memory Ensembles. Neuron , 2016, vol. 89, no. 5, p. 1074-1085

DOI : 10.1016/j.neuron.2016.01.024 PMID : 26875623

Available at:

http://archive-ouverte.unige.ch/unige:86309

Disclaimer: layout of this document may differ from the published version.

Article Hippocampal Somatostatin Interneurons Control the Size of Neuronal Memory Ensembles

Highlights

d

Active neuronal populations inhibit non active neurons during memory formation

d

Optogenetic activation of GCs creates an artificial memory and abolishes natural recall

d

Non active neurons are excluded from the memory trace via lateral inhibition

d

Excitatory neurons activate SST+ interneurons and engage dendritic lateral inhibition

Authors

Thomas Stefanelli, Cristina Bertollini, Christian Lu¨scher, Dominique Muller, Pablo Mendez

Correspondence

pablo.mendez@unige.ch

In Brief

Stefanelli et al. show that the size of the cellular engram is determined by a competitive process in which active neurons inhibit the recruitment of neighboring cells by engaging somatostatin-expressing dendrite- targeting interneurons.

Stefanelli et al., 2016, Neuron89, 1074–1085 March 2, 2016ª2016 Elsevier Inc.

http://dx.doi.org/10.1016/j.neuron.2016.01.024

Neuron

Article

Hippocampal Somatostatin Interneurons

Control the Size of Neuronal Memory Ensembles

Thomas Stefanelli,¹Cristina Bertollini,¹Christian Lu¨scher,^1,2Dominique Muller,^1,3,4and Pablo Mendez^1,4,*

1Department of Basic Neurosciences, Faculty of Medicine, University of Geneva, Michel-Servet 1, 1211 Geneva-4, Switzerland

2Service of Neurology, Geneva University Hospital, Gabrielle-Perret-Gentil 4, 1211 Geneva-4, Switzerland

3Deceased, April 29, 2015

4Co-senior author

*Correspondence:pablo.mendez@unige.ch http://dx.doi.org/10.1016/j.neuron.2016.01.024

SUMMARY

Hippocampal neurons activated during encoding drive the recall of contextual fear memory. Little is known about how such ensembles emerge during acquisition and eventually form the cellular engram.

Manipulating the activity of granule cells (GCs) of the dentate gyrus (DG), we reveal a mechanism of lateral inhibition that modulates the size of the cellular engram. GCs engage somatostatin-positive interneurons that inhibit the dendrites of surrounding GCs. Our findings reveal a microcircuit within the DG that controls the size of the cellular engram and the stability of contextual fear memory.

INTRODUCTION

Memory acquisition starts when exploration of a new environ- ment drives synaptic activity in a small fraction of hippocampal neurons, triggering synaptic plasticity mechanisms that are believed to underlie long-term storage of memory (Martin et al., 2000; Malenka and Bear, 2004; Matsuzaki et al., 2004). Although synaptic plasticity occurring during memory formation has been extensively investigated in many brain areas (Whitlock et al., 2006; Maren and Quirk, 2004; Frankland et al., 2001; Nabavi et al., 2014), it remains unknown how memory is assigned to selected populations of neurons. How neuronal ensembles are chosen to encode a particular memory has implications for sta- bility and specificity of memory and ultimately determines the storage capacity of neuronal networks (Olshausen and Field, 2004).

Excitatory neurons in the mouse dentate gyrus (DG) are essen- tial for memory formation and retrieval (Pierson et al., 2015;

Kheirbek et al., 2013). The immediate early genecFoshas widely been used as a marker for neuronal activity in this brain region (Smeyne et al., 1992; Reijmers et al., 2007; Morgan and Curran, 1991) since its rapid and transient upregulation correlates with experience driven synaptic activity. New environment explora- tion induces the formation of an active neuronal ensemble defined by cFos expression in a distinct fraction of granule cells (GCs) (Deng et al., 2013; Tashiro et al., 2007; Liu et al., 2012).

When associated with an aversive stimulus such as in fear con-

ditioning paradigms, retrieval of the contextual memory requires the reactivation of the cFos-expressing ensemble of neurons (Tayler et al., 2013). As context information is permanently stored, the active neuronal ensemble that emerged during the exploration becomes both necessary and sufficient for the mnemonic representation of the context (Ramirez et al., 2013;

Tanaka et al., 2014; Denny et al., 2014) and is then referred to as the cellular engram.

The mechanisms ruling how fear memory is assigned to excitatory neurons in the amygdala involve the activity of the transcription factor cAMP response element-binding protein (CREB) that modulates cellular excitability ultimately governing memory allocation (Han et al., 2007; Yiu et al., 2014). Although much evidence has identified cell autonomous mechanisms that regulate the selection of neuronal ensembles (Rogerson et al., 2014), network mechanisms may also contribute to the for- mation of the cellular engram. Do active ensembles of neurons interact with silent neurons during memory acquisition? What factors limit the size of the engram? What are the consequences of this restricted recruitment for memory stability?

The activity of hippocampal excitatory projection neurons is controlled by a diverse population of inhibitory interneurons (INs) (Markram et al., 2004). For example, parvalbumin (PV)-ex- pressing inhibitory cells target soma and axon initial segment and thus control the action potential output of excitatory cells (Freund and Katona, 2007). In contrast, somatostatin (SST)-ex- pressing INs target dendrites and filter synaptic input to principal cells (Miles et al., 1996; Maccaferri, 2005). Recent work has pro- posed a role of inhibition in the neuronal computations per- formed in the hippocampus during fear memory formation (Lovett-Barron et al., 2014). Dendritic- and somatic-targeting INs are ideally situated to control synaptic activity flow in the hip- pocampal network (Mendez and Bacci, 2011), yet whether they play a role in determining the cellular engram and by extension in memory formation, remains elusive.

Here we investigate network mechanisms that govern the for- mation of the cellular engram and test its effects on memory stability. We find that memory is assigned to hippocampal GC populations by an activity-dependent process that regulates the size of the cellular engram and the stability of the associated memory. Active neurons engage local INs and inhibit surround- ing projection neurons excluding them from the engram. Alto- gether, our results reveal a role of specific inhibitory circuits in the network plasticity responsible for contextual fear memory.

RESULTS

Neuronal Activity during Spatial Exploration Determines the Active Ensemble

In order to test if neuronal activity affects the formation of the cellular engram, we bi-directionally controlled the activity of a random population of GCs in mice exploring a new environment.

We then quantified the size and distribution of the active neuronal ensemble using cFos.

We unilaterally infected the DG with adeno-associated viruses (AAVs) expressing the recombinase Cre under the control of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) promoter and a Cre-dependent Channelrhodopsin 2 (ChR2) and implanted a fiber optic cannula (seeExperimental Proceduressection). The use of the viral mix with the CaMKII promoter led to selective expression of ChR2 in a small fraction of dorsal DG excitatory cells (12.8% ± 1.3% of GCs at –2.0 mm from bregma, n = 12, Figures S1A and S1B) whose firing rate was reliable controlled by blue light (Figure S1C). Mice performed spatial exploration (SE) for 15 min in the presence (SE-Stimulated) or absence (SE-Control) of optogenetic stimulation (10 Hz pulses of 470 nm light during the entire duration of the exploratory phase).

