Volume 89, Issue 5, Pages 1074-1085 (March 2016) Hippocampal Somatostatin Interneurons Control the Size of Neuronal Memory Ensembles  Thomas Stefanelli, Cristina Bertollini, Christian Lüscher, Dominique Muller, Pablo Mendez  Neuron  Volume 89, Issue 5, Pages 1074-1085 (March 2016) DOI: 10.1016/j.neuron.2016.01.024 Copyright © 2016 Elsevier Inc. Terms and Conditions

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: 10 μm. (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: 10 μm. (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 also Figure S1. Neuron 2016 89, 1074-1085DOI: (10.1016/j.neuron.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Optogenetic Activation of a Fraction 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 optogenetic 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 also Figure S2. Neuron 2016 89, 1074-1085DOI: (10.1016/j.neuron.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Inactivation of a Fraction of GCs during Training Improves Memory and Increases 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 population 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 increases 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 also Figure S3. Neuron 2016 89, 1074-1085DOI: (10.1016/j.neuron.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

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 population. 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: 5 μm. (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: 5 μm. (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: 5 μm. Error bars, SEM. See also Figures S4 and S5. Neuron 2016 89, 1074-1085DOI: (10.1016/j.neuron.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Sparse Population of DG Excitatory Cells Activate Strong Dendritic Targeting 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 amplitude 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], 10μM), tetrotodotoxin ([TTX], 1μM), 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 also Figure S5. Neuron 2016 89, 1074-1085DOI: (10.1016/j.neuron.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Dendritic but Not Somatic Targeting INs Control the Size of the Active Neuronal Population and Memory Formation (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 activation (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, Bonferroni 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 conditioning. (E) No difference was observed in freezing responses 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 significant (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 activity 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 uninfected mice perfused 1 hr after the training session. Error bars, SEM. See also Figures S5 and S6. Neuron 2016 89, 1074-1085DOI: (10.1016/j.neuron.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions