Differential Coupling of Adult-Born Granule Cells to Parvalbumin and Somatostatin Interneurons

<jats:title>ABSTRACT</jats:title><jats:p>The dentate gyrus of the hippocampus is dominated by a strong GABAergic tone that maintains sparse levels of activity. Adult neurogenesis disrupts this balance through the continuous addition of new granule cells (GCs) that display high excitability while develop and connect within the preexisting host circuit. The dynamics of the connectivity map for developing GCs in the local inhibitory networks remains unknown. We used optogenetics to study afferent and efferent synaptogenesis between new GCs and GABAergic interneurons expressing parvalbumin (PV-INs) and somatostatin (SST-INs). Inputs from PV-INs targeted the soma and remained immature until they grew abruptly in &gt;4-week-old GCs. This transition was accelerated by exposure to enriched environment. Inputs from SST-INs were dendritic and developed slowly until reaching maturity by 8 weeks. Synaptic outputs from GCs onto PV-INs matured faster than those onto SST-INs, but also required several weeks. In the mature dentate network, PV-INs exerted an efficient control of GC spiking and were involved in both feedforward and feedback loops, a mechanism that would favor lateral inhibition and sparse coding. Our results reveal a long-lasting transition where adult-born neurons remain poorly coupled to inhibition, which might enable a parallel streaming channel from the entorhinal cortex to CA3 pyramidal cells.</jats:p>

section_content: Activity-dependent changes in synaptic connectivity are thought to underlie learning and long-term memory storage. In the dentate gyrus of the mammalian hippocampus, including humans, plasticity also involves the generation of new neurons that develop, integrate and contribute to information processing (Goncalves et al., 2016; Mongiat and Schinder, 2011; Moreno-Jimenez et al., 2019; van Praag et al., 2002; Zhang et al., Retroviral labeling was used to express red fluorescent protein (RV-RFP) in newly generated GCs of the same mice. PV Cre ; CAG floxStopChR2EYFP mice labeled a homogeneous neuronal population that expressed the calcium buffer parvalbumin. Their bodies were localized primarily in the GCL, their axons spread along the GCL, and they displayed high spiking frequency (>100 Hz), typical of GABAergic basket interneurons (Fig. 1A, B ; Fig. S1 ). 

SST Cre ; CAG floxStopChR2EYFP mice labeled neurons that expressed the neuropeptide somatostatin, localized primarily in the hilar region, and displaying variable spiking patterns, corresponding to a heterogeneous population of GABAergic interneurons (Fig. 1E, F ; Fig. S2 ). 

Stereotaxic surgery was performed in 6-7-week-old mice to deliver a RV-RFP in a cohort of new GCs. 

ChR2-expressing INs were reliably activated using brief laser pulses (0. 2 ms), which elicited spikes with short onset latency (Fig. S3A-C, Fig. S4A-C ). Whole cell recordings were performed on RFP-GCs in acute slices at 2-8 weeks post injection (wpi). Laser stimulation of PV-INs elicited inhibitory postsynaptic currents (IPSCs) in RFP-GCs that were completely abolished by the GABAA receptor antagonist picrotoxin (100 µM), but were not affected by the ionotropic glutamate receptor blocker kynurenic acid (KYN, 6 mM) (Fig. 1C ; Fig. S3D, E ). 

Together with the fast IPSC onset, these data reveal that PV-INs make monosynaptic GABAergic contacts onto adult-born CGs (Fig. S3H ). Activation of ChR2-PVs reliably elicited IPSCs already in 2 wpi GCs, but responses displayed small amplitude and slow kinetics, typical of immature synapses (Fig. S3F, G ). As GC development progressed, the amplitude of postsynaptic responses increased and kinetics became substantially faster, as revealed by the reduction of half-width and rise time, particularly in the window between 4 and 6 wpi (Fig. 1D ; Fig. S3E-J ). In fact, 4 weeks can be visualized as a transition point with two split populations where some GCs display slow rise time and others have already became fast. Remarkably, while synapse formation from PV-INs to GCs was initiated early in development (before 2 wpi), synaptic maturation was only apparent at >6 wpi, when IPSCs reached fastest kinetics and maximal amplitude. 