When mice were killed and perfused 45 min later (Figure 1A), cFos+ neurons were more abundant in both SE groups compared to animals that remained in their home cage for the whole duration of the experiment (F(2,10) = 64.95, p < 0.001,Fig- ures 1B and 1C), confirming the induction of experience-depen- dent neuronal ensembles by SE. In addition, those mice that received optogenetic stimulation had a significantly higher num- ber of cFos+ GCs compared to unstimulated mice (Home cage 3 ± 2; SE-Control 27 ± 3; SE-Stimulated 39 ± 1 cFos+ cells/field, p < 0.01,Figures 1B and 1C). Without concomitant laser stimu- lation, most cFos+ neurons were ChR2 (Figure 1D, white bar), indicating a low degree of overlap between the two ensem- bles. In contrast, optogenetic stimulation during SE strongly enhanced cFos expression in ChR2+ GCs (F(3,14) = 93.56, p <

0.001,Figure 1D, striped bars). Importantly, this increase in the proportion of cFos+ neurons was at the expense of the cFos+

in ChR2 GCs that was reduced by optogenetic stimulation (Control 84% ± 3%, Stimulated 20% ± 5% of ChR2 /cFos+, p < 0.001). These results demonstrate that optogenetically acti- vated neurons add to the active neuronal ensemble while sup- pressing the recruitment of non-activated neighboring neurons.

We next silenced a fraction of GCs by unilateral stereotaxic in- jections of AAVs expressing the inhibitory designer receptor exclusively activated by designer drugs (DREADD) hM4D under the CamKII promoter. Clozapine-N-oxide (CNO) application silenced GCs expressing hM4D receptor hyperpolarizing the membrane by 7.9 ± 1.6 mV (Figures S1D and S1E). Mice ex- pressing hM4D receptor in GCs (25.7% ± 1.8% of GCs at –2.0 mm from bregma, n = 7,Figure S1D) received an intra- peritoneal (i.p.) injection of CNO (2 mg/kg) or saline 1 hr before being placed in the new environment. After 1 hr of SE, mice were killed and perfused and brains processed for cFos quanti- fication (Figure 1E). As in the previous experiments, SE increased the number cFos+ neurons in the GC layer of the DG compared with home cage animals (F(2,12) = 167.2, p < 0.001,Figures 1F and 1G) and even more so in mice where a fraction of GCs

were silenced (Home cage 3 ± 2; SE-Control 31 ± 1; SE-Silenced 50 ± 2 cFos+ cells/field, p < 0.001,Figure 1G). Despite this in- crease in absolute number of cFos+ neurons, the ratio of cells simultaneously expressing cFos and hM4D decreased (Figures 1F and 1H, striped bars,F(3,18) = 79.77, p < 0.001). These results indicate that silencing neuronal activity in a random set of GCs again increases the size of the active neuronal ensemble, but now by recruiting preferentially non-inactivated hM4D neu- rons. Altogether, these results show that altering the activity of a random set of neurons during SE increases the active neuronal ensemble size, either because cells are directly stimulated or because neighboring cells are silenced.

Manipulation of the Active Neuronal Ensemble Affects Contextual Fear Memory

We next determined if manipulating the active neuronal ensemble size is behaviorally relevant by testing for an effect on contextual fear memory and cellular engram activation.

Training for this memory test requires SE of a new context that is subsequently associated with an aversive stimulus (McHugh and Tonegawa, 2007). We bilaterally injected and cannulated mice for optogenetic activation of a random fraction of GCs dur- ing the training session of the contextual fear conditioning proto- col (Figure 2A, upper pannel). During recall, driven exclusively by contextual cues, mice that were stimulated during training showed much reduced freezing behavior compared with control mice (F(1,8) = 35.02, p < 0.001,Figure 2B). Optogenetic stimula- tion of GCs did not affect exploratory activity (Control 2.0 ± 0.12 m, Stimulation 1.75 ± 0.14 m, p = 0.28, n = 4 and 8,Fig- ure S2A), short-term memory, or shock reactivity (Figures S2B and S2C), and both groups spent equal time in freezing behavior during the training session (p = 0.99,Figure 2B). Moreover, opto- genetic stimulation in one context did not affect fear conditioning in another one (Figure S2D). These results suggest that optoge- netic activation of GCs during training disrupts long-term mem- ory performance.

To test if memory deficits arise from impaired activation of the cellular engram, we compared the size of the cFos+ ensemble induced after 1 week recall and after the training session.

Context-dependent recall 1 week after training induced similar number of cFos+ GCs compared to those detected immediately after training (Training 10 ± 2, Recall, control 11 ± 2,Figure 2C), suggesting comparable size of the acquisition active neuronal ensemble and recall cellular engram. In contrast, mice optoge- netically stimulated during training had lower number of cFos+

GCs after the 1 week recall session (Recall, stimulated 5 ± 2 cells/field, p < 0.05,Figure 2C). Consistent with the absence of optogenetic stimulation of GCs activity during recall, no differ- ence was observed in the number of ChR2+/cFos+ double stained GCs (Figure S2E) showing the lack of bias toward ChR2+ GC activation during context-dependent recall. These observations indicate that optogenetic stimulation of a random population of GCs during training impairs long-term memory recall by making contextual cues unable to drive cellular engram activation.

Is this memory trace irreversibly lost? To answer this question, we injected, cannulated, and trained as above another group of mice. However, in this case we performed on/off epochs of

additional optogenetic stimulation of GCs during the 1 week recall session (Figure 2A, lower pannel). Laser-mediated neu- ronal stimulation restored freezing behavior in mice that had received optogenetic stimulation of GCs also during training (Stimulated,F(1,7) = 120.10, p < 0.001,Figure 2E), although to slightly lower levels (Figure S2F). Light stimulation during recall had no effect in mice with unaltered neuronal activity during

training (Control,F(1,3) = 11.22, p < 0.05,Figure 2D). These re- sults suggest that optogenetic stimulation of a fraction of GCs during training disrupts natural memory but creates an artificial cellular engram by directing memory to activated neurons.

We next bilaterally silenced a fraction of excitatory neurons in the DG by injecting mice with AAV-CamKII-hM4D in the DG.

5 weeks later, mice received i.p. injections of saline (Control) or Figure 1. Neuronal Activity Regulates the Size of Active Neuronal Ensembles

(A) Mice expressing ChR2 in a fraction of GCs were placed in a novel cage with (Stimulated) or without (Control) optogenetic activation of a sparse population of excitatory cells of the DG. Mice performed spatial exploration (SE) for 15 min, returned to the home cage for 45 min, and were then perfused and brains processed for cFos analysis.

(B) Brain slices containing the DG upper blade were stained with cFos (green) to asses neuronal activation. ChR2 was tagged with mCherry (red) to allow visualization of infected neurons. Arrows: cFos+/ChR2+ GCs; arrowheads: cFos+/ChR2 GCs. Scale bar: 10mm.

(C) SE induced an increase in the number of cFos+ GCs compared with animals that remained in the home cage (gray bar), ***p < 0.001. Mice with optogenetic activation of GCs (SE-Stimulated, blue bar) showed increased number of cFos+ GCs with respect to control group (SE-Control, white bar), **p < 0.01, one-way ANOVA, Bonferroni corrected, four to five mice per group.

(D) Stimulated animals show a reduction in the fraction of non-co-localizing cFos+/ChR2 neurons (white bars) and an increase in the fraction of cFos+/ChR2+

neurons (striped bars), ***p < 0.001, one-way ANOVA, Bonferroni corrected, four to five mice per group.

(E) Mice expressing hM4D receptors in GCs were treated with saline (Control) or CNO (Silenced, 2 mg/kg) 1 hr before being placed in a novel cage. Immediately after 1 hr of SE, mice were perfused and brains were processed for histological analysis.

(F) Brain slices were stained with cFos (green) to asses neuron activation. hM4D+ GCs were filled with a soluble fluorescent protein (red) to allow visualization of infected neurons. Arrows: cFos+/hM4D+ GCs; arrowheads: cFos+/hM4D GCs. Scale bar: 10mm.

