# A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency

Xiao Min Zhang, Urielle François, Katlin Silm, Maria Florencia Angelo, Maria Victoria Fernandez-Busch, Mona Maged, Christelle Martin, Veronique Bernard, Fabrice Cordelières, Melissa Deshors, et al.

► To cite this version:

Xiao Min Zhang, Urielle François, Katlin Silm, Maria Florencia Angelo, Maria Victoria Fernandez-Busch, et al.. A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency. eLife, 2019, 8, pp.e50401. 10.7554/eLife.50401. hal-02365471

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Submitted on 15 Nov 2019

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<figure><img src="image_1.png" /></figure>
1 A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency

3

4 Xiao-Min Zhang<sup>1,2,3,§</sup>, Urielle François<sup>1,2,*</sup>, Katlin Silm<sup>4*†</sup>, Maria Florencia Angelo<sup>1,2</sup>,

5 Maria Victoria Fernández Busch<sup>1,2</sup>, Mona Maged<sup>1,2</sup>, Christelle Martin<sup>1,2</sup>, Véronique Bernard

6<sup>4</sup>, Fabrice P. Cordelières<sup>5</sup>, Melissa Deshors<sup>1</sup>, Stéphanie Pons<sup>6</sup>, Uwe Maskos<sup>6</sup>, Alexis-Pierre

7 Bemelmans<sup>7,8</sup>, Sonja M. Wojcik<sup>3</sup>, Salah El Mestikawy<sup>4,9</sup>, Yann Humeau<sup>1,2</sup>, Etienne

8 Herzog<sup>1,2,#</sup>

9

10<sup>1</sup> Interdisciplinary Institute for Neuroscience, Université de Bordeaux, UMR 5297, 33000

11 Bordeaux, France.

12<sup>2</sup> Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, 33000 Bordeaux, France.

13<sup>3</sup> Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, D-37075 Göttingen, Germany.

15<sup>4</sup> Neuroscience Paris Seine NPS, INSERM U1130, CNRS UMR8246, Université Pierre et Marie Curie UM119, 75252 Paris cedex 05, France.

17<sup>5</sup> Bordeaux Imaging Center, Université de Bordeaux, CNRS UMS 3420, INSERM US4, 33000, Bordeaux, France.

19<sup>6</sup> Institut Pasteur, CNRS UMR 3571, Unité NISC, Paris, France.

20<sup>7</sup> Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Direction de la Recherche Fondamentale (DRF), Institut de Biologie François Jacob (IBFJ), Molecular Imaging Research Center (MIRCen), Fontenay-aux-Roses, France.

23<sup>8</sup> Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud, Université Paris-Saclay, UMR 9199, Neurodegenerative Diseases Laboratory, Fontenay-aux-Roses, France.

26<sup>9</sup> Douglas Mental Health University Institute, Department of Psychiatry, McGill University, Montreal, QC, H4H 1R3, Canada.

28<sup>§</sup> *Present address:* Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, 1130033, Japan.

30<sup>†</sup> *Present address:* Department of Neurology and Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA.

32

33* These authors contributed equally to the work

34

35<sup>#</sup> Corresponding and lead contact author: Etienne Herzog; etienne.herzog@u-bordeaux.fr

36

37

38 **Keywords:** proline-rich domain; synaptic vesicle; synaptic vesicle super-pool; vesicular glutamate transporter; liquid phase separation; EndophilinA1; quantal neurotransmitter release; fluorescence live cell microscopy; synaptic vesicle cycle.

41
42 **ABSTRACT**

43 Glutamate secretion at excitatory synapses is tightly regulated to allow for the precise tuning
44 of synaptic strength. Vesicular Glutamate Transporters (VGLUT) accumulate glutamate into
45 synaptic vesicles (SV) and thereby regulate quantal size. Further, the number of release sites
46 and the release probability of SVs maybe regulated by the organization of active zone proteins
47 and SV clusters. In the present work, we uncover a mechanism mediating an increased SV
48 clustering through the interaction of VGLUT1 second proline-rich domain, endophilinA1 and
49 intersectin1. This strengthening of SV clusters results in a combined reduction of axonal SV
50 super-pool size and miniature excitatory events frequency. Our findings support a model in
51 which clustered vesicles are held together through multiple weak interactions between Src
52 homology 3 and proline-rich domains of synaptic proteins. In mammals, VGLUT1 gained a
53 proline-rich sequence that recruits endophilinA1 and turns the transporter into a regulator of
54 SV organization and spontaneous release.
55

55

56 **INTRODUCTION**

57 Synaptic vesicles (SVs) engage in multiple protein interactions at the presynaptic active zone
58 (1) and fuse with the presynaptic plasma membrane upon calcium influx, to release their
59 neurotransmitter content (2). Within axon terminals, SVs are segregated from other organelles
60 and grouped in a cluster behind the active zone (3). In adult neurons, SV supply at synapses
61 depends not only on *de novo* vesicle biogenesis, but also on the exchange of mobile SVs
62 between *en passant* boutons along the axon. This exchange pool has been named “SV super-
63 pool” (4-8) and is probably a feature of both glutamatergic and GABAergic axons (9). While
64 the last steps in the regulation of SV release have been studied intensively in different models,
65 the relationship between super-pool SVs, clustered SVs, and the fine-tuning of release at
66 terminals is much less well understood. However, synapsins, a family of SV associated
phospho-proteins, play a central role in the regulation of SV clustering and mobility (10,11).

A growing body of evidence furthermore suggests that SV cluster formation may result from a liquid phase separation from other cytoplasmic elements (12,13). Phase separation may be induced by the loose interaction of multiple proline-rich (or Poly-Proline; PRD) domains with multiple SH3 (Src Homology 3) domain proteins (14). Indeed, PRD/SH3 interactions are numerous among the actors of SV trafficking such as synapsins and dephosphins (15,16). In addition to these interactions, the actin cytoskeleton may contribute to the scaffolding of SV clusters and SV super-pool motility (5,17-20).

Besides SV dynamics and release competence, SV loading with neurotransmitter is another important parameter for the fine-tuning of neurotransmission. To fulfill this function, each excitatory SV may contain between 4 and 14 molecules of Vesicular Glutamate Transporters (21,22). Three isoforms of VGLUTs have been identified, and named VGLUT1-3 (23-28).

They share a nearly identical glutamate transport mechanism (29-31) but have distinct expression patterns (32). VGLUT1 is predominantly expressed in pathways of the olfactory bulb, neo-cortex, hippocampus and cerebellum and is associated with low release probability, while VGLUT2 is strongly expressed in sub-cortical pathways of the thalamus and brainstem, and is preferentially associated with high release probability projections (33,34). This observation raised questions regarding a potential role of the VGLUT transporters in tuning SV release probability.

Hence, soon after their initial characterization, VGLUT1 and -2 were suspected to bear additional functional features that influence neurotransmitter release beyond quantal size (34-37). We discovered that mammalian VGLUT1, but not -2 or -3, interacts with the SH3 domain of endophilinA1 via a proline-rich sequence (38-40). The VGLUT1/EndophilinA1 interaction reduces SV release probability (41) and increases the speed of endocytosis of several SV proteins upon long trains of stimulation (39,42). Furthermore, VGLUTs bear
several di-leucine motifs on their N- and C- terminal sequences, which are responsible for efficient internalization after exocytosis (39,42-44). The functional relevance of these additional properties of VGLUTs is underscored by the 40% reduction in the number of SVs at Slc17a7 (VGLUT1) knock-out (VGLUT1 KO) hippocampal Schaffer collateral and cerebellar parallel fiber terminals (34,45). Despite this reduction in clustered SV numbers, SV protein expression is not diminished nor displaced to other subcellular compartments (45). Yet, we unexpectedly discovered a significantly larger SV super-pool in the axons of VGLUT1 KO neurons (45). Also, VGLUT1 KO SVs appear pleomorphic under hyperosmotic chemical fixation (45,46), however this latter phenotype is most likely a direct consequence of a major change in the ionic composition of the SV lumen (29-31,47).