Interestingly, the age-dependent growth in IPSC amplitude was mainly due to an increased quantal size rather than changes in the number of synaptic contacts; no differences were found in the number of functional synapses between young and mature GCs, measured as the ratio between IPSC in saturation and unitary IPSC amplitude (Fig. S3K-M ). These results demonstrate a slow age-dependent maturation of the PV-IN to GC synapse. 

ChR2-SSTs also formed functional monosynaptic contacts onto new GCs as early as 2-3 wpi (Fig. 1G -J; Fig. L, M ). At these developmental stages, activation of SST-INs reliably elicited IPSCs with small amplitude and slow kinetics (Fig. S4H-O ). Two types of responses were distinguished based on kinetics, coefficient of variation of the amplitude (Fig. S4D, E ) and reversal potential; one slow component observed at a depolarized membrane potential, and one fast that was visualized at hyperpolarized potentials. To determine their nature, their amplitude and kinetics were measured by holding the membrane at the reversal potential of the alternate component. Both responses displayed age-dependent increase in IPSC amplitude (Fig. 1G-J ). However, the kinetic features for both components remained fundamentally unchanged through GC maturation (Fig. 1H,J; Fig. S4H-O ). Finally, mature synaptic properties were only observed in GCs at >8 wpi. Together, these results show that new GCs receive monosynaptic GABAergic inputs from PV-INs and SST-INs early in development, and both connections become gradually strengthened along maturation, acquiring mature synaptic properties at 6 to 8 weeks of age.
section_name: INTRODUCTION

section_content: In whole-cell recordings, the intracellular Cl -concentration ([Cl -]i) near the soma is imposed by the recording patch pipette, whereas Cl -transporters in distal dendritic compartments can overcome the pipette load and maintain physiological levels of [Cl -]i (Khirug et al., 2005). This gradient in [Cl -]i results in differences in the reversal potential of GABA-mediated currents along the somato-dendritic axis (Laplagne et al., 2007; Pearce, 1993). To reveal the subcellular localization of the PV-IN to GC synapse, we monitored the reversal potential of optogenetically activated currents by means of whole-cell recordings under conditions that resulted in an equilibrium potential for [Cl -]i of -30 mV at perisomatic compartments. Extracellular stimulation of GABAergic axons in the outer molecular layer (OML) was used to activate distal dendritic inputs (Laplagne et al., 2007) (Fig. 2A-E ). Thus, activation of PV-INs elicited fast IPSCs with depolarized reversal potential for all neuronal ages, suggesting that synaptic localization was close to the recording compartment (soma) and remained stable throughout GC development. In contrast, OML stimulation evoked slow IPScs with hyperpolarized reversal potentials as expected for the physiological [Cl -]i (~-70 mV), suggesting that they originated in the dendritic compartment, located distally from the recording site. In fact, fast-inward and slow-outward IPSCs were simultaneously observed at intermediate membrane potentials (Vh=-50 mV) when ChR2-PVs and OML stimulation were combined (Fig. 2B ). These results demonstrate a perisomatic origin for PV-IN-mediated IPSCs at all GC ages. Stimulation of ChR2-SSTs elicited mixed inward and outward IPSCs in adult-born GCs held at -50 mV (Fig. 2F-H ), arising from synaptic responses originated in compartments with different distances to the soma. Indeed, the fast current exhibited a depolarized reversal potential (~-30 mV), consistent with a proximal localization, whereas the slow current reversed at more negative potentials (up to ~-60 mV), suggesting a distal contact. Proximal IPSCs maintained similar values for reversal potential through GC development, while distal IPSCs showed a subtle but progressive hyperpolarization, consistent with the observation that control of [Cl -]i homeostasis improves during neuronal development (Khirug et al., 2005) (Fig. 2I ). We conclude that ChR2-SSTs establish functional synapses onto new GCs with distinct proximal and distal localizations.
section_name: Differential subcellular localization of synapses formed by PV-INs and SST-INs

section_content: During normal behavior, networks of principal neurons and interneurons exhibit complex patterns of activation and undergo spiking discharges in a wide range of frequencies. Under these conditions, synapses are subject to short-and long-lasting activity-dependent modifications of synaptic transmission (Hsu et al., 2016; Lee et al., 2016; Pardi et al., 2015). To investigate how repetitive activity impinges on postsynaptic responses in developing GABAergic synapses, ChR2-PVs or ChR2-SSTs were stimulated by brief trains (5 laser pulses at 20 Hz) and whole-cell recordings were performed in developing GCs. Responses to ChR2-PVs stimulation displayed short-term depression that became more pronounced as GCs matured (Fig. 3A-D ). 

