Supplementary Figures

Figure S1. Optogenetic and chemogenetic control of GC activity.

A. Dentate gyrus (DG) of wild type (WT) mice injected with AAV-CamKII-Cre+AAV-DIO-ChR2 (red) stained with the interneuron (IN) marker GAD67 (green). The vast majority of ChR2+ cells showed the typical morphology of granule cells (GCs) and only 1.1 ± 0.4%, 1.0 ± 0.5% and 0.5 ± 0.3% of infected cells were positive for the IN markers GAD67, SST or PV respectively (right graph). Scale bar 10 μm. Error bars, SEM.

B. Sparse ChR2 expression in GCs of WT mice DG after delivery of AAVs expressing ChR2 under the control of the CamKII promoter. Right graph shows the proportion of infected GCs (ChR2+) at different dorso-ventral coordinates (in -, from Bregma) expressed as percentage of total DAPI cell counts in the GC layer. C. Ex vivo current clamp recordings show that brief blue light pulses reliably induce tetrodotoxin (TTX) sensitive action potentials and command trains of APs during prolonged periods of time in ChR2 expressing granule cells. Left traces correspond to 15 trials before (black) and after (red) TTX (1mM) application. Bottom right trace corresponds to the last seconds of a 5 minute train of 10Hz action potentials stimulation protocol used for behavioral experiments. Scale bars: 10mV, 10ms and 50mV, 1s. D. DG infected with AAVs expressing hM4D receptor under the control of the CamKII promoter (AAV-CamKII-hM4D). Right graph shows the proportion of infected GCs (hM4D+) at different dorso-ventral coordinates (in -mm, from Bregma) expressed as percentage of total DAPI cell counts in the GC layer. Scale bar: 150 μm. E. Ex vivo current clamp recordings from a hM4D expressing GC shows that Clozapine-N-oxide (CNO) application causes significant hyperpolarization of the membrane potential (Vm) of GCs. Left, representative current clamp traces of a GC response to positive and negative 500 ms current injections (-40 and +160 pA, respectively).

Scale bars: 10mV, 100ms and 5mV, 2 min. Arrow marks the membrane resting potential of the recorded GC (-67mV). Graph shows membrane potential (Vm) changes before (Sal) and after CNO (1 μM) application, Paired t-test t(4)=11.21 ***, p<0.001.

Fig. S1. (Refers to Fig. 1) Optogenetic and chemogenetic control of GC activity.

A. Dentate gyrus (DG) of wild type (WT) mice injected with AAV-CamKII-Cre+AAV-DIO-ChR2 (red) stained with the interneuron (IN) marker GAD67 (green). The vast majority of ChR2+ cells showed the typical morphology of granule cells (GCs) and only 1.1 ± 0.4%, 1.0 ± 0.5% and 0.5 ± 0.3% of infected cells were positive for the IN markers GAD67, SST or PV respectively (right graph).

Scale bar 10 µm. Error bars, SEM.

B. Sparse ChR2 expression in GCs of WT mice DG after delivery of AAVs expressing ChR2 under the control of the CamKII promoter. Right graph shows the proportion of infected GCs (ChR2+) at different dorso-ventral coordinates (in -, from Bregma) expressed as percentage of total DAPI cell counts in the GC layer.

C. Ex vivo current clamp recordings show that brief blue light pulses reliably induce tetrodotoxin (TTX) sensitive action potentials and command trains of APs during prolonged periods of time in ChR2 expressing granule cells. Left traces correspond to 15 trials before (black) and after (red) TTX (1mM) application. Bottom right trace corresponds to the last seconds of a 5 minute train of 10Hz action potentials stimulation protocol used for behavioral experiments. Scale bars: 10mV, 10ms and 50mV, 1s.

D. DG infected with AAVs expressing hM4D receptor under the control of the CamKII promoter (AAV-CamKII-hM4D). Right graph shows the proportion of infected GCs (hM4D+) at different dorso-ventral coordinates (in -mm, from Bregma) expressed as percentage of total DAPI cell counts in the GC layer. Scale bar: 150 µm.