(G) SE induced an increase in the number of cFos+ GCs compared with animals that remained in the home cage (gray bar), ***p < 0.001. Mice with a silenced fraction of GCs (SE-Silenced, black bar) showed increased number of cFos+ GCs with respect to control mice (SE-Control, white bars), ***p < 0.001, one-way ANOVA, Bonferroni corrected, four to six mice per group.

(H) Silencing GCs decreased cFos expression among hM4D+ GCs and increased the fraction of hM4D /cFos+ neurons (white bars), **p < 0.01, one-way ANOVA, Bonferroni corrected, five to six mice per group. Error bars, SEM.

See alsoFigure S1.

CNO (Silenced) 1 hr before training in a contextual fear condition- ing chamber in order to silence a fraction of GCs exclusively during memory acquisition (Figure 3A). In the recall sessions per- formed 1 day and 1 week later, mice with silenced neurons dur- ing training showed increased freezing behavior (F(1,18) = 9.52, p < 0.01,Figure 3B).

CNO had no effect on freezing in uninfected mice (F(1,6) = 0.08, p = 0.78,Figure S3A). In addition, chemogenetic inactiva- tion of a fraction of GCs did not induce alteration in anxiety- related behaviors, such as the time spent in the open arms of the elevated plus maze (Figure S3B) and freezing levels during the training session (Figure 3B). Enhanced memory in mice with a silenced fraction of GCs at the time of training led to a larger proportion of cFos+ cells (p < 0.05,Figure 3C) but a decreased ratio of hM4D+/cFos+ double stained cells after recall session at 1 week (F(3,16) = 71.83, p < 0.001Fig- ure 3D). These experiments indicate that alterations of GC activity during training cause the artificial recruitment of behaviorally relevant neurons that contribute to contextual fear memory stability. Memory recall is driven by context- dependent neuronal activity when neurons were silenced during training but requires optogenetic stimulation of the engram in mice stimulated during training (see model onFig- ures S3C–S3E).

Interneurons Are Activated during SE

Our data suggest that the population of active GCs, which are re- cruited during memory formation, may suppress GCs in their vicinity (Figure S3F). The inhibitory effect of this interaction strongly suggests the involvement of GABAergic INs in a di-syn-

aptic network. According to this idea, silencing the activity of DG GABA neurons using hM4D receptors during SE increased the size of the active ensemble (Figure S3G). In order to identify the specific IN subtype activated during SE, we injected a virally encoded reporter of synaptic activity based on the promoter region of the Arg3.1/Arc gene (AAV-SARE-GFP) in the DG of mice (Figures S4A and S4B) (Kawashima et al., 2009). Compara- ble induction of endogenous Arg3.1/Arc protein was observed after brief periods of optogenetic-induced activity in SST and PV INs, validating the use of AAV-SARE-GFP to unbiasedly compare neuronal activity in different IN types (Figure S4C).

1 hr after SE, mice were perfused and the expression of the syn- aptic activity reporter GFP was assessed in GCs, PV+, and SST+

INs (Figure S4D). As expected, we found a consistent fraction of neurons in the GC layer expressing the synaptic activity reporter GFP (GC 7% ± 2% of infected neurons, n = 4,Figure 4A). For GABAergic INs, only a small percentage of PV+ neurons showed increased levels of GFP (3% ± 2% of PV+ neurons, n = 4,Fig- ure 4A) while a high proportion of SST+ cells expressed high levels of GFP (33% ± 7% of SST+ neurons, n = 4, Kruskal-Wallis test, p < 0.01,Figure 4A).

SST+ inhibitory neurons respond to the increase in synaptic activity induced by SE. Dendrites of SST+ INs are confined in the hilus where mossy axons from GCs form ‘‘en passant’’

synapses (Acsa´dy et al., 1998). In order to test if GCs activate microcircuits formed by SST+ INs, we optogeneti- cally stimulated a fraction of GCs during SE and analyzed cFos expression in SST+ cells. Most SST+ INs were cFos+

in stimulated mice in contrast with the low fraction in con- trol mice (Control 14 ± 3, Stimulated 77% ± 5% of SST+

Figure 2. Optogenetic Activation of a Frac- tion of GCs during Training Disrupts Natural Recall and Creates an Artificial Memory Trace

(A) Mice were trained in the fear conditioning set up with (Stimulated, blue bars) or without (Control, white bars) optogenetic stimulation of a sparse population of GCs. Mice were tested in the same context 1 day and 1 week after without opto- genetic stimulation (top, [B] and [C], contextual recall) or with epochs of light stimulation (30 s per minute, total test duration 3 min, bottom, [D] and [E], optogenetic recall).

(B) Quantitative analysis of fear responses driven by contextual cues during 1 day and 1 week recall sessions showed decreased freezing in mice with optogenetic activation of GC activity during training. Training p = 0.99, non-significant (ns); 1 Day: ***p < 0.001; 1 Week, ***p < 0.001, two-way ANOVA, Bonferroni corrected, five mice per group.

(C) Analysis of cFos expression GCs after the 1 week recall indicates that in mice trained with optogentic stimulation of GCs activity, recall of memory by contextual cues leads to a decreased number of cFos+ GCs. Shaded area represents the number of cFos+ (mean ± SD) in WT uninfected mice (n = 12) perfused 1 hr after the training session,t(8) = 3.33, *p < 0.05, five mice per group.

(D and E) Graphs show the freezing response during optogenetic stimulation epochs (blue circles) and non-stimulated epochs (white circles) 1 week after training.

Freezing behavior was increased during optogenetic stimulation with respect to non-stimulated epochs in ChR2 ([E], Stimulated, 1st epoch ***p < 0.001; 2nd epoch *p < 0.05; 3rd epoch **p < 0.01, two-way ANOVA, Bonferroni corrected, 8 mice) but not in mCherry expressing mice ([D], Control, 1st epoch, p = 0.81 non- significant (ns); 2nd epoch p = 0.08, ns; 3rd epoch p = 0.10, ns; two-way ANOVA, Bonferroni corrected, 4 mice). Error bars, SEM.

See alsoFigure S2.

population, p < 0.001,Figure 4B). This result suggests that SST+ INs are efficiently activated by sparse populations of GCs in the DG.

Chemogenetic effectors were then used to asses if perturbing the activity of SST+ INs had an effect on cFos expression in this IN population. SST-Cre mice performed 1 hr of SE while the ac- tivity of SST+ INs was increased or decreased by CNO-mediated activation of DREADD receptors expressed exclusively in DG SST+ INs (Figures S5A and S5B). High levels SST+ IN activity (Stimulated, hM3D) increased the expression of cFos in this IN population while decreased activity (Silenced, hM4D) lowered the proportion of cFos+ SST+ INs (Control 26 ± 6, Silenced 9 ± 4, Stimulated 47% ± 9% of SST+ neurons,F(2,17) = 6.75, p < 0.01, Figure 4C, left graph). Conversely, the number of cFos+ GCs was increased by SST+ IN silencing and decreased in mice with stimulated SST+ neurons (F(2,17) = 39.33, p < 0.01, Figure 4C, right graph). These results suggest that SST+neuronal activity bidirectionally controls gene expression and neuronal ensemble size.

SST+ INs Provide Inhibition to GCs Dendrites

We next characterized the connectivity of GCs in acute hippo- campal slice preparations using patch-clamp recordings.

Optogenetic stimulation of a sparse population of ChR2+ GCs produced a small glutamatergic response in ChR2 GCs, in line with weak recurrent excitatory connectivity. In contrast, light stimulation of ChR2+ GCs produced a large amplitude GABAergic response in the same neurons, suggesting the recruitment of INs (Mean amplitude GABA 163 ± 18, Glu 10 ± 3 pA,Figure 5A). The GABAergic response was abolished by the sodium channel blocker tetrodotoxin (TTX) (1mM) and the GABAA receptor antagonist SR 95531 (GBZ) (10mM) and

largely reduced by the wide-spectrum glutamate receptor blocker Kynurenic acid (Kyn) (6 mM,F(4,32) = 16.70, p < 0.001, Figures 5B and 5C). These results suggest that optogenetic stim- ulation of GCs activates downstream GABAergic neurons, which in turn inhibit neighboring GCs.

We next wanted to establish the origin of this form of lateral inhibition. We optogenetically stimulated GCs to induce poly- synaptic GABAergic responses (Lateral inhibition, CamKII) or PV+ or SST+ INs to elicit mono-synaptic inhibition from periso- matic or dendrite targeting INs, respectively (Figure S5C). In all three conditions, stimulation produced time-locked GABAergic responses of similar amplitude in the recorded ChR2 GC (PV 181 ± 55, SST 194 ± 44, CamKII 163 ± 18 pA;F(2,28) = 0.24, p = 0.78, Figure 5D). However, the kinetic of the responses was different. GABAergic currents originating in PV+ and SST+ INs had a shorter delay than those recorded after stimula- tion of GCs (PV 5.6 ± 0.5, SST 5.8 ± 0.3, CamKII 7.8 ± 0.5 ms;

F(2,28) = 6.72, p < 0.01,Figure 5D), accordingly with the poly- synaptic nature of the latter. The rise time of PV+ GABAergic current was shorter while rise slope was more than 2-fold higher when compared with SST+ currents (Rise time: PV 1.0 ± 0.1, SST 4.8 ± 0.5 ms; rise slope: PV 77.6 ± 14.3, SST 30.4 ± 10.2 pA/ms, Figure 5D), consistent with large electrotonic filtering of dendrite (SST) but not somatic (PV) originated cur- rents. GABAergic responses resulting from stimulation of excit- atory cells (CamKII) showed slower rise times compared with currents arising from PV+ INs but similar to those from SST+

INs (Rise time CamKII 6.0 ± 0.7 ms, rise slope CamKII 23.1 ± 4.4pA/ms; Kruskal-Wallis test, rise time p < 0.001, rise slope p < 0.01,Figure 5D). These results suggest that lateral inhibition elicited by optogenetic activation of GCs arises from SST+

dendrite targeting INs.

Figure 3. Inactivation of a Fraction of GCs during Training Improves Memory and In- creases the Size of the Recall Population by Preferentially Recruiting Non-Inactivated Neurons

(A) Mice with hM4D-expressing GCs were treated with saline (Control) or CNO (Silenced) 1 hr before fear conditioning. Mice were tested 1 day and 1 week later in the absence of the unconditioned stimulus and perfused for cFos analysis.

(B) Mice with a silenced fraction of GCs during training (Silenced, black bar) showed increased freezing levels with respect with saline (Control, white bar) injected mice, Training p = 0.99, non significant (ns); 1 Day, *p < 0.05; 1 Week, *p < 0.05, two-way ANOVA, Tukey HSD post hoc, 9, 11 mice per group.

(C) Mice with silenced GCs showed a larger pop- ulation of cFos+ GCs induced by 1 week memory recall,t(8) = 2.23, *p < 0.05, five mice per group.

Shaded area represents the number of cFos+

(mean ± SEM) in WT uninfected mice perfused 1 hr after the training session.

(D) Silencing a fraction of GC during training in- creases cFos expression among hM4D neurons (white bars) in the recall population, ***p < 0.001, one-way ANOVA, Bonferroni corrected, five mice per group. Error bars, SEM.

See alsoFigure S3.

SST+ INs Regulate Memory Strength and Distribution We next tested whether SST+ IN activity control experience- dependent ensemble size and memory formation by testing for an effect in contextual fear memory and cellular engram activa- tion. Chemogenetic modulation of SST+ IN activity during the training session had no effect on freezing behavior (Figures 6A and 6B, p > 0.99). In contrast, mice where SST+ INs were silenced during training showed enhanced freezing behavior

during recall 1 day and 1 week later (Figure 6B, p < 0.01, black bars). Conversely, when activity in SST+ INs was increased dur- ing training, freezing during recall was lower than in control mice during the 1 week test session (F(2,35) = 19.63, p < 0.001,Fig- ure 6B, gray bars).

Bidirectional modulation of behavior by SST+ IN activity was reflected by the number of cFos+ GCs detected after the last recall session. Mice with silenced SST+ INs showed a larger Figure 4. Dendrite Targeting Interneurons and GCs Are Preferentially Activated during Spatial Exploration

(A) Mice carrying a fluorescent reporter of synaptic activity (AAV-SARE-GFP) in the DG were perfused after 1 hr of SE. GFP expression was assessed by confocal imaging in immunohistochemically defined populations of PV+ (blue, left) and SST+(blue, right) INs. Two representative examples are shown for each IN pop- ulation. The graph shows quantitative analysis of the fraction of GCs (asterisks in images), PV+ (left), SST+ INs (right) that are positive for the synaptic activity marker GFP (arrows), **p < 0.01, one-way ANOVA, Dunn post hoc test, 4 mice per group. Scale bar: 5mm.

(B) WT mice performed SE with (Stimulated) or without (Control) stimulation of a fraction of GCs for 15 min and were perfused 45 min later. SST and cFos were simultaneously immunodetected. Optogenetic activation of GCs greatly increased cFos expression in hilar SST+ interneurons,t(7) = 8.16, ***p < 0.001, 3 and 6 mice per group. Scale bar: 5mm.

(C) SST-Cre mice expressing silencing hM4D or stimulating hM3Dq DREADD receptors in SST+ INs received saline (Control) or CNO injections and 1 hr before SE. Images show staining of cFos (green) in hilar SST INs. Silencing SST+ INs (Silenced, hM4D) decreased while activating SST+ IN (Activated, hM3D) increased cFos expression in this IN population (left graph). Conversely, the number of cFos+ GCs after 1 hr SE was higher in mice with silenced SST+ INs (hM4D) but was reduced in mice with increased SST+ IN activity (hM3D, right graph), ***p < 0.001, **p < 0.01, one-way ANOVA, Bonferroni post hoc test, 9, 5, 6 mice per group.

Scale bar: 5mm. Error bars, SEM.

See alsoFigures S4andS5.

population of cFos+ GCs while increased SST+ IN activity at the time of training reduced the number of cFos+ GCs when compared to control mice (F(2,30) = 19.93, p < 0.001,Figures 6C andS6E).

We performed similar experiments in PV-Cre mice to test the role of PV+ IN activity in memory formation. Chemogenetic acti- vation of hM3D expressing PV+ INs during SE increased the frac- tion of these neurons expressing cFos (Control 25 ± 10, Active 69% ± 6% of PV+ neurons, p < 0.01,Figure S5E) and caused sig- nificant membrane depolarization and spiking activity (Fig- ure S5D), suggesting effective modulation of neuronal activity in this IN population. However, increased PV+ IN activity during training in the contextual fear memory test resulted in no differ- ence in the time spent in freezing behavior in any of the recall sessions (1 day, 1 week,F(1,9) = 1.40, p = 0.27; Figure 6E).

Similar number of cFos+ neurons was counted in the GC layer after the last recall session in both groups (p = 0.27,Figure 6F).

In line with these results, optogenetic activation of PV+ neurons during training did not alter freezing levels during test sessions (F(1,12)<0.01 p = 0.99, Figure S6A) or the number of cFos+

GCs after SE (Figurse S6B and S6C). These results suggest that SST+ but not PV+ IN activity during training bidirectionally modulates long-term memory and the size of cellular engram.