These results reveal changes in presynaptic release machinery along synaptic maturation. 

SST-IN to GC synapses of proximal and distal locations were discriminated by their reversal potential and their responses upon repetitive stimulation were analyzed separately. Activation of ChR2-SSTs by brief trains (20 Hz) induced a marked short-term depression in proximal IPSCs, which became more pronounced in more mature GCs (Fig. 3E-H ). In contrast, distal IPSCs showed stable pulse amplitudes along the train and no signs of depression for any of the GC ages (Fig. 3I-L ). These results further support the conclusion that proximal and distal responses evoked by SST-INs belong to functionally different synapses.
section_name: Short-term plasticity of GABAergic responses

section_content: The impact of PV-INs and SST-INs on spiking activity of the granule cell layer (GCL) was monitored in field recordings of excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the medial performant path (mPP) (Fig. 4A ). In these recordings, the area of the population spike (pop-spike) is proportional to the number of active GCs, and the fEPSP slope reflects the strength of the synaptic input. Paired activation of ChR2-PVs with mPP stimulation modulated the fEPSP response; increasing laser power recruited more PV-INs, which resulted in a progressive and reliable reduction of the pop-spike (Fig. 4B ). SST-INs were also able to control GCL recruitment, but they exerted a smaller effect over the pop-spike than PV-INs. Maximum inhibitory effects were found when PV-INs or SST-INs and mPP axons were simultaneously stimulated (Fig.
section_name: GABAergic interneurons control activity in the granule cell layer

section_content: In addition, inhibition of the pop-spike by PV-IN activation was more efficient and acted over a broader time interval compared to SST-INs, in concordance with their larger IPSCs and perisomatic targeting. These data demonstrate that both types of INs can modulate spiking in the GCL, although control by PV-INs is more reliable, probably due to the somatic localization of their synapses.
section_name: 4C-F).

section_content: To map the networks of GABAergic interneurons activated by adult-born GCs, we used retroviruses to selectively express ChR2-GFP in cohorts of new GCs (ChR2-GCs) at different stages of development (3 to 11 wpi). Reliable activation of ChR2-GCs was achieved by laser stimulation (1-ms pulses; Fig. S5A-C ), allowing the study of synaptic responses in INs. PV Cre ;CAG floxStop-tdTomato and SST Cre ;CAG floxStop-tdTomato were used to label PV-INs and SST-INs, respectively, and perform whole-cell recordings of excitatory postsynaptic currents (EPSCs; Fig. 5A-J ). Activation of developing GCs elicited glutamatergic excitatory postsynaptic currents in both PV-INs and SST-INs, but no functional connections were detected before 4-6 wpi (Fig. S5D-Q ). When responses occurred, they displayed short onset latency (Fig. S5H, O ) and were blocked by KYN (not shown), indicating that these glutamatergic connections are monosynaptic. At early ages, GCs elicited a large proportion of transmission failures. As neurons became more mature, the proportion of failures decreased to reach a plateau that occurred at 6 weeks for PV-INs and >8 weeks for SST-INs (Fig. 5E, J ). 

GCs activation in awake behaving rodents can cover a broad range of discharge activity. To better characterize the physiological significance of GC to IN connections, we delivered brief trains of laser stimulation (5 pulses at 20 Hz) onto ChR2-GCs. In contrast to the depression that was typically observed in IPSCs (Fig. 3 ), EPSCs displayed strong facilitation at all developmental stages in both PV-INs and SST-INs (Fig. 5B-D,G-I ). Facilitation resulted in decreased failures in synaptic transmission along subsequent pulses within a train, suggesting that repetitive firing in GCs is more likely to activate GABAergic INs than individual spikes. 

