Regulation of the Hippocampal Network by VGLUT3-Positive CCK- GABAergic Basket Cells

Hippocampal interneurons release the inhibitory transmitter GABA to regulate excitation, rhythm generation and synaptic plasticity. A subpopulation of GABAergic basket cells co-expresses the GABA/glycine vesicular transporters (VIAAT) and the atypical type III vesicular glutamate transporter (VGLUT3); therefore, these cells have the ability to signal with both GABA and glutamate. GABAergic transmission by basket cells has been extensively characterized but nothing is known about the functional implications of VGLUT3-dependent glutamate released by these cells. Here, using VGLUT3-null mice we observed that the loss of VGLUT3 results in a metaplastic shift in synaptic plasticity at Shaeffer’s collaterals – CA1 synapses and an altered theta oscillation. These changes were paralleled by the loss of a VGLUT3-dependent inhibition of GABAergic current in CA1 pyramidal layer. Therefore presynaptic type III metabotropic could be activated by glutamate released from VGLUT3-positive interneurons. This putative presynaptic heterologous feedback mechanism inhibits local GABAergic tone and regulates the hippocampal neuronal network.


INTRODUCTION
The hippocampus is centrally engaged in the regulation of spatial and emotional memory. In the hippocampus, different classes of interneurons interact with specific cellular domains of pyramidal cells to modulate cell firing, plasticity and network activity. These interneurons form a complex group composed of up to 21 types of cells displaying a variety of molecular expression profiles and distinct morphological and physiological features (Somogyi and Klausberger, 2005;Klausberger and Somogyi, 2008). Basket cells are interneurons that form a dense plexus of inhibitory terminals on the soma of pyramidal cells. Two types of basket cells have been identified: the classic parvalbumin-positive (PV) fast-firing interneurons and the regular-firing cholecystokininexpressing (CCK) interneurons. PV basket interneurons contribute to the generation of oscillations and are considered the "clocks" of the hippocampus (Cobb et al., 1995;Bartos et al., 2007;Korotkova et al., 2010;Amilhon et al., 2015). The functions of CCK interneurons are less well known; however, anatomical and physiological evidence suggests that these interneurons are intensely innervated by neurons from the raphe and respond to cannabinoids (Freund and Katona, 2007). CCK interneurons are thought to be centrally engaged in mood regulation (Freund and Katona, 2007). Interestingly, some of the CCK interneurons express the atypical vesicular glutamate transporter 3 (VGLUT3) , which indicate that these interneurons may release both glutamate and GABA from their terminals contacting pyramidal cells. However, the role of this dual transmission of GABA and glutamate by basket cells remains unknown.
Unlike VGLUT1 and VGLUT2, VGLUT3 is found mostly in neurons that use transmitters other than glutamate, such as acetylcholine (ACh), serotonin (5-HT) or GABA (for review, see El Mestikawy et al., 2011). VGLUT3 facilitates the vesicular accumulation and transmission of ACh and 5-HT via a mechanism known as "vesicular synergy" (Gras et al., 2008;Amilhon et al., 2010) and VGLUT3-bearing terminals endow ACh and 5-HT fibers with the capacity to use glutamate in fast excitatory neurotransmission (Varga et al., 2009;Higley et al., 2011). Furthermore, it was recently established that glutamate released by VGLUT3-positive ACh interneurons negatively regulate dopamine release in the nucleus accumbens through metabotropic glutamate receptors (mGLURs), (Sakae et al., 2015). Overall, the expression of VGLUT3 in ACh-and 5-HT-expressing neurons contributes to both slow and fast neurotransmission and has significant effects on locomotor activity, reward and anxiety.
Little is known about the paradoxical co-transmission of GABA and glutamate and its putative effects on hippocampal network. The main goal of this work was to describe the molecular, cellular, and integrated properties of VGLUT3 in the hippocampus. We observed that VGLUT3 enhances vesicular GABA filling and endows these inhibitory interneurons with the ability to putatively co-release glutamate. In addition, we showed that VGLUT3 deletion alters synaptic plasticity and metaplasticity as well as theta oscillations in the hippocampus. These observations suggest that glutamate released by VGLUT3expressing basket cells could inhibit GABAergic synaptic transmission by activating presynaptic metabotropic glutamate receptors (mGLURs).

Animals
Animal care and handling was performed according to the guidelines of the Canadian Council on Animal Care 1 and approved by the Facility Animal Care Committee of the Douglas Research Center. The VGLUT3 −/− mouse line used in the present study was obtained as previously described (Gras et al., 2008;Amilhon et al., 2010) and backcrossed with 10 generations 1 http://ccac.ca/en_/standards/guidelines (F10) of mice with a C57BL/6N background (99% homozygous). Heterozygous mice were bred to generate VGLUT3 −/− mice and wild type littermates.
The VGLUT3 flox/flox mice Slc17a8 mutant mouse line was established at Phenomin -iCS (Phenomin-Institut Clinique de la Souris-, Illkirch, France 2 ). In brief, the targeting vector was constructed as follows ( Figure 4A). A 0.8 kb fragment encompassing exon 2 was amplified by PCR (from C57Bl/6N ES cells genomic DNA) subcloned in a Phenomin-iCS proprietary vector. This Phenomin-iCS vector contains a LoxP site as well as a floxed and flipped Neomycin resistance cassette. A 4.5 kb fragment (corresponding to the 5 homology arm) and 3.1 kb fragment (corresponding to the 3 homology arms) were amplified by PCR and subcloned in step1 plasmid to generate the final targeting construct. The linearized construct was electroporated in C57Bl/6N mouse embryonic stem (ES) cells. After selection, targeted clones were identified by PCR using external primers and further confirmed by Southern blot with 5 and 3 external probes. Two positive ES clones were injected into BALB/cN blastocysts, and male chimeras derived gave germline transmission. Mice lacking VGLUT3 selectively in GABAergic neurons (VGLUT3 VIAAT−Cre−flox/flox ) were generated by breeding VIAAT −ires−cre mice (The Jackson laboratory, stock No: 016962) with VGLUT3 flox/flox mice.
Mice were housed in groups of three to four animals per cage under standard conditions: 22 • C and a 12 h light/dark cycle (8:00-20:00 light period) with food and water provided ad libitum. All precautions were taken to minimize the number of animals used and their suffering they may have had to undergo.

Autoradiography of Type I Cannabinoid Receptor (CB1)
CB1 autoradiography was performed as previously described (Herkenham et al., 1991). Briefly, fresh frozen brain sections (10 µm) were incubated for 3 h at RT • in a buffer containing Tris HCl (50 mM, pH7.4), bovine serum albumin (5%) and [ 3 H] CP-55 940 (10 nM, NEN, Boston, MA, USA) in the absence or presence of AM251 (10 mM, Tocris Bioscience) to determine total and non-specific binding, respectively. Slides were washed in Tris HCl (50 mM) containing bovine serum albumin (1%, fatty acid-free) for three times (30 min each) at 4 • C dipped in ice-cold distilled water. Dried sections were apposed to tritiumsensitive screen (BAS-TR Fuji Imaging) for a period of 7 days and developed with the FujiBAS 7000 PhosphoImager. Densitometry was analyzed with MCID software, using the standard curve generated from [ 3 H]-standards. Specific binding measured in each structure was determined by subtracting the non-specific binding image from that of total binding.

Radioactive In Situ Hybridization
In situ hybridization was performed as previously reported (Gras et al., 2008;Vigneault et al., 2015). Shortly, sense or antisense oligonucleotides specific for CB1 or for the floxed exon 2 of VGLUT3 (Tables 1, 2, generated by Oramacell) were labeled with [ 35 S]-dATP (GE Healthcare) using terminal deoxynucleotidyl transferase (GE Healthcare) to a specific activity of 1-3 × 10 8 dpm/mg. Mouse brain fresh frozen sections (10 µm) were incubated with hybridization medium (Oramacell, Paris, France) containing 1.4 µl of the labeled oligonucleotides, washed, dried and exposed to a BAS-SR Fujifilm Imaging Plate for 5 days. Plates were scanned with a Fujifilm BioImaging Analyser BAS-5000.

Electron Microscopy
VGLUT3 was detected with an electron microscope with a post-embedding immunogold method as previously described (Gras et al., 2002(Gras et al., , 2008. Sagittal brain sections of the hippocampus were incubated with guinea pig anti-VGLUT3 antiserum (1:1000). Immunolabeling was detected with goat anti-guinea pig IgGs conjugated to gold particles (1.4 nm in diameter; Nanoprobes, NY, USA, 1:100). After post-fixation (1% glutaraldehyde), the immunogold signal was enhanced with a silver enhancement kit (HQ silver, Nanoprobes; NY, USA). Ultrathin sections were examined with a transmission electron microscope (EM 912 OMEGA, ZEISS).

Immuno-Isolation of Synaptic Vesicles, Western Blot, and [ 3 H]glutamate Uptake Assay
Synaptosomes from hippocampus were prepared, and the LS1 fractions containing synaptic vesicles (SVs) were isolated as described previously (Zander et al., 2010). The LS1 fractions were incubated with * superparamagnetic beads (Dynabeads Pan Mouse IgG, Invitrogen, Life Technologies Inc., Burlington, ON, Canada) coupled to anti-synaptophysin, anti-VGLUT1 or anti-VIAAT antisera (all from Synaptic Systems, Göttingen, Germany). Beads without primary antibodies were used as negative controls. Bead-bound SVs were dissolved in Laemmli buffer, and the resultant protein pattern was analyzed by SDS-PAGE/Western blot (Zander et al., 2010) using anti-VGLUT1, anti-VIAAT, anti-synaptophysin and anti-VGLUT3 (all from Synaptic Systems, Göttingen, Germany). Immuno-signals were visualized by enhanced chemiluminescence. Densitometric analyses were performed using LabImage 1D 2006 (Kapelan Bio-Imaging Solutions) and quantified using standard curves obtained from the initial LS1 fraction. Vesicular [ 3 H]glutamate uptake analyses were performed as described previously (Zander et al., 2010;Frahm et al., 2015). LS1-immunoisolated SVs were bound to M-280 sheep anti-mouse beads (Invitrogen). Beads coupled with non-specific mouse IgG (Santa Cruz Biotechnologies) were used as negative controls. Uptake assays were performed with immunoisolated SVs and initiated by the addition of L-glutamate (49.5 µM, Sigma-Aldrich) and [ 3 H]-glutamate (0.5 µM, Hartmann Analytic GmbH, Germany). Uptake was stopped after 10 min at 25 • C, and unbound radioactivity was removed by centrifugation (435,000 × g, 10 min).
For patch-clamp experiments, evoked and spontaneous miniature inhibitory postsynaptic potentials (eIPSCs and mIPSCs, respectively) were recorded in CA1 pyramidal neurons. Recordings were performed using the whole cell configuration of the patch-clamp technique with borosilicate glass electrodes (tip resistance: 2.5-3.5 M ) filled with a "high chloride" intracellular solution (70 mM K-gluconate, 70 mM KCl, 2 mM NaCl, 2 mM MgCl 2 , 10 mM HEPES, 1 mM EGTA, 5 mM QX-314, 2 mM Mg-ATP, and 0.3 mM Na-GTP (Manseau et al., 2010)). The E Cl estimated using the Nernst equation was approximately −16 mV. Action potentials were blocked with QX-314 (5 mM). Under these conditions, monosynaptically elicited eIPSCs in a pyramidal neuron held at −70 mV were represented by an inward current. Fast glutamatergic transmissions were blocked with DNQX and AP5 for all patch-clamp recordings. The perfusion of bicuculline (5 µM) completely abolished the recorded inward current, which confirmed the GABAergic nature of the postsynaptic events (see Supplementary Figure S1). An inhibitory square pulse was applied before each stimulation to measure the input resistance and assess the quality of the patch. Recordings were excluded if the input resistance varied more than 20%. Although the theoretical ECl − was estimated to be approximately −16 mV by the Nernst equation, the measured ECl − was approximately −8 mV on the IV curve of GABAergic currents (n = 5, see Supplementary Figure S1). The membrane potential was clamped at −70 and −80 mV for eIPSC and mIPSC recordings, respectively. Spontaneous mIPSCs were recorded in the presence of tetrodotoxin (TTX, 0.5 µM). The eIPSCs were evoked monosynaptically by placing a borosilicate glass pipette in the stratum pyramidale 50 µm away from the recorded neuron. The signals were amplified with a Multiclamp 700B patch-clamp amplifier (Axon Instruments, Foster City, CA, USA), sampled at 20 kHz and filtered at 10 kHz using a Multiclamp 700B commander (Axon Instruments). The data were analyzed using pClamp (Axon Instruments) and Origin (Microcal Software, Northampton, MA, USA) software. A custom-written software was used for the mIPSC analysis (Manseau et al., 2010).
For long term potentiation/depression (LTP/LTD) recordings, field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the medial CA1 region with a borosilicate glass electrode (tip resistance: 2.7-3.3 M ) filled with ACSF at RT. The fEPSPs were evoked every 20 s with a 80 µsec stimulation at afferent Schaffer collateral fibers delivered through a stimulus isolator (WPI) with a bipolar tungstenbased electrode. The stimulation intensity was adjusted to obtain amplitudes close to 50% of the maximum response. The fEPSP baseline was monitored for 20 min. The synaptic transmission was potentiated either by a high-frequency tetanic stimulation (HFS) consisting of three trains of 100 pulses 20 s apart at a frequency of 100 Hz or a theta-burst stimulation (TBS) consisting of 3 trains 20 s apart of 10 bursts of 5 pulses at 100 Hz at a burst frequency of 5 Hz. The fEPSP slope is defined as the slope of the linear fit of the portion of the field curve between 10 and 60% of maximum peak amplitude. The synaptic transmission was depressed by a low-frequency stimulation consisting of 900 pulses at either 1 Hz (LFS-1 Hz) or 3 Hz frequency (LFS-3 Hz). When stated, bicuculline (5 µM) was added to the ASCF throughout the experiment to block GABA A receptor-mediated fast inhibitory transmission. Longterm potentation (LTP) or long-term depression (LTD) are defined as the comparison of the mean fEPSP slope over the last 10 min of recording after stimulation (30-40 min. for LTP, 50-60 min. for LTD) with the mean fEPSP slope over the 10 min prior to stimulation.

Oscillation Recordings in the Entire Hippocampus Preparation In Vitro
Intact hippocampi were isolated from 21-to 25-day-old mice as previously described (Khalilov et al., 1997;Tretter et al., 2012;Amilhon et al., 2015). Briefly, the hippocampi were dissected and placed in an oxygenated high-sucrose solution before use. Recordings were made with 4.0 mM KCl ACSF filled borosilicate pipette (Goutagny et al., 2009). Local field potentials (LFP) recorded in the CA1 stratum radiatum of the temporal part of the hippocampus were filtered online (0.1-500 Hz) and sampled at 5 kHz. Rhythmic oscillations ranging from 3 to 12 Hz were considered to represent theta oscillations.

Statistics
Statistical analyses were performed using Prism 4 software (Graph Pad). Each statistical test was appropriately chosen for the relevant experimental design. To compare two groups, a twotailed paired Student t-test or a non-parametric Mann-Whitney test was performed according to normality of the group distributions. For multiple group comparisons, a one-or twoway ANOVA or repeated measure ANOVA was performed followed by a post hoc Bonferroni comparison of the group means. Data are expressed mean ± SEM. * P < 0.05, * * P < 0.01, * * * P < 0.001. A descriptive table of all the statistics is provided in the Supplementary Tables S1-S5.

VGLUT3 Is Expressed by a Subclass of GABA Terminals in CA1
In the hippocampus, VGLUT3 is expressed by CCK-positive and more rarely by calbindin-positive interneurons Klausberger and Somogyi, 2008). We first determined the percentage of GABAergic interneurons expressing VGLUT3. A double FISH technique was used to label the mRNA encoding VGLUT3 and VIAAT ( Figure 1A) or glutamic acid decarboxylase (GAD, the GABA-synthesizing enzyme, Figure 1B). We found that 8.7 ± 0.3% of all GAD expressing GABAergic neurons in the hippocampus expressed VGLUT3 (Figure 1B). Similar results were obtained with VIAAT-expressing GABAergic neurons (not shown). Once synthesized, VGLUT3 is transported to the axon terminals surrounding the somata of neurons in the pyramidal layer of the hippocampus (Figures 1C,D). We determined by three-dimensional non-biased counting that 98 ± 1% of VGLUT3-positive terminals co-expressed VIAAT but only 13 ± 2% of VIAAT-positive terminals contained VGLUT3 in the CA1 pyramidal layer of the hippocampus. Electron microscopy showed that VGLUT3-immunoglod puncta were present over synaptic vesicle clusters in axon terminals forming axo-somatic symmetrical synapses ( Figure 1E). VGLUT3-positive terminals co-localized with gephyrin ( Figure 1F), a major scaffolding protein found at inhibitory synapses (Freund and Buzsaki, 1996). These results demonstrated that only a minority of functional GABAergic synapses in the pyramidal cell layer expressed VGLUT3.
We next assessed whether VGLUT3 and VIAAT were co-localized within the same SVs. After immunoisolation of VGLUT1-or VIAAT-containing SVs, VGLUT3 was recovered only from VIAAT-isolated SVs (Figures 1G,H). Moreover, VIAAT-positive SVs were able to accumulate [ 3 H]glutamate (VIAAT, n = 4; IgG, n = 4, Student t-test, unpaired, P = 0.0028, t = 2.65, Figure 1I). These results demonstrated that VGLUT3 was co-expressed with VIAAT in SVs and that these SVs could readily concentrate glutamate, and might have the ability to release both glutamate and GABA.

VGLUT3 Ablation Modified the Network Activity and the Plasticity of the Hippocampus
GABAergic inputs inhibit individual pyramidal cells and synchronize large groups of excitatory neurons. PV-positive basket cells may play an important role in generating hippocampal rhythms, and CCK-positive basket cells may modulate network activity in a behaviorally and environmentally contingent manner (Bliss and Collingridge, 1993;Klausberger and Somogyi, 2008;Lapray et al., 2012). We examined whether the absence of VGLUT3 significantly affected theta rhythms and long-term synaptic plasticity, two important phenomena involved in learning, memory and emotional processing.
Long-term plasticity of synaptic transmission is a molecular and cellular process that underlies learning and memory through the fine-tuning of synaptic strength FIGURE 1 | VGLUT3 is present in the GABAergic terminals and synaptic vesicles of basket cells of the CA1 layer. (A) Double FISH labeling of mRNA encoding VIAAT (red) and VGLUT3 (green) on a coronal section from WT mouse hippocampus. Nuclei were stained with DAPI (blue). An enlargement of the CA1 area is shown on right panels. (B) Double FISH labeling of mRNA encoding VGLUT3 (red) and GAD (green) in CA1 pyramidal cell layer. Nuclei are stained with DAPI (blue). Quantification of neurons synthesizing GAD (green bar) or VGLUT3 and GAD (yellow bar) (C) Immunofluorescence labeling of VGLUT3 (red), VIAAT (green), and DAPI (blue). Note the dense VGLUT3 labeling in the pyramidal cell layer (delineated by a dashed line). Histogram represents the quantification of VGLUT3 and VIAAT co-localization in GABAergic terminals from CA1 pyramidal layer. (D) 3D reconstruction of VIAAT (green)-and/or VGLUT3 (red)-positive terminals surrounding a pyramidal cell detected by its nucleus (blue). (E) VGLUT3-positive gold immunoparticles detected by electron microscopy in 2 side-by-side axo-somatic terminals (light yellow) making symmetrical synapses. (F) Frequent apposition of VGLUT3-(red) and gephyrin-(green) positive terminals. (G) Immunoisolation of synaptic vesicles from hippocampal LS1 fractions. Immunoisolated VIAAT-positive vesicles co-express VGLUT3. Synaptophysin-containing vesicles express both synaptophysin and VGLUT3 (positive control). No VGLUT3 vesicles were immunoisolated in the absence of antibodies (negative control). (H) Immunoisolated VIAAT-containing vesicles co-express VGLUT3 and synaptophysin, whereas vesicles immunoisolated with VGLUT1 co-express synaptophysin but not VIAAT or VGLUT3, which demonstrates the specificity of the immunoisolation technique. (I) Glutamate uptake measured in immunoisolated glutamatergic (VGLUT1-positive) or GABAergic (VIAAT-positive) vesicles. VIAAT-isolated vesicles take up glutamate to a lesser extent than pure glutamatergic vesicles but to a significantly greater extent than the negative control (IgG). Scale bars in (A): 500 µm in A (insets: 150 µm); 40 µm in (B) and (F); 30 µm in (C), 10 µm in (D); 0.250 µm in (E). * * P < 0.01. (Cooke and Bliss, 2006;Omiya et al., 2015). Using CA1 field potential recordings and extracellular stimulations we examined whether VGLUT3 loss modulates the expression of LTP and LTD. Under conditions of normal glutamate and GABA transmission, tetanic high-frequency stimulation (HFS; see Materials and Methods) of Schaffer's collateral-CA1 connections led to a long-term potentiation of +24 ± 5% of field EPSP (fEPSP) slope in WT mice (n = 9, Wilcoxon test, P = 0.0039, W = −45, Figure 2C). In VGLUT3 −/− mice no LTP was significantly induced by HFS stimulation (+5 ± 7%, n = 10, Wilcoxon test, P = 0.4922, W = −15, Figure 2C). Therefore, the absence of VGLUT3 resulted in a significant difference in HFS-induced LTP between genotypes (Mann-Whitney test, P = 0.0350, U = 19).
Theta burst stimulation (TBS; see Materials and Methods) is a pattern of activity of milder intensity but considered more physiologically relevant than tetanic HFS. We wondered whether the loss of VGLUT3 would also affect TBS-induced LTP. TBS induced a significant LTP in WT Significant LTD was induced in the WT mice (n = 7), whereas no significant LTD was induced in the VGLUT3 −/− mice (n = 7). (H) Schematic representation of the plot of plasticity amplitude as a function of the strength of the stimulation protocol in the presence or absence of VGLUT3. The curve obtained with VGLUT3 −/− mice is shifted toward lower frequencies. This indicates that VGLUT3 −/− mice are still capable of bidirectional plasticity in the hippocampus. However, the loss of VGLUT3 strongly increases local GABA transmission and hence slows down the entire network and shifts metaplasticity toward the left. * P < 0.05, * * P < 0.01, * * * P < 0.001. mice of +16,6 ± 5% (n = 9, Wilcoxon test, P = 0.0039, W = −49, Figure 2E) as well as in VGLUT3 −/− mice: +16,8 ± 4% (n = 8, Wilcoxon test, P = 0.0078, W = −36, Figure 2E). The LTP elicited in both groups were very similar in amplitude (Mann-Whitney test, P > 0.99, U = 36). These results suggest that the loss of VGLUT3 affected specific patterns of activity rather than blunting synaptic plasticity in general.
The theoretical curve shown on Figure 2H summarizes the long-term plasticity results. Levels of LTP and LTD were plotted in regard to the intensity of the stimulation protocol. These results suggest that the removal of VGLUT3 induces a metaplastic shift of synaptic plasticity toward lower frequencies of induction. Taken together, these results demonstrate the impact of VGLUT3 on the excitatory-inhibitory balance and demonstrate that VGLUT3 is an important player in the regulation of theta oscillations and synaptic plasticity.

Role of VGLUT3 in GABAergic Transmission
We next investigated how VGLUT3 regulated the excitatoryinhibitory balance of the hippocampal network. In CCK-basket cells, VGLUT3-dependent glutamate could act directly as a neurotransmitter of excitatory synaptic transmission and/or indirectly as a neuromodulator of the inhibitory synaptic transmission.
To test VGLUT3-dependent glutamate activity on basal glutamatergic synaptic transmission, we recorded fEPSPs at various stimulation intensities. We built up an input/output curve of fEPSP slopes obtained in the CA1 region of WT and VGLUT3 −/− mice ( Figure 3A). The average maximal amplitude of fEPSPs obtained in each genotype was similar (Mann-Whitney test, P = 0,6182, U = 43, not shown). When normalized to the maximal amplitude of each slice, the mean slope recorded at each intensity of stimulation was also similar between WT and VGLUT3 −/− mouse groups [RM-ANOVA, P = 0.6765, F(1,18) = 0,1799, Figure 3A]. This result suggests that VGLUT3-dependent glutamate did not directly regulate fast glutamatergic transmission.
These results demonstrate that VGLUT3-dependent glutamate acts as a neuromodulator that strongly inhibits GABAergic transmission.

VGLUT3-Dependent Glutamate Modulating GABA Transmission Originates from GABAergic Basket Cells
To confirm the GABAergic origin of this VGLUT3-dependent regulatory mechanism, we developed mutant mice selectively lacking VGLUT3 in GABAergic neurons. The exon 2 of Slc17a8, the gene coding for VGLUT3, was flanked by LoxP sites (Figure 4A). VGLUT3 flox/flox mice were bred with VIAAT-CRE mice. In situ hybridization was performed with antisense oligonucleotides targeting exon 2 of VGLUT3 in the hippocampus of WT and VGLUT3 VIAAT−Cre−flox/flox mice (Figures 4B,C, left hemisphere). These experiments showed that VGLUT3 exon 2 was absent from the hippocampus of VGLUT3 VIAAT−Cre−flox/flox mice.
This result demonstrates that VGLUT3-dependent glutamate release by GABAergic basket cells is a potent inhibitory modulator of GABAergic transmission.
To further confirm this hypothesis we examined whether mGLURs and VGluT3 were co-localized on perisomatic basket cell terminals in the CA1 pyramidal layer. Among the group-III mGluRs, mGluR4, mGluR7 and mGluR8 are known to be present on presynaptic elements of the hippocampus (Shigemoto et al., 1996;Fremeau et al., 2002;Somogyi et al., 2003). Using immunofluorescence, we found that mGluR4a (but not mGLUR7) was expressed in a subset of VGLUT3-immunopositive interneurons (Figures 5I-L). Indeed, some pyramidal cells were surrounded by VGLUT3and mGluR4-positive boutons ( Figure 5L). Observations along the Z-axis confirmed that VGLUT3 and mGluR4 were co-localized and not superimposed ( Figure 5L3). Interestingly, most of the VGLUT3-positive boutons surrounding pyramidal cells co-expressed mGLUR4 (stars); however, in other cases, VGLUT3-and mGluR4-positive terminals were juxtaposed (arrow heads). Taken together, these results suggest that VGLUT3-containing terminals are physically co-localized or closely localized with mGluRs to modulate the release of GABA (proposed model Figure 6).

DISCUSSION
Using double FISH and immunofluorescent experiments we extend previous findings establishing that in the hippocampus, VGLUT3 is exclusively expressed in GABAergic interneurons (Gras et al., 2002;Herzog et al., 2004;Katona et al., 2006). In the hippocampus, a majority of CCK-positive basket cell terminals are CB1-positive (Hnasko et al., 2010;Lasztoczi et al., 2011;Albayram et al., 2016). In agreement with previous findings, we confirm that VGLUT3-positive interneurons are also CB1-positive (Hnasko et al., 2010;Albayram et al., 2016). (1) Homosynaptic inhibition limited to CCK-VGLUT3 interneurons (blue, inset 1). Our results suggest that glutamate putatively released by CCK/VGLUT3-positive basket cells terminals could activate mGluRs (possibly group-III metabotropic glutamate receptors) located on the presynaptic side of the synaptic boutons. Their activation could inhibit the release from GABAergic synaptic vesicles release resulting in a decrease of the IPSC amplitude.
Taken together our observations and previous publications suggest that VGLUT3 is present in CCK-positive baskets and more rarely in calbindin-positive interneurons in the hippocampus Hnasko et al., 2010). Therefore, VGLUT3-CCK-positive basket cells (VCB) have the potential to release both glutamate and GABA, the main excitatory and inhibitory neurotransmitters in the brain, respectively.
Interestingly, only a minority of GABAergic interneurons and terminals expresses VGLUT3 (9-13%) in the CA1 region of the ventral hippocampus. Furthermore, VGLUT3 and VIAAT are present on the same synaptic vesicles, and VIAATimmunopurified vesicles are able to accumulate [ 3 H]glutamate. It has been shown that VGLUTs facilitate the storage of ACh, 5-HT and DA in vesicles, a phenomenon named vesicular synergy (Ferraguti and Shigemoto, 2006;Gras et al., 2008;Amilhon et al., 2010;Frahm et al., 2015). Data from the literature suggest that VGLUTs can also promote GABAergic transmission through vesicular synergy (Zander et al., 2010). Our finding that VGLUT3 ablation led to a 17% decrease in the amplitude of mIPSCs suggests that VGLUT3-dependent vesicular synergy could also operate in GABAergic basket cell terminals.
However, increasing the load of GABA into synaptic vesicles is not the prevailing effect of VGLUT3 on GABAergic synaptic transmission.
In this study we revealed a new VGLUT3-dependent glutamatergic modulation of GABAergic transmission through an autoreceptor-mediated presynaptic mechanism (Figure 6). Indeed, one of the most striking observation was the marked increase in GABAergic transmission onto CA1 pyramidal cells in the VGLUT3 −/− mice strain. Using a VGLUT3 VIAAT−Cre−flox/flox mouse line with a specific ablation of VGLUT3 in GABA neurons, we further confirmed that this effect is indeed related to the expression of VGLUT3 in GABAergic neurons.
Moreover, we also found a significant increase in the frequency of unitary IPSCs (+76%) in the VGLUT3 −/− mice and an increase in monosynaptic-evoked IPSCs amplitude in both VGLUT3 −/− (+68%) and VGLUT3 VIAAT−Cre−flox/flox (+55%) mice. Thus, despite their low abundance, VGLUT3-positive terminals exert a robust inhibitory control on the local release of GABA. Previous anatomical and pharmacological data have shown that group III mGLUR4, mGLUR7 and mGLUR8 can be found on GABAergic terminals in the hippocampus (for review, see Semyanov and Kullmann, 2000). The leading theory explaining the role of mGluR activation was that principal neurons or astrocytes might be the source of glutamate that binds to mGluRs and regulates GABA release (Kullmann and Asztely, 1998). Here, we present evidence that mGluR4 are present on VGLUT3-positive terminals and on the perisomatic terminals of other neighboring basket cells. From a mechanistic point of view, our results suggest that VCB cells are a likely important source of the glutamate that activates these mGluRs (Figure 6). Furthermore, it is noticeable that 10% of terminals can inhibit 70% of the local GABAergic transmission. One speculative explanation could be that VCB synapses may exert inhibitory control over neighboring GABAergic terminals in a spillover phenomenon (see Ren et al., 2011 and Figure 6 inset 2).
As for the VGLUT3-dependent postsynaptic actions, data from the literature have shown that the expression of a VGLUT also provides modulatory neurons with the ability to mediate fast, glutamatergic, AMPA receptor-mediated postsynaptic depolarizations (Ferraguti and Shigemoto, 2006;Karson et al., 2009;Varga et al., 2009;Higley et al., 2011). Previous studies using paired recording experiments have not described the occurrence of glutamatergic-dependent postsynaptic responses in pyramidal cells or in PV interneurons in response to CCK interneurons stimulation (Losonczy et al., 2004;Colgin, 2013); however, it was unclear whether the CCK interneurons evaluated in these studies expressed VGLUT3. Therefore, it remains unclear whether VCB cells can directly depolarize postsynaptic neurons following the release of glutamate.
With respect to synaptic plasticity and network activity, the disinhibition of hippocampal GABAergic transmission associated with the absence of VGLUT3 altered the oscillatory activity of synchronized networks and induced a metaplastic shift of synaptic plasticity in the ventral hippocampus. The frequency of theta oscillations was significantly reduced in the VGLUT3 −/− mice. Hippocampal theta oscillations are thought to contribute to learning and memory and the expression of anxiety in rodents (Hasselmo et al., 2002;Gordon et al., 2005;Jutras et al., 2013). Many theories suggests that coordinated activity of hippocampal neurons in the frequency range of 4-8 Hz is implicated in the separation between encoding and retrieval of memory traces trough the oscillation phaselocking with either entorhinal inputs or CA3 inputs at CA1 pyramidal neurons (Huerta and Lisman, 1995). Consequently, phasic changes in synaptic transmission induced by theta oscillations have been proposed to regulate the gating of bidirectional synaptic plasticity at CA1 pyramidal neurons (Huerta and Lisman, 1995). The relationship between local peaks and sinks of theta waves and LTP and LTD could particularly be observed on slice preparation when theta rhythm was induced by cholinergic agonists (Seal et al., 2008). Consequently, any shift or alteration in the oscillatory properties of pyramidal neurons could induce perturbations of synaptic plasticity mechanisms. These changes could also lead to an alteration of learning and memory processes accompanied or not with an impaired expression of anxiety. In line with these observations an increased anxiety was previously reported in VGLUT3 null mice (Amilhon et al., 2010).
It has been reported that constitutive deletion of VGLUT3 results in generalized epilepsy with very little change of motor behavior (Boyce et al., 2016). It can therefore not be ruled out that some of the hippocampal phenotypes herein reported (such as theta oscillation modifications) could result from these non-convulsive seizures in VGLUT3 −/− mice. However, the ≈50% increased GABA tone should on the contrary tone down epileptic activity. These conflictual observations will deserve future investigation.
We observed that the disinhibition of hippocampal GABAergic transmission associated with VGLUT3 loss altered the metaplastic properties of the hippocampal network. In VGLUT3 −/− mice, spontaneous theta oscillations of ventral CA1 significantly shifted to lower frequencies. They were accompanied by a different profile of synaptic plasticity in ventral slice preparation. Under classical conditions, low frequency stimulations (1 to 10 Hz) generally induce LTD of fEPSPs at Schaffer-CA1 synapses. As expected, both stimulations at 1 and 3 Hz induced significant LTD in WT mice. However, in VGLUT3 −/− mice low frequency stimulation at 1 Hz induced LTD but not at 3 Hz (the frequency of spontaneous theta oscillations we observed in vitro). This is in accordance with the previously mentioned theory that stimulation of Schaffer's collaterals in a frequency range close to the theta synchronicity of the network would rather be in favor of potentiation. In hippocampus slice preparations at Schaffer-CA1 synapses, LTP is induced by protocols using a high-frequency stimulation (50-100 Hz). In the absence of VGLUT3, tetanic HFS-induced LTP was markedly absent. However, when GABA A blockers were used to inhibit fast GABAergic transmission, LTP was restored to control level. Interestingly, similar expression of TBS-induced LTP was observed in WT and VGLUT3 −/− mice slices. TBS is composed of both theta and tetanic high frequency components. We hypothesize that while at high frequencies of stimulation, the strong disinhibition of GABAergic transmission in VGLUT3 −/− mice prevails on postsynaptic excitability -an effect that is abolished with the GABA-A antagonist bicuculline -the 5 Hz component of TBS still enables VGLUT3 −/− mice synapses to undergo sufficient potentiation to express significant LTP. Taken together, the field recording results show that VGLUT3 −/− mice still express a significant amount of bidirectional synaptic plasticity but the frequency-plasticity relationship in VGLUT3 −/− mice appears shifted toward the lower frequencies of stimulation. The implication of VGLUT3 in such metaplastic shifting could have complex but significant effects on hippocampal related processes such as learning and memory.
Our data suggest that the VGLUT3-dependent release of glutamate by hippocampal interneurons could finely tune GABAergic transmission onto principal cells and modulate metaplasticity at glutamatergic synapses. If our working hypothesis depicted in Figure 6 is correct, this tuning could be mediated by a presynaptic mechanism involving the activation of type III mGluR autoreceptors. The long-term plasticity of hippocampal synapses is currently thought to underlie the cellular basis of learning and memory (Cooke and Bliss, 2006;Omiya et al., 2015). Among their many other functions, theta oscillations are thought to contribute to learning and development of anxiety in rodents (Hasselmo et al., 2002;Gordon et al., 2005;Jutras et al., 2013). Therefore, it can be speculated that VGLUT3 could play a key role in emotion-associated memories in the ventral hippocampus.

AUTHOR CONTRIBUTIONS
CF, SW, and SEM designed the study and wrote the manuscript with inputs from BG, GA-H, SD, JR, and KP. CF, JR, KP, MM-S, RG, and FM performed electrophysiology experiments and analyzed the data. CF, DS, MM-S, DC, LR, and SB performed immunohistochemistry experiments and analyzed the data. VB performed electron microscopy experiments. SD designated oligonucleotides and designed in situ hybridization protocols. EV performed in situ hybridization, double FISH and analyzed the data. J-FZ performed Immuno-isolation of synaptic vesicles, western blot and [ 3 H]glutamate uptake assay and analyzed the data. LM performed autoradiography experiments.