In the present work, we determined the minimal domain responsible for the influence of VGLUT1 on the axonal super-pool in mammals. To this end, we generated mutants that disconnect the transport function of VGLUT1 from its trafficking function. We observed that VGLUT1 reduces SV super-pool size as well as the frequency of miniature Excitatory Post-Synaptic Currents (mEPSC) exclusively through the interaction of its PRD2 motif with EndophilinA1. These effects were further mediated by the non-canonical interaction of the VGLUT1/EndophilinA1 complex with the SH3B domain of the presynaptic scaffold protein intersectin1 (15). Taken together, our data support the idea that VGLUT1 fine-tunes SV release by strengthening the liquid phase-separation between clustered and super-pool SVs.

# RESULTS

## A specific and dose dependent reduction of super-pool size by VGLUT1

We first compared SV exchange between clusters and the axonal super-pool in wild type and VGLUT1 KO littermate primary neuron cultures. To this end, we expressed a tagged synaptobrevin protein (Syb2<sup>EGFP</sup>) as a reporter (Figure 1A). Through FRAP (fluorescence recovery after photo-bleaching) of boutons we measured higher exchange rates of Syb2<sup>EGFP</sup>
with the axonal compartment, for VGLUT1 KO neurons compared to wild-type neurons (Figure 1B, C, supplementary file 1). A higher exchange rate of VGLUT1 KO SVs with the axonal super-pool is in line with our previous findings (45). We then transduced VGLUT1<sup>Venus</sup> cDNA (8) to rescue VGLUT1 KO neurons or over-express VGLUT1 in wild type neurons (Figure 1D). FRAP of VGLUT1<sup>Venus</sup> fluorescence revealed that VGLUT1 overexpression further reduces SV exchange with axonal pools compared to the rescue of the knock-out to endogenous levels (Figure 1E). Finally, VGLUT2<sup>Venus</sup> expression didn't reduce the VGLUT1 KO larger super-pool phenotype (Figure 1-figure supplement 1, supplementary file 1). Therefore, VGLUT1 expression in neurons reduces the size of the SV super-pool in a dose dependent manner.

## 128 Structure analysis of VGLUT1

To uncover the molecular mechanism by which VGLUT1 regulates SV super-pool size, we generated a series of mutants spanning the sequence of the transporter (Figure 2A and Table 1). VGLUT1 contains 12 trans-membrane domains with both termini on the cytoplasmic side and N-glycosylation on the first luminal loop (56). As vesicular glutamate transport strongly impacts SV tonicity, we first aimed at determining whether the SV loading state impacts SV mobility between SV clusters and the axonal super-pool. A triple point mutant R80Q, R176K, R314Q was generated to produce a glutamate transport deficient transporter as previously reported for VGLUT2 (46,56,sVGLUT1 for silent VGLUT1; Figure 2A blue residues; 57).
We then focused our efforts on several conserved patterns at the VGLUT1 C-terminus. Indeed, mammalian VGLUT1 displays a unique double proline-rich (PRD1 530-540; PRD2 550-556) pattern conserved in all mammals and absent in VGLUT2 and -3 or in invertebrate orthologs of VGLUT1 (38). We thus generated deletions and point mutations to test the function of the PRD motifs (Figure 2A and Table 1). A conserved 540SYGAT sequence between PRD1 and PRD2 is present in all VGLUT isoforms including invertebrates (38). In addition to the
143 deletion mutants, we also generated S540 and T544 to alanine mutations (Figure 2A and 144 Table 1). We furthermore tested the full deletion of the C-terminus and point mutations in a 145 putative PDZ-type3 binding domain (DQL514; PDZ: Post-synaptic density 146 protein/Drosophila disc large tumor suppressor/Zonula occludens-1; see Figure 2A and Table 1). 147

148 All mutants were tagged using our successful c-terminal strategy (8) and the expression level 149 after transduction was monitored to match the endogenous levels of VGLUT1 using 150 immunoblot (not shown). During FRAP and time-lapse experiments, the first frame of each 151 sequence was used to further check the expression level of each mutant compared to the wild- 152 type control (see examples in Figure 2B and quantification in Figure 2C, D). None of the 153 mutants displayed a significant qualitative or quantitative difference in expression compared 154 to wild-type controls. 155

Vesicular glutamate uptake function does not influence SV super-pool size.