In fact, train stimulation revealed connections that remained silent when assessed by individual stimuli (Fig. 5E, J ). Taking into account the EPSC success rate, which represents the likelihood of finding functional synaptic connections, our data indicate that immature GCs are reliable in establishing connections onto PV-INs, while SST-INs receive sparse inputs.
section_name: Functional synaptogenesis of GC outputs onto local interneurons

section_content: Dentate gyrus INs participate in feedforward (FFI) and feedback (FBI) inhibitory microcircuits, with functional impact in both the GCL and CA3. To dissect the participation of PV-INs and SST-INs in those inhibitory loops, we designed an experiment that allowed both an efficient recruitment of IN spiking and the assessment of feedback and feedforward pathways. 

We thus combined whole-cell recordings in PV-or SST-INs with simultaneous field recordings in the GCL, and measured responses to electrical stimulation of the mPP to a level that evoked a reliable pop-spike (~50 % of maximum response). When recording from PV-INs, mPP activation typically elicited two action potentials, one occurring before the peak of the pop-spike and another occurring after a brief delay (Fig. 6A-C ). This sequence suggests that the first spike was evoked directly by mPP activation, while the second one was evoked by activation of the heterogeneous GC population (including both mature and developing neurons). To test this possibility, we used DCG-IV, an agonist of group II metabotropic glutamate receptors that reduces release probability in mossy fiber terminals and in mPP terminals in the GCL (Kamiya et al., 1996; Macek et al., 1996). DCG-IV reduced the amplitude of the fEPSP response, eliminating the pop-spike, which in turn abolished the second PV-IN spike without altering the first one (Fig. 6D, E ). Subsequent application of KYN blocked the first spike. Together, these results demonstrate that the same individual PV-INs are recruited by mPP axons that activate a feedforward inhibitory loop and by GCs that recruit a feedback loop in the GCL. 

In contrast, the same assay showed that SST-INs were primarily recruited to trigger action potentials after the pop-spike (12/12 neurons), with only a small proportion activated before (3/12, Fig. 6F-H ). Thus, SST-INs mainly participate in feedback inhibition, while their participation in the feedforward inhibitory loop is scarce. Together, these results demonstrate that cortical activity reaching the dentate gyrus through mPP axons recruit feedforward inhibition through PV-INs that exert tight control over GC spiking (Fig. 4 ). In turn, GCs activate a feedback inhibitory loop by PV-INs, now acting in concert with SST-INs to provide finely tuned activation of the GCL.
section_name: Contribution of PV-INs and SST-INs to inhibitory loops

section_content: We have previously shown that experience in enriched environment (EE) can promote early development of newly generated GCs, with PV-INs acting as key transducers from behavior to local circuit rearrangement (Alvarez et al., 2016). We now investigated whether experience can also influence synaptic connections of PV-INs onto more developed GCs that may already be involved in information processing. RV-GFP was delivered into PV Cre ; CAG floxStopChR2EYFP mice that were then exposed to regular cages or switched to EE for 2 weeks. Synaptic transmission in response was analyzed at 4 wpi (Fig. 7A, B ). Single laser pulses elicited IPSCs of similar amplitude in both conditions, but responses obtained from EE mice displayed faster kinetics, consistent with a more mature synapse (Fig. 7C-F ). This difference was more evident when ChR2-PVs were stimulated with 50-Hz trains. In control mice, GCs presented individual responses to repetitive pulses that accumulate along the train, finalizing with a slow decay after the last stimulus. In contrast, signals from mice exposed to EE displayed faster kinetics, resulting in a progressive depression of the synaptic response (Fig.
section_name: Modulation of perisomatic inhibition by experience

section_content: These results demonstrate that transmission in this developing synapse is sensitive to experience in a manner that favors a mature behavior.
section_name: 7G-I).

section_content: The function that neurons acquire in a given circuit depend on their intrinsic properties, relevant for signal integration, and their connections, which determine network dynamics. It has been proposed that developing GCs play unique functional roles in DG computation (Clelland et al., 2009; Kim et al., 2012; Kropff et al., 2015). We found a slow development of connectivity between new GCs and GABAergic INs, which conveys immature GCs the property to behave as computational modules with rules that vary along development and are different to those of mature GCs. PV-IN synapses onto new GCs are formed early (Song et al., 2013) but, as shown here, synaptic responses mature during several weeks, becoming increasingly stronger and faster. The slow IPSC kinetics exhibited by young synapses may well explain the hyperexcited neuronal behavior described previously in 4-week old GCs (Marin-Burgin et al., 2012; Pardi et al., 2015). 