The effect of SST+ IN activity in freezing behavior after condi- tioning could reflect an influence of this IN type on fear generalization. We addressed this question by testing whether hM4D-mediated inactivation of this IN type during training affects freezing behavior in a different context than the one used for training. Although inactivation of SST+ INs increased freezing levels during subsequent exposition to fear conditioning chamber, it did not affect fear response when mice were exposed to a different unrelated context (Figure S6D). Chemoge- netic modulation of SST+ IN activity did not alter performance of mice in the elevated plus maze, excluding a role of anxiety like

behavior in the increased freezing response observed in mice with decreased SST activity (Figure S6D). These results show that enhanced memory performance upon SST+ INs inactivation during training is specific for the training context and suggest that SST+ INs do not influence fear generalization.

DISCUSSION

Our study identifies lateral inhibition between GCs as a mecha- nism that constrains the ensemble of activated neurons during SE of a new environment. In contextual fear paradigm, the lateral inhibition determines the size of the cellular engram and the sta- bility of the memory trace.

Several factors may limit the size of the active neuronal en- sembles that emerge in the DG during SE. First, the number of GCs is several fold higher than the number of upstream excit- atory neurons in the entorhinal cortex (Schmidt et al., 2012). Sec- ond, GCs’ dendrites strongly attenuate synaptic inputs so that the cell fires only when many inputs are concomitantly active (Krueppel et al., 2011). In addition, our results now reveal the ex- istence of lateral inhibition among GCs. This inhibitory interaction maintains the low level of neuronal excitation in cells not imme- diately active during SE that is typically required for DG function (Treves and Rolls, 1994).

As memory formation progresses, naturally formed neuronal ensembles acquire engram properties (i.e., they become neces- sary and sufficient to evoke specific memories) (Tanaka et al., 2014; Denny et al., 2014). Previous studies have manipulated the memory trace associated with naturally formed neuronal en- sembles (Han et al., 2009; Ramirez et al., 2013; Cowansage et al., 2014). Here, we succeed to assign fear memory to a neuronal ensemble that was artificially generated in the hippo- campus without specific context representation. Optogenetic stimulation of this subset of neurons at the time of training Figure 5. Sparse Population of DG Excit- atory Cells Activate Strong Dendritic Target- ing Lateral Inhibition

(A) Brief light stimulation of a sparse population of ChR2+ GCs induced a large amplitude GABAergic current (GABA) and little glutamatergic response (Glu) in ChR2 GCs. Quantification of the ampli- tude of GABAergic and glutamatergic currents is shown in C (black bars). Scale bars: 20 pA, 50 ms.

(B) GABAergic response before (black trace) or after pharmacological treatment with SR95531 (Gabazine, [GBZ], 10mM), tetrotodotoxin ([TTX], 1mM), and kynurenate ([Kyn], 6 mM) (gray traces).

Scale bars: 20 pA, 50 ms.

(C) Quantification of GBZ, TTX, and Kyn treatment on GABAergic current amplitude (white bars).

***p < 0.001, n = 17, 5, 4, 4, and 7 for GABA, Glu, GBZ, TTX, and Kyn, respectively; one-way ANOVA, Bonferroni post hoc test.

(D) Representative normalized traces of GABAergic response induced by optogenetic activation of PV (light gray), SST (dark gray), and CamKII population (Black). Blue arrow shows the onset of light stimulation. Graphs show the average peak amplitude, delay, rise time, and slope of responses obtained upon stimulation of three different neuronal populations. *p < 0.05, **p < 0.01, ***p < 0.001, n = 7, 7, and 17 for PV, SST, and CamKII, respectively; one-way ANOVA, Bonferroni (peak amplitude and delay) or Dunn (rise time and slope) post hoc tests. Scale bar: 5 ms. Error bars, SEM.

See alsoFigure S5.

determines the allocation of an artificial memory trace to the active fraction of neurons. However, changes in reactivation of the ensembles due to rapid uncoupling of the context and the fear response by the non-naturalistic stimulation pattern may cause memory extinction and reduced optogenetic memory retrieval (Figure S2F).

Figure 6. Dendritic but Not Somatic Target- ing INs Control the Size of the Active Neuronal Population and Memory For- mation

(A) Mice expressing DREADD receptors in SST+

INs received saline (Control) or CNO injections in order to decrease (Silenced, hM4D) or increase (Stimulated, hM3D) SST+ IN activity during training. Mice were tested 1 day and 1 week later.

1 hr after the last test, mice were perfused for cFos analysis.

(B) Chemogenetic inactivation of SST INs during training (Silenced, hM4D) increased while activa- tion (hM3D) decreased fear responses. Training hM4D p = 0.99, non significant (ns); hM3D p = 0.99, ns; 1 Day: hM4D **p < 0.01; hM3D p = 0.99, ns;

1 Week, hM4D, hM3D, ***p < 0.001, one-way ANOVA, Bonferroni post hoc test; 18,11, 9 mice per group.

(C) The number of cFos+ GCs after the 1 week recall session was larger in mice with silenced SST+ INs (hM4D) but was reduced in mice with increased SST+ IN activity during training (hM3D),

*p < 0.05, ***p < 0.001, one-way ANOVA, Bonfer- roni post hoc test, 16, 8, 9 mice per group. Shaded area is the number of cFos+ GCs (mean ± SEM) in mice perfused 1 hr after the training with unaltered neuronal activity.

(D) PV-Cre mice and wild-type litter mates with bilateral infection of AAV-DIO-hM3D in the DG were treated CNO (2 mg/kg) 1 hr before fear con- ditioning.

(E) No difference was observed in freezing re- sponses during 1 day and 1 week recall sessions between control wild-type (Control) and PV-Cre mice with increased PV+ IN activity (hM3D).

Training, 1 Day and 1 Week, p = 0.99, non signifi- cant (ns); two-way ANOVA, Bonferroni post hoc test, 5, 6 mice per group.

(F) The number of cFos+ GCs observed after the last test session did not differ between control (Ctrl) mice and mice with enhanced neuronal ac- tivity in PV+ INs (hM3D) at the time of training,t(9) = 1.18, p = 0.27, 5–6 mice per group. Shaded area is the number of cFos+ (mean ± SEM) in WT unin- fected mice perfused 1 hr after the training ses- sion. Error bars, SEM.

See alsoFigures S5andS6.

At the same time, the active ensemble inhibits the recruitment of neighboring cells avoiding retrieval of memory by nat- ural cues and providing specificity to the artificial cellular engram. How is this achieved? Since the sizes of both the ChR2-tagged population of GCs (13%) and the fraction of GCs receiving strong synaptic input from the EC (8% of GC express cFos+ after SE) are small, the chances for a neuron to belong simultaneously to both pools (that would allow association of sensory information with the ChR2 induced activity) are negligible. Optogenetic activation of GCs abolishes the formation of an ensemble that could guide

recall by natural cues since cFos staining is almost absent in ChR2 GCs (Figures 1D andS2E). Context information would be then represented exclusively by ChR2+ GCs whose activity, transmitted to downstream brain regions (for example, amyg- dala), would be associated with information from the uncondi- tioned stimulus. On the other hand, optogenetic activation of GCs may cause homeostatic reductions of neuronal activity (Goold and Nicoll, 2010) that may decrease reactivation of the ChR2+ ensemble by context-dependent inputs, preventing nat- ural recall.