E. Ex vivo current clamp recordings from a hM4D expressing GC shows that Clozapine-N-oxide (CNO) application causes significant hyperpolarization of the membrane potential (Vm) of GCs. Left, representative current clamp traces of a GC response to positive and negative 500 ms current injections (-40 and +160 pA, respectively). Scale bars: 10mV, 100ms and 5mV, 2 min. Arrow marks the membrane resting potential of the recorded GC (-67mV). Graph shows membrane potential (Vm) changes before (Sal) and after CNO (1 µM) application, Paired t-test t(4)=11.21 ***, p<0.001.

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Figure S2. Optogenetic modulation of excitatory neuron in the DG does not affect exploration, shock reactivity or memory of an unrelated context. Measurement of freezing behavior from averaged epochs of on/off light.

A. WT mice were infected with AAV-CamKII-Cre+AAV-DIO-ChR2. Graph shows distance travelled during the 3 minutes of cage exploration before the delivery of the unconditioned stimulus (foot shock) in the training session of the fear conditioning protocol. No difference was observed between stimulated and non stimulated mice suggesting that optogenetic activation of a sparse population of granule cells did not alter exploratory behavior during the training session t(10)=1.13, non significant (ns); n=4, 8 mice. B. Time spent in freezing behavior during 1 minute immediately after shock delivery did not differ between control and stimulated mice, suggesting that both group of mice remember similarly for short periods of time after fear conditioning. t(20)=1.37, non significant (ns); n=9, 13 mice. C. Distance travelled during 4 seconds epochs before, during and immediately after shock delivery in the training session of the fear conditioning protocol.

The large increase in travelled distance during the shock epoch corresponds to mice escape response. No difference was observed between stimulated and non stimulated mice suggesting that optogenetic activation of a sparse population of granule cells did not alter shock reactivity. 1-way ANOVA, F(5, 57)=73.30, p<0.001, non significant, ns; n=9, 12 mice. D. Mice expressing ChR2 in a fraction of GCs were placed in a novel cage (triangle) with (Stimulated) or without (Control) optogenetic activation of a sparse population of excitatory cells of the DG. The following day, mice were trained in the fear conditioning set up (squares) without optogenetic stimulation. Mice were tested in the fear conditioning chamber 1 day and 1 week after without optogenetic stimulation. Optogenetic activation of a sparse population of GCs in a context non associated to fear memory did not affect subsequent fear conditioning in a different context. The levels of freezing behavior during the training, 1 day and 7 day test sessions of the fear conditioning protocol did not differed between control and stimulated animals. F(1, 12)=0.72, non significant (ns), n=6, 8 animals per group. E. Analysis of c-fos+/ChR2+ double stained cells after the 1 week recall session in which only contextual cues were present. No difference was observed in c-fos expression in ChR2+ GCs. 1-way ANOVA followed by Bonferroni correction, F(3,16)=60.94 , p<0.001; ns, non significant effect of stimulation, p>0.99. F. Graphs showing the averaged time spent in freezing behavior in all 3 on/off light epochs during recall sessions using optogenetic stimulation of GCs (1 week). Light stimulation significantly increased freezing behavior in ChR2 but not in mCherry expressing animals. Freezing levels during light on epochs in stimulated mice were reduced with respect to control mice, 2-way ANOVA, F(1,10)=12.45, p<0.01, ***p<0.001, *p<0.05; non significant, ns, n=4, 8 mice per group. Error bars, SEM.

Fig. S2. (Refers to Fig. 2) Optogenetic modulation of excitatory neuron activity in the DG does not affect exploration, shock reactivity or memory of an unrelated context. Measurement of freezing behavior from averaged epochs of on/off light.

A. WT mice were infected with AAV-CamKII-Cre+AAV-DIO-ChR2. Graph shows distance travelled during the 3 minutes of cage exploration before the delivery of the unconditioned stimulus (foot shock) in the training session of the fear conditioning protocol. No difference was observed between stimulated and non stimulated mice suggesting that optogenetic activation of a sparse population of granule cells did not alter exploratory behavior during the training session t(10)=1.13, non significant (ns); n=4, 8 mice.