When IN activity occurs in bursts instead of single spikes (train stimulation in our experiments), synaptic maturation results in a reduction of the integration time, transforming slow responses with sustained postsynaptic currents (in 3 to 4-week-old GCs), into faster postsynaptic responses with pronounced depression (8-week-old GCs). We found that this synapse is sensitive to behavioral stimuli; experience in EE accelerated the transition to the fast responsiveness typical of a mature synapse. Because PV-INs are involved both in FBI and FFI, a change in kinetics like the one reported here would produce substantial changes at the network level (Hu et al., 2014). 

Optical stimulation of SST-INs generated two types of responses that differed in kinetics and reversal potential when measured in mature GCs. We observed a fast response with a depolarized reversal potential that revealed proximal localization, and a slower response with a more hyperpolarized reversal potential that corresponded to dendritic (distal) distribution. ChR2-SST stimulation using 20 Hz trains resulted in substantial synaptic depression in proximal responses but stable amplitude in distal synaptic currents, which strengthened the idea of separate proximal and distal responses. It is unclear whether they correspond to different axons of the same INs or different INs altogether. Electrophysiological characterization of intrinsic properties revealed four groups of SST-INs with distinctive spiking patterns and input resistance (Fig. S2 ). In this context, we speculate that proximal and distal synapses derive from individual populations of SST-INs that target different cellular compartments. In fact, two subtypes of SST-INs were recently reported in the DG and might underlie the responses observed here; hilar-perforant-path-associated INs with axon fibers in the molecular layer that make distal synapses, and INs with axons in the hilus that provide perisomatic inhibition (Yuan et al., 2017). 

Activation of mature GCs recruit feedback loops that limit activation of the GCL through lateral inhibition (Espinoza et al., 2018; Temprana et al., 2015). They also recruit mossy cells that activate a range of interneuron cell types, with a preference for basket cells (Scharfman, 2018). As shown here using direct optogenetic activation, both PV-INs and SST-INs can limit activation of the GCL and could mediate the FBI triggered by GCs, although PV-INs are more efficient (Fig. 4 ), probably due to the localization and strength of their output contacts and their high degree of network connectivity (Espinoza et al., 2018). We performed two experiments to monitor FBI in the network. First, optogenetic activation of adult-born GCs revealed that both PV-INs and SST-INs are direct targets of new GCs with increasing synaptic strength as they approach maturation. Second, activation of PV-INs and SST-INs occurred following the GCL pop-spike elicited by stimulation of mPP axons, and did not occur when the pop-spike was blocked by DGC IV (Fig. 6 ). Together, these results show that the FBI loop that controls activity of the GCL involves both INs. As expected, the feedforward loop was activated by mPP stimulation independently of the presence of the pop-spike and primarily involved PV-INs rather than SST-INs, consistent with a higher efficacy of the connectivity of mPP axons towards basket PV-INs (Hsu et al., 2016). 

Jonas and collaborators have recently obtained a thorough map of the dentate gyrus network assessing the connectivity between mature GCs and different types of GABAergic INs. They found that PV-INs are the most extensively connected type of GABAergic IN and, in this network, inhibition is much more abundant than excitation (Espinoza et al., 2018). They also showed that PV-INs preferably contact GCs from which they receive no input, thus favoring lateral over recurrent inhibition by about 10 fold. It was proposed that such architecture favors a winner-takes-all model in which GCs that are strongly recruited during a particular behavior will dominate activity in the dentate gyrus. This model would be compatible with pattern separation, a network computation where similar inputs are converted into non-overlapping patterns of network spiking and might be crucial for hippocampal functions that include spatial navigation and contextual discrimination (Drew et al., 2013; McAvoy et al., 2015). Interestingly, lateral inhibition would require the coincident activation of a number of excitatory inputs from GCs to reach spiking threshold of PV-INs (Espinoza et al., 2018). Our finding that the same individual PV-INs participate in feedforward and feedback inhibition suggests that excitation from mPP axons might contribute to lower the threshold for efficient activation of PV-INs by a sparse population of active GCs. 

Using a simple computational model, we have proposed that adult neurogenesis may favor the acquisition of non-overlapping input spaces through the delayed coupling to inhibition of developing GCs (Kropff et al., 2015; Temprana et al., 2015). Our new results demonstrate that, during several weeks, developing GCs remain poorly coupled to the IN networks both at the input and output levels, escaping lateral inhibition and creating a parallel channel for the information flow from entorhinal cortex to CA3 (Marin-Burgin et al., 2012; Temprana et al., 2015). During this period, activity-dependent synaptic modifications might refine input and output connections required to encode relevant information on the acquired task (Ge et al., 2007b; Gu et al., 2012). As we have shown here, experience modulates this network at the level of the PV-IN synapse during a critical period of high excitability in new GCs. With time, inhibition becomes more efficient and new GCs are more sparsely activated.
section_name: DISCUSSION

section_name: MATERIALS AND METHODS

section_content: Genetically modified mice Pvalb tm1(cre)Arbr mice (Hippenmeyer et al., 2005), kindly provided by S. Arber, Sst tm2. 1(cre)Zjh/J mice (Taniguchi et al., 2011), and CAG floxStop-tdTomato (Ai14) (B6;129S6-Gt(ROSA)26Sor tm14(CAG- tdTomato)Hze /J) conditional reporter line (Madisen et al., 2010), obtained from Hongkui Zeng, were crossed to generate PV Cre ; CAG FloxStopTom mice and SST Cre ; CAG FloxStopTom mice to label PV-and SST-expressing GABAergic interneurons (Tom-PV and Tom-SST), respectively. Pvalb tm1(cre)Arbr and Sst tm2. 1(cre)Zjh/J mice were also crossed with CAG floxStopChR2-EYFP (Ai32) (Gt(ROSA)26Sor tm32(CAGCOP4*H134R/EYFP)Hze /J) mice from Jackson Laboratories, to generate PV Cre ;CAG FloxStopChR2 and SST Cre ; CAG FloxStopChR2 mice. Mice were maintained in C57Bl/6J background. 

Genetically modified mice of either sex were used at 6 -7 weeks of age, housed at 2 -4 mice per cage. Running wheel housing started 2-3 days before surgery and continued until the day of slice preparation, to maximize the number of retrovirally transduced neurons. For surgery, mice were anesthetized (150 µg ketamine/15 µg xylazine in 10 µl saline/g), and virus (1 -1. 2 µl at 0. 15 µl/min) was infused into the dorsal area of the right dentate gyrus using sterile microcapillary calibrated pipettes and stereotaxic references (coordinates from bregma: -2 mm anteroposterior, -1. 5 mm lateral, -1. 9 mm ventral).
section_name: Animals and surgery for retroviral delivery

section_content: A replication-deficient retroviral vector based on the Moloney murine leukemia virus was used to specifically transduce adult-born granule cells as done previously (Marin-Burgin et al., 2012; Piatti et al., 2011). Retroviral particles were assembled using three separate plasmids containing the capside (CMVvsvg), viral proteins (CMV-gag/pol) and the transgenes: CAG-RFP or channelrhodopsin-2 (ChR2; Ubi-ChR2-EGFP retroviral plasmid, kindly provided by S. Ge, SUNY Stony Brook). Plasmids were transfected onto HEK 293T cells using deacylated polyethylenimine. Virus-containing supernatant was harvested 48 h after Whole-cell voltage-clamp recordings were performed at a holding potential (Vh) of -70 mV, except for the experiment to study the reversal potential of GABAergic current onto GCs (Fig. 2 ). For GCs, series resistance was typically 10-20 MΩ, and experiments were discarded if higher than 25 MΩ. For INs, series resistance was typically 5-10 MΩ, and experiments were discarded if higher than 15 MΩ. 

Recording target. Adult-born GCs expressing RFP or ChR2 were binned in the following age groups: 13-14 dpi (2 wpi), 20-22 dpi (3 wpi), 27-30 dpi (4 wpi), 40-44 dpi (6 wpi), 54-60 dpi (8 wpi) and 75-77 dpi (11 wpi). In previous work we have compared mature neurons born in 15-day-old embryos (which populate the outer granule cell layer), 7-day-old pups and adult mice, finding no functional differences among neuronal groups (Laplagne et al., 2006). Therefore, unlabeled neurons localized in the outer third of the granule cell layer were selected here as mature controls. Recorded neurons were visually identified in the granule cell layer Field recordings. Medial perforant path (mPP) stimulation was performed by placing a steel monopolar electrode in the middle of the molecular layer, and current pulses ranging from 10 to 150 μA (0. 2 ms) were applied at 0. 07 Hz. The recording microelectrode was placed in the GCL to record the population spike (popspike) in response to mPP stimulation (Marin-Burgin et al., 2012). Experiments were performed at stimulus intensities that evoked 30 -55 % of maximal pop-spike amplitude. Population activity was recorded by several subsequent trials until stable pop-spike amplitude was obtained. At that moment, a laser pulse (0. 2 ms) was paired to mPP stimulation at different times (as indicated), alternating 5 consecutives trials with the laser on and 5 trials off. 

Reversal potential of GABAergic currents onto GCs. Outer molecular layer (OML) stimulation was performed by placing a steel monopolar electrode in the outer third of the molecular layer, at least 300 µm away from the recording site. Current pulses ranging from 40 to 100 μA (0. 2 ms) were applied at 0. 05 Hz to recruit GABAergic current of dendritic origin. In addition, IPSCs evoked onto GCs in response to optogenetics stimulation of ChR2-INs were measured. This study was performed in presence of kynurenic acid.
section_name: Retroviral vectors

section_content: Two weeks after RV infusion, mice were exposed for two weeks to an EE consisting of a large cage (80 cm x 40 cm x 20 cm) containing tunnels of different lengths, toys, and two running wheels. The location of the objects in the EE were changed after a week of exposure. Control mice were left in a regular cage with two running wheels (consistent with our experiments). At 4 wpi, animals were prepared for electrophysiological recordings
section_name: In vivo assays. EE Exposure

section_content: Immunostaining was performed in 60 µm free-floating coronal sections throughout the brain from six weeks old PV Cre and SST Cre ; CAG FloxStopChR2 mice. Antibodies were applied in TBS with 3% donkey serum and 0. 25% Triton X-100. Triple labeled immunofluorescence was performed using the following primary antibodies: GFP (Green Fluorescent Protein, Chicken antibody IgY Fraction 1:500, Aves Labs Inc. ), PV (mouse anti-Parvalbumin monoclonal antibody, 1:3000, Swant) and SOM (rat-anti Somatostatin monoclonal antibody 1:250, Millipore). The following corresponding secondary antibodies were used: donkey anti-chicken Cy2, donkey anti-mouse Cy5 and donkey anti-rat Cy3, (1:250; Jackson ImmunoResearch Laboratories). Incubation with Dapi (10 minutes) was applied to avoid fluorescence bleaching when slice characterization was performed.
section_name: Immunofluorescence

section_content: Sections from the hippocampus (antero-posterior, -0. 94 to -3. 4 mm from bregma) according to the mouse brain atlas (Paxinos and Franklin, 2004) were included. Images were acquired using Zeiss LSM 510 Meta microscope (Carl Zeiss, Jena, Germany). Analysis of antibody expression was restricted to cells with fluorescence intensity levels that enabled clear identification of their somata. Images were acquired (40X, NA 1. 3, oil-immersion) and colocalization for the three markers was assessed in z-stacks using multiple planes for each cell. Colocalization was defined as positive if all markers were found in the same focal plane.
section_name: Confocal microscopy

section_content: Analysis of all recordings was performed off-line using in-house made Matlab routines. Intrinsic Properties. Membrane capacitance and input resistance were obtained from current traces evoked by a hyperpolarizing step (10 mV, 100 ms). Spiking profile was recorded in current-clamp configuration (membrane potential was kept at -70 mV by passing a holding current) and the threshold current for spiking was assessed by successive depolarizing current steps (10 pA for GCs and 50 pA for INs; 500 ms) to drive the membrane potential (Vm) from resting to 0 mV. Action potential threshold was defined as the point at which the derivative of the membrane potential dVm/dt was 5 mV/ms (data not shown). AP amplitude was measured from threshold to positive peak and after-hyperpolarization amplitude, from threshold to negative peak during repolarization. Time between consecutive spikes (interspike interval, ISI) was measured from peak to peak. Instantaneous frequency was calculated from ISI and adaptation ratio was defined as the ISI ratio between the third spike and the last spike. To perform the whole spiking characterization, we measured the threshold current intensity and a stimulus intensity three times higher than the threshold was used to evaluate all the parameters. Postsynaptic Currents. Statistical methods were used to differentiate laser-responsive cells and laser-evoked events from spontaneous activity using in-house Matlab routines. Events were identified as peaks in the lowpass filtered current (<250 Hz) when exceeded 4 standard deviations of the noise level (measured at >500 Hz high-pass filtered current). The onset of an event was defined as the time in which 10 % of the maximum amplitude was reached in the unfiltered signal. Once all events were identified, a cell was classified as responsive to laser stimulation if there was a tendency greater than chance for events to accumulate within a time window of 12 ms after laser stimulation (p < 0. 05). In order to achieve such a classification, the probability distribution of a similar accumulation of spontaneous events happening by pure chance was determined for each cell using a 2000 step shuffling procedure. Once a cell was classified as responsive to laser, spontaneous and laser-evoked events were differentiated. 

In all cases, reported PSCs values for peak amplitude correspond to the product of the mean value for positive trials and the probability of success, taken as the fraction of trials in which an evoked response was observed. 

The rise time was calculated from 20% to 80% (EPSC) or 70% (IPSC) of peak amplitude, and decay time was calculated from 80% (EPSC) or 70% (IPSC) to 30%. 

Response to repetitive stimulation. The charge of laser evoked events during repetitive stimulation was measured within a time window equal to the distance between two consecutive laser pulses, starting at the corresponding pulse. To analyze short-term plasticity, we calculated the charge ratio during repetitive stimulation. To perform this normalization, we used the response evoked by the first pulse for INs onto GCs synapses and the charge related to the last pulse for GCs onto INs synapses.
section_name: Data analysis

section_content: Unless otherwise specified, data are presented as mean ± SEM. Normality was assessed using Shapiro-Wilk's test, D'Agostino & Pearson omnibus test, and Kolmogórov-Smirnov's test, at a significance level of 0. 05. A distribution was considered as normal if all tests were passed. When a data set did not satisfy normality criteria, non-parametric statistics were applied. Two-tailed Mann-Whitney's test was used for single comparisons, two-tailed Wilcoxon matched pairs signed rank test was applied for paired values, Kruskal-Wallis test by ranks was employed to compare multiple unmatched groups and Friedman test followed by Dunn's post test was used to compare multiple matched groups. For normal distributions, homoscedasticity was assessed using Bartlett's test and F-test, at a significance level of 0. 05. For homogeneous variances, twotailed t-test was used for single comparisons, and ANOVA test followed by post-hoc Bonferroni's test was used for multiple comparisons. Two sample Kolmogorov-Smirnov test was applied to compare cumulative distributions. Two-tailed Fisher's exact test (small sample size) or Chi-square test were used in the analysis of contingency tables.
section_name: Statistical analysis

section_name: ACKNOWLEDGMENTS

section_content: We thank Verónica Piatti, the members of the A. F. S. lab and Guillermo Lanuza lab for insightful discussions. A. F. S. is investigator in the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). S. M. Y. and A. I. G. were supported by CONICET fellowships. This work was supported by grants from the Argentine Agency for the Promotion of Science and Technology ( PICT2015-3814 and PICT2016-0675 ), Howard Hughes Medical Institute (# 55007652 ), and NIH ( R01NS103758 ) to A. F. S.
section_name: ACKNOWLEDGMENTS

section_content: Chi-square test (p=0. 748). (I) The spatial distribution for INs presenting each spiking profile is similar except for FS, which is mainly located in the GCL.