Our results suggest a competitive distribution of the memory trace among hippocampal neurons, akin to other brain regions (Han et al., 2007). However, our data also indicate that acute modulation of neuronal activity during training bidirectionally af- fects the size of the cellular engram through a network mecha- nism. Manipulations that increase the number of GCs recruited in the active ensemble led to a larger size of the cellular engram and increased performance in memory tests while decreasing the size of the active ensemble had the opposite effect (Fig- ure S6E). During SE, active neurons inhibit the recruitment of neighboring cells confining to the engram neurons that receive strong synaptic input from multiple entorhinal afferents. Interest- ingly, the mechanisms that govern the recruitment of neurons during memory acquisition resemble those engaged during memory recall, suggesting a common form of long-term plas- ticity of the network that would be implemented during engram formation.

The lateral inhibition between neighboring GCs is di-synaptic, mediated by GABAA transmission, and observed in almost all GCs. Among GABAergic INs in the DG, SST+ cells, which target dendrites of neighboring CGs, constitute the link between two excitatory neurons. Dendritic lateral inhibition has also been observed in the cerebral cortex (Kapfer et al., 2007; Silberberg and Markram, 2007) where it is also mediated by SST+ INs that correlate pyramidal neuron activity (Berger et al., 2010) to silence excitatory transmission (Urban-Ciecko et al., 2015).

SST+ INs projecting to dendrites fire at high rates during move- ment (Katona et al., 2014), which may help to maintain low levels of DG activity and make manipulations aimed to increase their activity less effective and require longer time to become evident (Figure 6B). Dendrite GABAergic synapses can shunt excitatory currents generated by excitatory afferents allowing SST+ INs to modulate synaptic plasticity, intracellular signaling, and gene expression required for memory formation (Chiu et al., 2013;

Chalifoux and Carter, 2010).

Although acute modulation of hippocampal PV+ INs during training does not prevent fear memory formation (Lovett-Barron et al., 2014), PV+ INs may also contribute to information storage by adapting network plasticity (and subsequent memory forma- tion) to previous experience (Donato et al., 2013). Our study thus suggests that SST+ INs play a role in memory analogous to the sharpening of orientation tuning in the visual cortex (Wilson et al., 2012). It is important to notice that the limited temporal res- olution of chemogenetics does not allow for exclusion of a role for SST+ INs in fear memory formation that goes beyond the training phase (for example, during memory consolidation). In addition, neuronal activity regulates cFos levels in SST+ INs.

Thus, activity-dependent modulation of gene expression may

control memory-related plasticity also in SST+ INs by altering input and output connectivity in this IN type (Spiegel et al., 2014).

Beyond physiology, altered lateral inhibition may be associ- ated with several brain diseases. Accumulating evidence sup- ports the involvement of abnormalities of GABAergic circuits in schizophrenia (Lewis et al., 2005; Marı´n, 2012), epilepsy (Cossart et al., 2001), and Alzheimer disease (Verret et al., 2012). Our find- ings may set the stage for future investigations with the goal to understand the molecular and cellular basis for the associated memory deficits and eventually propose rational therapies.

EXPERIMENTAL PROCEDURES Animals

Group housed adult (12–15 weeks) wild-type, SST-Cre (Ssttm2.1(cre)Zjh/J, RRID:MGI_4838419), and PV-Cre (Pvalb tm1(cre)Arbr/J, RRID:IMSR_

JAX:017320) mice maintained in a 12 hr light/dark cycle and with unlimited ac- cess to food were used for the study. All mice lines were kept in a C57BL/6J genetic background. Experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Geneva and the Geneva Veterinary office.

Viruses

AAVs used in this study were produced by the University of North Carolina and University of Pennsylvania vector cores. AAV-EF1a-DIO-hChR2(H134R)- mCherry, AAV-hSyn-DIO-hM4D(Gi)-mCherry, AAV-hSyn-DIO-hM3D(Gq)- mCherry, and AAV-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine constructs were used with serotype 5 AVVs. In order to obtain sparse optogenetic activation of DG excitatory cells, diluted (1:5,000) AAV-CamKII0.4-Cre (serotype 1) was coinjected with AAV-EF1a-DIO-hChR2(H134R)-mCherry. AAV-SARE-GFP was constructed by subcloning the EcoRI-KpnI fragment of the CSIV-FII- PGK-FP635-SARE-ArcMin-d2EGFP vector (courtesy of H. Bito, University of Tokyo) in an emptied AAV vector backbone. The resulting AAV (serotype 2) en- codes a bidirectional expression cassette that expresses a red fluorescent protein (TurboFP635) under the control of the constitutive promoter from mouse PGK and a destabilized version of the GFP (d2EGFP) under the control of a synaptic activity responsive promoter (SARE).

Surgery

Analgesic treatment (paracetamol 0.2 g/kg) was administered for 2 days around surgery. Anesthesia was induced at 5% and maintained at 1.5%–

2.0% isoflurane (w/v) (Baxter AG). Mice were placed in a stereotaxic frame (Angle One), and craniotomies were performed using stereotaxic coordinates adapted from a mouse brain atlas to target the dorsal DG: –2.2 anterior–

posterior; ± 1.4 medial–lateral; –1.9 dorsal–ventral (from the surface of the brain). Injections of virus (0.4ml per injection site) were performed using grad- uated pipettes (Drummond Scientific Company), broken back to a tip diameter of 10–15mm, at an infusion rate of0.05ml/min. Micropipettes were left in place an additional 5 min following microinjection and slowly retracted (0.4 mm/min) to avoid reflux of the viral solution. Behavioral or electrophysio- logical experiments were performed on the fifth week after the injection. All injection sites were verified histologically. As criteria, we only included in behavioral and histological analysis mice with robust expression of viral en- coded products in the DG in at least three sections spaced 0.6 mm across the anterior-posterior axis.

Optical fiber implants were constructed and positioned above the DG ac- cording to the protocol described elsewhere (Sparta et al., 2012). Optical fibers were constructed by gluing a piece of multimode optical fiber (0.39 NA 200mm core diameter, Thor Labs) to a 1.25 mm diameter 6.4 mm long ceramic ferrule (Thor Labs) using epoxy resin. Fibers were cut leaving approximately 2 mm beyond the end of the ferrule and polished on both sides using polishing sheets of decreasing grit size. Implants were discarded if light transmission was below 80%. All optical fibers were inserted with the help of a stereotaxic frame 0.5 mm above the DG and firmly attached to the skull using screws and dental cement darkened with charcoal powder to prevent light efflux through the implant.

In Vivo Remote Control of Neuronal Activity

1 hr before the behavioral test, mice expressing DREADD receptors were intraperitoneally injected with CNO 2 mg/kg in saline solution (3.3 ml/kg).

ChR2-expressing neurons were activated by attaching a customized fiber patch cord (0.39 NA 200mm core diameter, Thor Labs) to the implanted ferrules. DPSS blue light lasers (MBL-473/50 cmW; CNI Lasers) connected to a single or double rotary joint (Doric Lenses) allowed mice to move freely during stimulation. The laser was triggered to deliver 20 ms pulses at 10 Hz calibrated to an output power of 6–8 mW at the fiber end. The same stimulation protocol was used during training and recall sessions of the fear conditioning protocol.

Spatial Exploration

Behavioral experiments were performed during the light period of the cycle.

1 week before the experiments, all animals were habituated to the experi- menter by one or two daily sessions of 5 min handling.

SE was performed in a large (46.5336.5331 cm) opaque plastic cage.

Several objects (open tubes, bead filled objects, and floor grills) were placed at different locations inside the cage. Mice were placed at random location and allowed to explore the cage for 15 min (optogenetic experiments) or 1 hr (chemogenetic experiments) in the dark. The different duration of SE in opto- genetic (Figures 1A,4B, andS6C) with respect to chemogenetic experiments (Figures 1E,4C,S3G,S5E, andS6B) arises from the need to reduce unspecific effects of light delivery to brain tissue and to prevent abnormal network activity that may result from prolonged excitatory neuron stimulation. In all cases, mice were perfused 1 hr after the start of the exploration session.