B. Time spent in freezing behavior during 1 minute immediately after shock delivery did not differ between control and stimulated mice, suggesting that both group of mice remember similarly for short periods of time after fear conditioning. t(20)=1.37, non significant (ns); n=9, 13 mice.

C. Distance travelled during 4 seconds epochs before, during and immediately after shock delivery in the training session of the fear conditioning protocol. The large increase in travelled distance during the shock epoch corresponds to mice escape response. No difference was observed between stimulated and non stimulated mice suggesting that optogenetic activation of a sparse population of granule cells did not alter shock reactivity. 1-way ANOVA, F(5, 57)=73.30, p<0.001, non significant, ns; n=9, 12 mice.

D. Mice expressing ChR2 in a fraction of GCs were placed in a novel cage (triangle) with (Stimulated) or without (Control) optogenetic activation of a sparse population of excitatory cells of the DG. The following day, mice were trained in the fear conditioning set up (squares) without optogenetic stimulation. Mice were tested in the fear conditioning chamber 1 day and 1 week after without optogenetic stimulation. Optogenetic activation of a sparse population of GCs in a context non associated to fear memory did not affect subsequent fear conditioning in a different context. The levels of freezing behavior during the training, 1 day and 7 day test sessions of the fear conditioning protocol did not differed between control and stimulated animals. F(1, 12)=0.72, non significant (ns), n=6, 8 animals per group.

E. Analysis of cFos+/ChR2+ double stained cells after the 1 week recall session in which only contextual cues were present. No difference was observed in cFos expression in ChR2+ GCs. 1-way ANOVA followed by Bonferroni correction, F(3,16)=60.94 , p<0.001; ns, non significant effect of stimulation, p>0.99.

F. Graphs showing the averaged time spent in freezing behavior in all 3 on/off light epochs during recall sessions using optogenetic stimulation of GCs (1 week). Light stimulation significantly increased freezing behavior in ChR2 but not in mCherry expressing animals. Freezing levels during light on epochs in stimulated mice were reduced with respect to control mice, 2-way ANOVA, F(1,10)=12.45, p<0.01, ***p<0.001, *p<0.05; non significant, ns, n=4, 8 mice per group. Error bars, SEM.

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Figure S3. CNO treatment specifically affect memory formation only in mice expressing the DREADD receptors.

A. Uninfected WT mice were treated with saline (Sal) or CNO (2mg/kg) 1 hour before fear conditioning. Mice were tested 1 day and 1 week later in the absence of the unconditioned stimulus. CNO treated mice (black bars) were indistinguishable from saline treated mice (white bars) regarding freezing levels during training, 1 day and 1 week after fear conditioning, 2-way ANOVA F(1,6)=0.08, p=0.78 followed by Bonferroni correction, Training, 1 day and 1 week p=0.99, non significant (ns); n=4 mice. B. WT mice infected with AAV-CamKII-hM4D were tested in the elevated plus maze (EPM, see experimental procedures section) one hour after being injected with saline or CNO (2mg/kg). No differences were observed in the amount of time spent in the open arms of the maze suggesting no major alteration of anxiety-related behavior by acute chemogenetic silencing of GCs, t(7)=0.24, p= 0.81, non significant (ns); n=4, 5 mice. C. Contextual memory formation drives synaptic input to the hippocampus activating a granule cell (GC) ensemble responsible for encoding sensory and spatial information. Memory recall requires the re-activation of the same neuronal population. D. When a fraction of GCs are silenced (hM4D+, red contoured cells), synaptic input to the hippocampus drives a larger ensemble recruiting preferentially non silenced hM4D- GCs. More GCs become involved in information encoding, increasing the chance for reactivation during memory recall and enhancing memory stability. E. Optogenetic activation of a GC ensemble during memory formation directs information encoding to optogenetically active cells and inhibits synaptic activation of non stimulated (ChR2-) GCs. In the absence of light stimulation (light OFF), natural cues are unable to reactivate the cellular engram and retrieval is only possible by light activation of ChR2+ cells (light ON). F. The altered formation of the cellular engram under conditions of increased or reduced neuronal activity can be explained by an inhibitory interaction between active and inactive ensembles of GCs during memory formation. G. Mice expressing Cre recombinase under the promoter of Glutamic Acid Decarboxilase (GAD-Cre) were injected in the DG with AAVs encoding Cre dependent hM4D receptors. Five weeks after infection, mice received CNO or saline injections and 1 hour later explored an enriched cage for 1 hour. Chemogenetic silencing of all DG interneurons during SE resulted in increased number of GCs expressing c-fos; t-test t(8)=3.38, **p<0.01; n=5 mice per group. Scale bar, 25 µm. Error bars, SEM.