156 VGLUT1 KO neurons display hypotonic SVs due to the loss of glutamate transport function 157 (44). To monitor a potential effect of SV lumen tonicity on SV mobility, we transduced 158 VGLUT1<sup>mCherry</sup> or the triple mutation sVGLUT1<sup>mCherry</sup> together with Syb2<sup>EGFP</sup> in VGLUT1 159 KO neurons (Figure 3A) and probed SV turn over at synapses using Syb2<sup>EGFP</sup> FRAP. While 160 we had a very high percentage of co-transduced neurons, we could find fibers with no 161 VGLUT1<sup>mCherry</sup> signal and used these as a negative control. Both rescue conditions lowered 162 the exchange of SVs compared to VGLUT1 KO synapses of the same cultures significantly 163 and to the same degree (Figure 3B, supplementary file 1). In patch clamp experiments, a 164 remaining spontaneous activity was found in the knock-out cultures that can be attributed to a 165 minor but significant expression of VGLUT2 in hippocampal neurons (35,58,59). To 166 minimize this contribution we monitored VGLUT2 levels in the culture at several ages and 167 established that the lower plateau is reached between DIV17 and DIV22 when we performed
our imaging and patch clamp experiments (see Figure 3-figure supplement 1). As anticipated, sVGLUT1 was unable to rescue the mEPSC amplitude to the same level as the wild type control (Figure 3C, D). The deficiency in the rescue of glutamate transport by sVGLUT1 is also apparent in the frequency of mEPSC events, as empty SVs also cycle (35,60). No impact of sVGLUT1 was seen on mIPSC features (Figure 3- figure supplement 2, supplementary file 1). Hence, the triple mutation sVGLUT1 failed to rescue SV loading with glutamate, but reduced SV super-pool size to the same extent as the wild-type control.

**VGLUT1 PRD2 domain reduces SV super-pool size and the frequency of miniature EPSCs.**

We then tested a series of VGLUT1<sup>venus</sup> mutations spanning the C-terminal cytoplasmic tail of the transporter to rescue the VGLUT1 KO large SV super-pool phenotype (see Figure 2 and Table 1). In the FRAP paradigm, only the mutations disrupting the PRD2 sequence were not able to reduce SV exchange down to WT levels (Figure 4A, B). A point mutation in PRD2 (P554A) that blocks the interaction with endophilin is sufficient to increase both the plateau of recovery and the rate of exchange of SVs (shorter slow half-life for P554A, Figure 4A, supplementary file 1). Similar shifts were observed upon deletion of PRD2 (not shown). To further measure SV super-pool size, we performed time-lapse imaging at high sampling rates (5 images per seconds) and tracked VGLUT1<sup>venus</sup> axonal transport between synaptic boutons (Figure 4C-E). This assay confirmed a significantly larger mobile axonal super-pool of SVs for VGLUT1<sup>P554A-venus</sup> but not for VGLUT1<sup>S540A-venus</sup> compared to WT rescue (Figure 4D). Yet, no difference in axonal transport speed could be measured between the 3 constructs (Figure 4E). We also investigated whether PRD2 function could be regulated by phosphorylation of the conserved 540-SYGAT sequence. We thus implemented the PhosTag assay that specifically shifts the electrophoretic mobility of phospho-proteins (55). VGLUT1<sup>venus</sup> constructs displayed an additional slow band in PhosTag gel migration, except
when samples were digested with alkaline phosphatase (Figure 4-figure supplement 1C). All
mutants including ΔC-term displayed this second slower band. Thus, VGLUT1 appears to be
phosphorylated, but not at the C-terminal tail, and the function of the extremely conserved
540-SYGAT sequence therefore remains unclear.