Fear Conditioning

Contextual fear conditioning chamber consisted in a 15318 cm methacry- late cage with a metallic grid floor. The bottom of the cage was scented with 1% ammoniac, and the back was covered with a black/white stripe pattern to produce additional contextual cues. Mice were placed in the cage and allowed 3 min exploration. Three mild electric shocks (0.5 mA, 2 s) were then delivered through the floor grid with 30 s interval. Mice stayed 1 min additionally before being returned to the home cage (Total training time:

5 min). Recall sessions (5 min except when noted) were performed 1 day and 1 week later in the same cage without electric shocks. Mice were perfused 1 hr after the training or last test session (1 week) for cFos analysis. Freezing behavior was monitored by video recordings that were digitized using Any- maze software (Stoelting). Light leakage produced during optogenetic stimu- lation reduced the sensitivity of the freezing detection software. For this reason, freezing behavior in experiments with light stimulation of neuronal activity during training and recall sessions were manually scored by an exper- imenter unaware of the condition tested.

Elevated Plus Maze

A plus shaped maze with one 35-cm-long open arm and one 35-cm-long en- closed arm (height of the enclosure 10 cm), elevated 50 cm from the floor, was used to monitor potential anxiogenic or anxiolitic consequences of optoge- netic and chemogenetic manipulations of neuronal activity. Thigmotaxis was quantified by video tracking mice position in the maze and software-based calculations of the proportion of time spent in the open arms (time in open arms/total test time). Total test time was 10 min.

Immunostaining and Cell Counting

Mice were injected with a lethal dose of pentobarbital (150 mg/kg) and perfusedtrans-cardiacally with cold PBS and 4% paraformaldehyde solution.

Brains were extracted and submerged in fixative for 24 hr at 4C. Six series of coronal 50-mm-thick sections were cut in a vibratome. Immunostaining started by blocking slices in PBS 10% BSA and 0.3% Triton X-100 followed by over- night incubation in PBS 3% BSA and 0.3%Triton X-100 with primary antibody:

cFos (rabbit polyclonal, Santa Cruz, RRID: AB_2106783), Arg3.1/Arc (mono- clonal, Santa Cruz, RRID:AB_626696), SST (rat monoclonal, Millipore, RRID:

AB_2255365), GAD67 (mouse monoclonal, Millipore, RRID:AB_2278725), and PV (rabbit polyclonal, Swant, RRID: AB_10000343). After three 15 min washes in PBST at room temperature, slices were incubated with 1:500 Alexa-conjugated secondary antibodies against the corresponding species

(Alexa-Fluor 488, 555, 645, Life Technologies). After three more steps of washing in PBST, slices were mounted and covered on microscope slides us- ing mounting medium. DAPI containing mounting medium was used to esti- mate the fraction of GCs infected in behavioral experiments (Figures S1B and S1D).

Images were obtained in a confocal laser-scanning microscopy with a Fluo- view 300 system (Olympus) using a 488-nm argon laser and a 537-nm helium- neon laser or in Leica SP5 confocal microscope using additional 350-nm laser with a 203/0.7 NA oil immersion or 403/0.8 NA water immersion objectives.

A semi-automated method was used to quantify viral infection and cFos expression in confocal images of brain slices containing the upper blade of the DG (0.430.3 mm,2.5 pixel/mm, 4mm step size). Equally thresholded im- ages were subjected to multiparticle analysis (NIH ImageJ). Region of interest (ROI) intensity values were obtained from the z stack of raw images by using Multi Measure tool. Colocalization was determined by overlap of the ROI ob- tained from the two independent fluorescence signals. Analysis was per- formed in at least three sections per animal.

Electrophysiology

Acute brain slices were used for electrophysiological experiments. Mice were deeply anesthetized with isofluorane inhalation and decapitated. Brains were quickly removed and immersed in ‘‘cutting’’ solution (4C) containing the following (in mM): 234 sucrose, 11 glucose, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, and 0.5 CaCl2(equilibrated with 95% O2 and 5%

CO2). Coronal slices (300mm) were cut with a vibratome (Leica) and then incu- bated in oxygenated artificial cerebrospinal fluid (ACSF) containing the following (in mM): 126 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, and 10 glucose (pH 7.4) at room temperature, before being trans- ferred to the recording chamber. Recordings were obtained at room temper- ature from visually identified GCs in the upper blade of the DG using infrared video microscopy. For current clamp experiments, GCs or SST+ INs were re- corded using patch-clamp electrodes (tip resistance, 2–3 MU) filled with an intracellular solution containing (in mM) 70 K-gluconate, 70 KCl, 2 NaCl, 2 MgCl2, 10 HEPES, 1 EGTA, 2 MgATP, and 0.3 Na2GTP (pH 7.3) corrected with KOH (290 mOsm). The intracellular solution experiments involving IPSCs and EPSC isolation at different holding potentials, contained the following (in mM): 125 CsMeSO3, 2 CsCl, 10 HEPES, 5 EGTA, 2 MgCl2, and 4 MgATP.

Reversal potentials for glutamate- and GABA-mediated responses were deter- mined in separate sets of experiment, in which EPSCs and IPSCs were iso- lated pharmacologically, using gabazine (10mM) and DNQX (10mM), respec- tively (not shown). CNO, TTX (Latoxan), SR95531 (Gabazine), and kynurenic acid (Sigma) were dissolved to the final concentration in ACSF and delivered through the perfusion system. Optogenetic stimulation during recordings was performed using a LED source of 470 nM coupled to a fiber guide and a collimator lens to focus light spot around the recorded neuron. Light pulses were of 10 ms duration with a nominal power at the exit of 0.35–0.79 mW. Se- ries and membrane resistance were monitored regularly during experiments and recordings were discarded when these parameters changed significantly (>20%). Signals were amplified using a Multiclamp 200B patch-clamp ampli- fier (Molecular Devices), sampled at 20 kHz, filtered at 10 kHz, and stored on a PC. Data were analyzed using pClamp (Axon Instruments) software. Except when noted, an average trace of at least ten trials was used for illustration.

Statistical Analysis

All values are given in mean ± SEM. Standard t tests were performed to compare Gaussian distributions while Mann-Whitney tests were used for non-Gaussian distributions. One- or two-way ANOVA with repeated-measures followed by Bonferroni post hoc test or Kruskal-Wallis followed by Dunn post hoc test were used when noted. For all tests, we adopted an alpha level of 0.05 to assess statistical significance. Statistical analysis was performed using Prism (Graphpad software).

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures and can be found with this article online athttp://dx.doi.org/10.1016/j.neuron.2016.01.024.

AUTHOR CONTRIBUTIONS

P.M. and D.M. conceived and design the experiments. T.S., P.M., and C.B.

performed the experiments and analyzed the data. P.M. and C.L. wrote the manuscript with help from all authors. C.L. supervised the project after the disappearance of D.M.

ACKNOWLEDGMENTS

We dedicate this article to the memory of Dominique Muller, an exceptional scientist, enthusiast mentor, and devoted teacher. We thank H. Bito (University of Tokyo) for sharing SARE-GFP constructs and A. Holtmaat (University of Geneva) and A. Bacci (ICM, Paris) for providing PV-Cre mice and SST-Cre mice. We are grateful to Lorena Jourdain for excellent technical assistance and Lu¨scher lab members (University of Geneva) for their initial assistance with in vivo optogenetic experiments and critical discussions during manu- script preparation. Work in D.M.’s lab is financed by SNF grant Sinergia and grant 310030B-144080. The authors declare no conflict of interest.