Fig. S3. (Refers to Fig. 3) CNO treatment does not affect memory formation in WT uninfected mice. Chemogenetic modulation of excitatory neurons activity in the DG does not affect mice performance in the elevated plus maze (EPM). Proposed model: Inhibitory interaction among ensembles of granule cells underlays contextual memory allocation in granule cells. Chemogenetic inactivation of DG GABA-neurons during spatial exploration using GAD-Cre mice increases engram size.

A. Uninfected WT mice were treated with saline (Sal) or CNO (2mg/kg) 1 hour before fear conditioning. Mice were tested 1 day and 1 week later in the absence of the unconditioned stimulus. CNO treated mice (black bars) were indistinguishable from saline treated mice (white bars) regarding freezing levels during training, 1 day and 1 week after fear conditioning, 2-way ANOVA F(1,6)=0.08, p=0.78 followed by Bonferroni correction, Training, 1 day and 1 week p=0.99, non significant (ns); n=4 mice.

B. WT mice infected with AAV-CamKII-hM4D were tested in the elevated plus maze (EPM, see experimental procedures section) one hour after being injected with saline or CNO (2mg/kg). No differences were observed in the amount of time spent in the open arms of the maze suggesting no major alteration of anxiety-related behavior by acute chemogenetic silencing of GCs, t(7)=0.24, p= 0.81, non significant (ns); n=4, 5 mice.

C. Contextual memory formation drives synaptic input to the hippocampus activating a granule cell (GC) ensemble responsible for encoding sensory and spatial information. Memory recall requires the re-activation of the same neuronal population.

D. When a fraction of GCs are silenced (hM4D+, red contoured cells), synaptic input to the hippocampus drives a larger ensemble recruiting preferentially non silenced hM4D- GCs. More GCs become involved in information encoding, increasing the chance for reactivation during memory recall and enhancing memory stability.

E. Optogenetic activation of a GC ensemble during memory formation directs information encoding to optogenetically active cells and inhibits synaptic activation of non stimulated (ChR2-) GCs. In the absence of light stimulation (light OFF), natural cues are unable to reactivate the cellular engram and retrieval is only possible by light activation of ChR2+ cells (light ON).

F. The altered formation of the cellular engram under conditions of increased or reduced neuronal activity can be explained by an inhibitory interaction between active and inactive ensembles of GCs during memory formation.

G. Mice expressing Cre recombinase under the promoter of Glutamic Acid Decarboxilase (GAD-Cre) were injected in the DG with AAVs encoding Cre dependent hM4D receptors. Five weeks after infection, mice received CNO or saline injections and 1 hour later explored an enriched cage for 1 hour. Chemogenetic silencing of all DG interneurons during SE resulted in increased number of GCs expressing cFos; t-test t(8)=3.38 **, p<0.01; n=5 mice per group. Scale bar, 25 µm. Error bars, SEM.

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Figure S4. Construction of the virally encoded reporter of synaptic activity based on the promoter region of the Arg3.1/Arc gene (AAV-SARE-GFP).

A. The SARE-GFP plasmid encodes an inducible reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP) under the control of Synaptic Activity-Responsive Element (SARE) and a constitutively expressed infection marker Red Fluorescent Protein (TurboFP635) under the control of phosphoglycerate kinase (PGK) promoter (described in ref). Two inverted terminal repeat sequences (ITR, black boxes) flank the expression cassette and allow packing of the sequence for AAV production. B. Spatial exploration drives SARE activity.

Left, low magnification image of a brain section 3 weeks after in vivo viral transduction of the SARE-GFP construct in the DG. Right, representative images of synaptic activity reporter Green Fluorescence Protein (GFP) in DG sections of home cage (HC) and mice that performed 1 hour of spatial exploration (SE). Scale bars 0.5 mm, 10 μm. C. Representative images of endogenous Arg3.1/Arc protein expression in PV+ and SST+ after brief optogenetic stimulation. ChR2 expressing PV+ and SST+ neurons were activated with identical protocols of blue light illumination (10 Hz for 15 minutes). Mice were perfused 45 minutes later and Arg3.1/Arc expression was quantified in PV+ and SST+ INs. Upper graphs: Arg3.1/Arc protein was induced in similar fractions of PV+ (t(5)=3.13, *p<0.05) and SST+ neurons (t(6)=3.38, *p<0.05). Lower graphs:

expression levels (arbitrary unit of fluorescence intensity) of Arg3.1/Arc where significantly increased in both PV+ (t(5)=2.57) and SST+ populations (t(6)=2.50), *p<0.05, n=3,4 animals per group. Scale bar 10 μm. D.

Representative low magnification images of the DG of AAV-SARE-GFP injected mice after spatial exploration. Section were stained with PV+ (top) or SST (bottom) antibodies to asses recent synaptic activity levels in these two INs subtypes. Boxed areas are magnified in figure 4A. Stars mark neurons with typical granule cell morphology and location that express high levels of the synaptic activity reporter protein GFP.

Scale bar 20 μm. Error bars, SEM.

Fig. S4. (Refers to Fig. 4) Construction of the virally encoded reporter of synaptic activity based on the promoter region of the Arg3.1/

Arc gene (AAV-SARE-GFP). Endogenous Arg3.1/Arc protein is similarly induced by neuronal activity in PV+ and SST+ neurons.

A. The SARE-GFP plasmid encodes an inducible reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP) under the control of Synaptic Activity-Responsive Element (SARE) and a constitutively expressed infection marker Red Fluorescent Protein (TurboFP635) under the control of phosphoglycerate kinase (PGK) promoter (described in ref). Two inverted terminal repeat sequences (ITR, black boxes) flank the expression cassette and allow packing of the sequence for AAV production.

B. Spatial exploration drives SARE activity. Left, low magnification image of a brain section 3 weeks after in vivo viral transduction of the SARE-GFP construct in the DG. Right, representative images of synaptic activity reporter Green Fluorescence Protein (GFP) in DG sections sections of home cage (HC) and mice that performed 1 hour of spatial exploration (SE). Scale bars 0.5 mm, 10 µm.

C. Representative images of endogenous Arg3.1/Arc protein expression in PV+ and SST+ after brief optogenetic stimulation. ChR2 expressing PV+ and SST+ neurons were activated with identical protocols of blue light illumination (10 Hz for 15 minutes). Mice were perfused 45 minutes later and Arg3.1/Arc expression was quantified in PV+ and SST+ INs. Upper graphs: Arg3.1/Arc protein was induced in similar fractions of PV+ (t(5)=3.13, *p<0.05) and SST+ neurons (t(6)=3.38, *p<0.05). Lower graphs: expression levels (arbitrary unit of fluorescence intensity) of Arg3.1/Arc where significantly increased in both PV+ (t(5)=2.57) and SST+

populations (t(6)=2.50), *p<0.05, n=3,4 animals per group. Scale bar 10 µm.

D. Representative low magnification images of the DG of AAV-SARE-GFP injected mice after spatial exploration. Section were stained with PV (top) or SST (bottom) antibodies to asses recent synaptic activity levels in these two INs subtypes. Boxed areas are magnified in figure 4A. Stars mark neurons with typical granule cell morphology and location that express high levels of the synaptic activity reporter protein GFP. Scale bar 20 µm. Error bars, SEM.

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Figure S5. Chemogenetic control of inhibitory neuron activity.

A. SST-Cre (left panel) and PV-Cre (right panel) mice DG infected with an AAV expressing Cre dependent hM3D receptor were stained with antibodies against somatostatin (SST, left panel, green) and parvalbumin (PV, right panel, green) respectively. The graphs show the proportion of infected neurons in SST-Cre (SST) and PV-Cre (PV) mice immunopositive for the correspondent IN marker (white bars) and the proportion of neurons immunopositive for SST or PV antibodies expressing the infection marker (black bars). The high proportion of double stained cells (arrows) suggest reliable targeting of protein expression to defined IN populations. Scale bar 5 μm. B. Ex vivo current clamp recordings from hM4D expressing SST+ INs show that Clozapine-N-oxide (CNO, 1μM) application causes significant hyperpolarization (9.5±1.9 mV) of the membrane potential (Vm) in this IN subtype. Left, representative current clamp traces of a SST+ IN response to positive and negative 500 ms current injections (-40 and +160 pA, respectively). Scale bars: 10 mV, 100 ms and 5 mV, 2 min. C. Infection of PV and SST Cre mice with an AAV-DIO-mCherry reveals differential segregation of axonal projections in the DG. While axons of SST+ cells cover the molecular layer (ML) and spare the granule cell layer (GCL), axons of perisomatic targeting PV INs are confined in GCL with little ramifications in the ML. Scale bar 50 μm. D. Ex vivo current clamp recordings from hM3D expressing PV+

neuron show that CNO application caused significant membrane depolarization and induced spiking activity (n=3). Left, representative current clamp traces of a PV+ IN response to positive and negative 500 ms current injections (-40 and +235 pA, respectively). Scale bars: 10 mV, 100 ms and 5 mV, 25 s. E. PV-Cre and WT litter mates mice injected with AAV-DIO-hM3Dq in the DG received CNO injections and 1 hour later explored an enriched cage for 1 hour. Images show representative staining of c-fos protein in the DG of animals with increased PV+ IN activity (hM3D) or unaltered (control). A larger fraction of PV+ cells are c-fos+

when PV INs were chemogenetically activated through hM3D receptors t(7)=3.85, **p<0.01, n=4,5 (Left graph). C-fos levels (arbitrary units of fluorescence intensity) were higher in PV+ IN population after chemogenetic activation, right graph t(7)=4.73, n=4,5. Scale bar 10 μm. Error bars, SEM.

Figure S6. Optogenetic and chemogenetic control of PV+ INs activity does not modify engram size and function.

A. PV-Cre and WT litter mates mice bilaterally injected with AAV-DIO-ChR2 in the DG and cannulated, were trained in the fear conditioning chamber without (Control) or with (Stimulated) optogenetic activation of PV+

INs. Both groups of animals showed similar levels of freezing behavior during the 1 day and 1 week test sessions. F(1,12)<0.01, training, 1 day and 1 week, p>0.99; ns, non significant, n=4, 10 mice per group. B.

PV-Cre mice and WT litter mates were injected with AAV-DIO-hM4D receptors in the DG. Five weeks after infection, mice received CNO injections and 1 hour late explored an enriched cage for 1 hour. Chemogenetic silencing of PV+ INs during SE resulted in no difference in the number of GCs expressing c-fos, t(6)=0.50;

ns, non significant, n=3, 5 mice. C. Optogenetic stimulation of PV+ INs during spatial exploration did not alter the number of c-fos+ GCs, ns, non significant, Mann-Whitney test, n=4 mice per group. D. SST-Cre mice

ns, non significant, n=3, 5 mice. C. Optogenetic stimulation of PV+ INs during spatial exploration did not alter the number of c-fos+ GCs, ns, non significant, Mann-Whitney test, n=4 mice per group. D. SST-Cre mice