In patch-clamp experiments, we tested the effect of VGLUT1<sup>P554A-venus</sup> rescue on miniature
events, compared to WT and empty vector controls (sham; Figure 4F). VGLUT1<sup>P554A</sup> rescued
miniature EPSC amplitudes to a similar level as the wild type transporter (Figure 4G).
Interestingly, VGLUT1<sup>P554A</sup> increased the frequency of miniature events significantly more
than the WT rescue (Figure 4H). In contrast to this, mIPSC frequencies were similar in all
three conditions (Figure 4I and Figure 4-figure supplement 2, supplementary file 1). As
intended in our rescue strategy, VENUS fluorescence intensity at boutons was not
significantly different between groups tested by electrophysiology (Figure 4J). Therefore, the
PRD2 sequence of mammalian VGLUT1 is sufficient to reduce SV super-pool size and
mEPSC frequency.

**Intersectin1 interaction with endophilin A1 mediates the reduction of the SV super-pool
promoted by VGLUT1 expression.**

Finally, we performed competition experiments with 3 SH3 domains to investigate the
VGLUT1<sup>PRD2</sup> dependent pathway mediating SV super pool reduction (see Table 1 and Figure
5A). SH3 domains fused to the cyan fluorescent protein mCERULEAN3 (50) were
overexpressed under control of the human synapsin promoter in neuron cultures from
VGLUT1<sup>venus</sup> mice (Figure 5B). Single boutons from fibers coexpressing mCERULEAN3
and VENUS were selected for FRAP experiments. An empty mCerulean3 sham construct was
used as negative control and had a VGLUT1<sup>venus</sup> FRAP curve similar to our previous
measurements (Figure 5B, C). The SH3 domain of EndophilinA1 was used to displace
endogenous full-length EndophilinA1 to test the contribution of the membrane binding Bin1,
218 Amphiphysin, RVs (BAR) domain. The FRAP curve in this experiment matched the sham
219 control recovery (Figure 5B, C). The SH3 domain of EndophilinA1 mutated at E329K and
220 S336K was over-expressed to displace endogenous EndophilinA1 and block the interaction
221 with the SH3B domain of intersectin1 (61). Endo-SH3E329K,S336K shifted the FRAP recovery to
222 a significantly higher plateau, creating a phenocopy of the VGLUT1P554A and VGLUT1 KO
223 mutants (Figure 5B, C, supplementary file 1). Similarly, the SH3B domain of intersectin1 was
224 over-expressed to displace endogenous intersectin1 from EndophilinA1. ITSN1-SH3B also
225 shifted the FRAP recovery to a higher plateau. Hence, full length intersectin1 may be required
226 for the VGLUT1 mediated reduction of SV super pool (Figure 5B, C). Taken together, these
227 competition experiments reveal the involvement of EndophilinA1 and intersectin1 in a
228 complex with VGLUT1 in the negative regulation of SV super pool size in mammals.
229

229

230 DISCUSSION

231 In the present study we could observe that VGLUT1 PRD2 motif is a negative regulator of
232 SV mobility and mEPSC frequency. Our data support that SV super-pool reduction is
233 mediated by an interaction of endophilinA1 with both VGLUT1 PRD2 and intersectin1 SH3B
234 domains.

235 Molecular dissection of VGLUT1 functions

236 Previous works had shown that several dileucine-like motifs are involved in VGLUT1
237 endocytosis (39,42,43) and that the PRD2 of VGLUT1 regulates endocytosis during long
238 trains of stimulation (39). The present data provide the first molecular separation of glutamate
239 transport and SV trafficking functions of VGLUT1. The sVGLUT1 mutant allowed us to test
240 the function of the VGLUT1 backbone structure without potential interference brought by the
241 complex flow of ions occurring through glutamate loading (29-31,57,62). sVGLUT1 revealed
242 that glutamate transport is not related to the negative regulation of SV super-pool size. Instead,
our detailed structure-function analysis of VGLUT1 C-terminus showed that the PRD2 motif is necessary and sufficient to inhibit SV exchange between boutons and the axonal super-pool.
We further tested a conserved putative type 3 PDZ binding motif (DQL514) and the PRD1 pattern but found no significant effect on SV super-pool size. Though we brought evidence for VGLUT1 phosphorylation, putative phosphorylation is not at the C-terminus. It thus remains to be seen whether additional features of the C-terminus such as 540SYGAT can be shown to regulate VGLUT1 tuning of SV mobility.