Received: July 7, 2015 Revised: October 15, 2015 Accepted: January 10, 2016 Published: February 11, 2016 REFERENCES

Acsa´dy, L., Kamondi, A., Sı´k, A., Freund, T., and Buzsa´ki, G. (1998).

GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus. J. Neurosci.18, 3386–3403.

Berger, T.K., Silberberg, G., Perin, R., and Markram, H. (2010). Brief bursts self-inhibit and correlate the pyramidal network. PLoS Biol.8, e1000473.

Chalifoux, J.R., and Carter, A.G. (2010). GABAB receptors modulate NMDA re- ceptor calcium signals in dendritic spines. Neuron66, 101–113.

Chiu, C.Q., Lur, G., Morse, T.M., Carnevale, N.T., Ellis-Davies, G.C., and Higley, M.J. (2013). Compartmentalization of GABAergic inhibition by dendritic spines. Science340, 759–762.

Cossart, R., Dinocourt, C., Hirsch, J.C., Merchan-Perez, A., De Felipe, J., Ben-Ari, Y., Esclapez, M., and Bernard, C. (2001). Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy. Nat. Neurosci.4, 52–62.

Cowansage, K.K., Shuman, T., Dillingham, B.C., Chang, A., Golshani, P., and Mayford, M. (2014). Direct reactivation of a coherent neocortical memory of context. Neuron84, 432–441.

Deng, W., Mayford, M., and Gage, F.H. (2013). Selection of distinct popula- tions of dentate granule cells in response to inputs as a mechanism for pattern separation in mice. eLife2, e00312.

Denny, C.A., Kheirbek, M.A., Alba, E.L., Tanaka, K.F., Brachman, R.A., Laughman, K.B., Tomm, N.K., Turi, G.F., Losonczy, A., and Hen, R. (2014).

Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis. Neuron83, 189–201.

Donato, F., Rompani, S.B., and Caroni, P. (2013). Parvalbumin-expressing basket-cell network plasticity induced by experience regulates adult learning.

Nature504, 272–276.

Frankland, P.W., O’Brien, C., Ohno, M., Kirkwood, A., and Silva, A.J. (2001).

Alpha-CaMKII-dependent plasticity in the cortex is required for permanent memory. Nature411, 309–313.

Freund, T.F., and Katona, I. (2007). Perisomatic inhibition. Neuron56, 33–42.

Goold, C.P., and Nicoll, R.A. (2010). Single-cell optogenetic excitation drives homeostatic synaptic depression. Neuron68, 512–528.

Han, J.H., Kushner, S.A., Yiu, A.P., Cole, C.J., Matynia, A., Brown, R.A., Neve, R.L., Guzowski, J.F., Silva, A.J., and Josselyn, S.A. (2007). Neuronal competi- tion and selection during memory formation. Science316, 457–460.

Han, J.H., Kushner, S.A., Yiu, A.P., Hsiang, H.L., Buch, T., Waisman, A., Bontempi, B., Neve, R.L., Frankland, P.W., and Josselyn, S.A. (2009).

Selective erasure of a fear memory. Science323, 1492–1496.

Kapfer, C., Glickfeld, L.L., Atallah, B.V., and Scanziani, M. (2007). Supralinear increase of recurrent inhibition during sparse activity in the somatosensory cortex. Nat. Neurosci.10, 743–753.

Katona, L., Lapray, D., Viney, T.J., Oulhaj, A., Borhegyi, Z., Micklem, B.R., Klausberger, T., and Somogyi, P. (2014). Sleep and movement differentiates actions of two types of somatostatin-expressing GABAergic interneuron in rat hippocampus. Neuron82, 872–886.

Kawashima, T., Okuno, H., Nonaka, M., Adachi-Morishima, A., Kyo, N., Okamura, M., Takemoto-Kimura, S., Worley, P.F., and Bito, H. (2009).

Synaptic activity-responsive element in the Arc/Arg3.1 promoter essential for synapse-to-nucleus signaling in activated neurons. Proc. Natl. Acad. Sci.

USA106, 316–321.

Kheirbek, M.A., Drew, L.J., Burghardt, N.S., Costantini, D.O., Tannenholz, L., Ahmari, S.E., Zeng, H., Fenton, A.A., and Hen, R. (2013). Differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus. Neuron 77, 955–968.

Krueppel, R., Remy, S., and Beck, H. (2011). Dendritic integration in hippo- campal dentate granule cells. Neuron71, 512–528.

Lewis, D.A., Hashimoto, T., and Volk, D.W. (2005). Cortical inhibitory neurons and schizophrenia. Nat. Rev. Neurosci.6, 312–324.

Liu, X., Ramirez, S., Pang, P.T., Puryear, C.B., Govindarajan, A., Deisseroth, K., and Tonegawa, S. (2012). Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature484, 381–385.

Lovett-Barron, M., Kaifosh, P., Kheirbek, M.A., Danielson, N., Zaremba, J.D., Reardon, T.R., Turi, G.F., Hen, R., Zemelman, B.V., and Losonczy, A. (2014).

Dendritic inhibition in the hippocampus supports fear learning. Science343, 857–863.

Maccaferri, G. (2005). Stratum oriens horizontal interneurone diversity and hip- pocampal network dynamics. J. Physiol.562, 73–80.

Malenka, R.C., and Bear, M.F. (2004). LTP and LTD: an embarrassment of riches. Neuron44, 5–21.

Maren, S., and Quirk, G.J. (2004). Neuronal signalling of fear memory. Nat.

Rev. Neurosci.5, 844–852.

Marı´n, O. (2012). Interneuron dysfunction in psychiatric disorders. Nat. Rev.

Neurosci.13, 107–120.

Markram, H., Toledo-Rodriguez, M., Wang, Y., Gupta, A., Silberberg, G., and Wu, C. (2004). Interneurons of the neocortical inhibitory system. Nat. Rev.

Neurosci.5, 793–807.

Martin, S.J., Grimwood, P.D., and Morris, R.G. (2000). Synaptic plasticity and memory: an evaluation of the hypothesis. Annu. Rev. Neurosci.23, 649–711.

Matsuzaki, M., Honkura, N., Ellis-Davies, G.C., and Kasai, H. (2004). Structural basis of long-term potentiation in single dendritic spines. Nature429, 761–766.

McHugh, T.J., and Tonegawa, S. (2007). Spatial exploration is required for the formation of contextual fear memory. Behav. Neurosci.121, 335–339.

Mendez, P., and Bacci, A. (2011). Assortment of GABAergic plasticity in the cortical interneuron melting pot. Neural. Plast. http://dx.doi.org/10.1155/

2011/976856.

Miles, R., To´th, K., Gulya´s, A.I., Ha´jos, N., and Freund, T.F. (1996). Differences between somatic and dendritic inhibition in the hippocampus. Neuron16, 815–823.

Morgan, J.I., and Curran, T. (1991). Stimulus-transcription coupling in the ner- vous system: involvement of the inducible proto-oncogenes fos and jun. Annu.

Rev. Neurosci.14, 421–451.

Nabavi, S., Fox, R., Proulx, C.D., Lin, J.Y., Tsien, R.Y., and Malinow, R. (2014).

Engineering a memory with LTD and LTP. Nature511, 348–352.

Olshausen, B.A., and Field, D.J. (2004). Sparse coding of sensory inputs. Curr.

Opin. Neurobiol.14, 481–487.

Pierson, J.L., Pullins, S.E., and Quinn, J.J. (2015). Dorsal hippocampus infu- sions of CNQX into the dentate gyrus disrupt expression of trace fear condi- tioning. Hippocampus25, 779–785.