Hence, distinctively from the dileucine based endocytosis of VGLUTs, the C-terminal proline-rich domain PRD2 supports the negative regulation of SV mobility and mEPSC frequency in mammals. SV mobility inhibition is operated independently of glutamate loading processed by the core trans-membrane domains. The structure-function relationship of other conserved sequences remain to be established.

## 255 Mammalian VGLUT1 is a molecular player of SV clustering at synapses

Synaptic vesicles are segregated from other organelles in the nerve terminal, and grouped in a cluster (3). It has been proposed that SVs are recruited to the cluster by synapsins to an actin based cytoskeleton (63). Yet, several lines of evidence suggest a different mechanism involving the low affinity binding of many partners forming a liquid phase separation to the rest of the cytosol (12,16,18). Indeed, endophilinA1, intersectin, amphiphysin are abundant within the vesicle cluster (Figure 5-figure supplement 1 and, 16,64) while the binding of SH3 domains to proline-rich motifs has been shown to generate liquid phase separations in a synthetic assay (14). Further, synapsins were recently shown to induce liquid phase separation with lipid vesicles (13). Hence, the best model to date infers the liquid phase separation of SVs in a dynamic array of labile interactions between synapsins, dephosphins (endophilins, amphiphysins, EPS15, synaptojanins...) and intersectins.
Previously, a slower re-acidification of PRD2 deleted VGLUT1-phluorin probes during long
trains of stimulation suggested that the recruitment of endophilinA1 increases the endocytosis
efficiency of VGLUT1 SVs (39). However, recent reports indicate that endophilinA1
interaction with intersectin1 favors clathrin uncoating (61,65), whereas the clathrin coat was
shown to inhibit SV acidification (66). Therefore, the recruitment of endophilinA1 and
intersectin1 at SVs may speed up clathrin uncoating and SV acidification. This advocates for
new experiments designed to discriminate between an impact of VGLUT1 PRD2 on SV
endocytosis versus SV uncoating, consecutive acidification kinetics and clustering. More
recently, intersectin1 (ITSN1) SH3B was shown to interact with the SH3 domain of
endophilinA1 while preserving the interaction of endophilinA1 with its PRD targets on
Dynamin and VGLUT1 (61). Furthermore, intersectin1 may as well interact directly with
VGLUT1 through the SH3A domain(67,68). Our present data provide evidence for a
reduction of the SV super-pool size to the benefit of the SV clusters mediated by a tripartite
complex between VGLUT1 PRD2, endophilinA1 and intersectin1 (VGLUT1/EndoA1/ITSN1,
Figure 5). The increase in SV exchange rate when PRD2 is mutated strongly suggests a
reduced strength in the interactions scavenging SVs in the cluster (7,8). Yet further
investigations using single particle tracking methods will be necessary to address whether
VGLUT1 PRD2 reduces SV mobility within synaptic clusters as well (20). Our data also fit
well with recent reports of intersectin1 function in the re-clustering of newly endocytosed
SVs, and on the nano-scale organization of synapsins at terminals (69,70). Downstream of
VGLUT1/EndoA1/ITSN1, the SV super-pool reduction may be driven by interactions of
intersectin1 with synapsins (13,70) and/or through remodeling of the actin cytoskeleton
surrounding the cluster (71). Yet further analysis will be required to discriminate between
these pathways and evaluate a possible regulation by phosphorylation/dephosphorylation
cycles.
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Mammalian VGLUT1 acts as a dual regulator of glutamate release

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Vesicular glutamate transporters are necessary and sufficient to generate a glutamatergic phenotype in neurons by loading secretory organelles with glutamate (23). Quantal size modulation has been reported upon changes in the level of VGLUT expression (35,36,72) and is reproduced in our present work (Figure 3 and 4). Furthermore, EndophilinA1 binding to the VGLUT1 PRD2 sequence was shown to reduce SV release probability (41). Our dataset now supports the role of VGLUT1/EndoA1/ITSN1 in the reduction of mEPSC frequency and SV exchange between clusters and super-pool (Figure 4 and 5). A discrepancy may arise from the fact that Weston et al. did not see an effect of VGLUT1 ΔPRD2 on mEPSC frequency but only on SV release probability. However, this may be explained by two differences in our recording conditions. First, the autaptic culture system from Weston et al. prevents the formation of a network. Autapses are powerful tools to dissect evoked activity, but mEPSCs in autaptic conditions arise from a single cell (the recorded cell), while in continental cultures they are generated by multiple cells targeting the recorded neuron. Additionally, continental cultures build a network that generates activity, which most likely leads to different set-points of SV super-pool sizes and homeostatic plasticity compared to autaptic micro-islands (73). Second, Weston and colleagues worked before 14 days in culture, whereas we worked after 17 days in culture when VGLUT2 expression reached a lower plateau and may generate less background mEPSC activity (Figure 3- figure supplement 1; 35,59).

The molecular mechanism of SV super-pool reduction that we uncovered requires the EndoA1 SH3 domain, but not the BAR domain (Figure 5). Previously, Weston et al. showed that endophilinA1 promotes SV release probability through dimerization and BAR domain mediated membrane binding (41). Our current results complement this model by adding a pathway through which VGLUT1/EndoA1 recruit intersectin1 to promote SV clustering and mEPSC frequency reduction in a BAR domain independent fashion. Hence, free
317 EndophilinA1 may actively promote SV exocytosis through membrane binding mechanisms
318 while VGLUT1-bound EndophilinA1 may actively reduce SV mobility and exocytosis
319 through an intersectin1 dependent pathway.

320 Beyond setting the quantal size, VGLUT1 influences glutamate release parameters most
321 likely by changing the strength of SVs phase separation in clusters. It remains to establish
322 how neurons take advantage of this feature to locally modulate the parameters of quantal
323 release at selected synapses (7,8,74). In this line, neuromodulation has been very recently
324 proposed to tune SV pools in the axon through GPCR signaling and synapsin phosphorylation
325 (75).

326 MATERIALS & METHODS

<table><caption>Key Resources Table</caption><thead><tr><th>Reagent type (species) or resource</th><th>Designation</th><th>Source or reference</th><th>Identifiers</th><th>Additional information</th></tr></thead><tbody><tr><td>strain, strain background (Mus musculus)</td><td>Slc17a7<sup>-/-</sup> (VGLUT1 KO) mice</td><td>PMID: 15103023</td><td>available upon request to Dr Sonja M. Wojcik wojcik@em.mp.g.de</td><td></td></tr><tr><td>strain, strain background (Mus musculus)</td><td>Slc17a7<sup>+/v</sup> (VGLUT1venus KI) mice</td><td>PMID: 22031900</td><td>available upon request to corresponding author</td><td></td></tr><tr><td>transfected construct (Homo sapiens)</td><td>F(syn)W-RBN::Synaptobervin2-EGFP</td><td>PMID: 23581566</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig1, Fig3 and related results part. Lentiviral vector expressing Syb2EGPF.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT1-venus</td><td>PMID: 23581566</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig1, Fig4 and related results part. Lentiviral vector expressing VGLUT1venus.</td></tr></tbody></table>
<table><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT2-venus</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig1-S1 and related results part. Lentiviral vector expressing VGLUT2venus.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT1 ΔC-term-venus</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig4 and related results part. Lentiviral vector expressing VGLUT1ΔC-term-venus.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT1 DQL514AQA-venus</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig4 and related results part. Lentiviral vector expressing VGLUT1DQL514A QA-venus.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT1 ΔPRD1+2-venus</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig4 and related results part. Lentiviral vector expressing VGLUT1ΔPRD1+2-venus.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT1 PP534AA-venus</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig4 and related results part. Lentiviral vector expressing VGLUT1PP534AA-venus.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT1 S540A-venus</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig4 and related results part. Lentiviral vector expressing VGLUT1S540A-venus.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT1 T544A-venus</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig4 and related results part. Lentiviral vector expressing VGLUT1T544A-venus.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT1 ΔPRD2-venus</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig4 and related results part. Lentiviral vector expressing VGLUT1ΔPRD2-venus.</td></tr></table>
<table><tr><td>transfected construct (Rattus norvegicus)</td><td>F(syn)W-RBN::VGLUT1 P554A-venus</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig4 and related results part. Lentiviral vector expressing VGLUT1P554A-venus.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>AAV9::VGLUT1mCherry-miniSOG</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig3 and related results part. Lentiviral vector expressing VGLUT1mCherry-miniSOG.</td></tr><tr><td>transfected construct (Rattus norvegicus)</td><td>AAV9::sVGLUT1mCherry-miniSOG</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig3 and related results part. Lentiviral vector expressing sVGLUT1mCherry-miniSOG.</td></tr><tr><td>transfected construct (Homo sapiens)</td><td>AAV9::mCerulean3-EndophilinA1SH3</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig5 and related results part. Lentiviral vector expressing mCerulean3-EndophilinA1SH3.</td></tr><tr><td>transfected construct (Homo sapiens)</td><td>AAV9::mCerulean3-EndophilinA1SH3<sup>E329K, S336K</sup></td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig5 and related results part. Lentiviral vector expressing mCerulean3-EndophilinA1SH3<sup>E329K, S336K</sup>.</td></tr><tr><td>transfected construct (Homo sapiens)</td><td>AAV9::mCerulean3-IntersectinSH3B</td><td>This paper</td><td>available upon request to Dr. Etienne Herzog</td><td>Fig5 and related results part. Lentiviral vector expressing mCerulean3-IntersectinSH3B.</td></tr><tr><td>antibody</td><td>GFP, Mouse, monoclonal</td><td>Roche</td><td>Cat. 11814460001 RRID: AB_390913</td><td>1:1000</td></tr><tr><td>antibody</td><td>VIAAT, Guinea pig, polyclonal</td><td>SYSY</td><td>Cat. 131004 RRID:AB_887873</td><td>1:1000</td></tr></table>
<table><tr><td>antibody</td><td>VGLUT2, Guinea pig, polyclonal</td><td>Millipore</td><td>Cat. AB2251<br>RRID:AB_1587626</td><td>1:2000</td></tr><tr><td>antibody</td><td>VGLUT1, guinea pig, polyclonal</td><td>Merck</td><td>Cat. AB5905<br>RRID:AB_2301751</td><td>1:5000</td></tr><tr><td>antibody</td><td>EndophilinA1, rabbit, polyclonal</td><td>PMID:<br>16606361</td><td>available upon request to Dr. Etienne Herzog</td><td>1:500</td></tr><tr><td>peptide, recombinant protein</td><td>FastAP<br>Thermosensitive Alkaline Phosphatase</td><td>Thermo Scientific</td><td>Cat. EF0651</td><td></td></tr><tr><td>peptide, recombinant protein</td><td>Halt™<br>phosphatase inhibitor cocktail</td><td>Thermo Scientific</td><td>Cat. 78420</td><td></td></tr><tr><td>chemical compound, drug</td><td>Phos-tag<br>Acrylamid</td><td>Wako</td><td>Cat. AAL-107</td><td></td></tr><tr><td>software, algorithm</td><td>FRAP Analysis Plugin</td><td>This paper</td><td>The plugin is available at: https://github.com/fabricecordelieres/IJ-Macro_FRAP-MM</td><td></td></tr><tr><td>software, algorithm</td><td>KymoToolbox Plugin</td><td>PMID:<br>23374344</td><td>The plugin is available at: https://github.com/fabricecordelieres/IJ-Plugin_KymoToolbox</td><td></td></tr></table>

327
328

Animals

All Slc17A7⁻ (VGLUT1 KO) (35) and Slc17A7⁺/⁺ (VGLUT1⁺velus knock-in) (8) and mice were maintained in C57BL/6N background and housed in 12/12 LD with ad libitum feeding.

Every effort was made to minimize the number of animals used and their suffering. The experimental design and all procedures were in accordance with the European guide for the
care and use of laboratory animals and approved by the ethics committee of Bordeaux
Universities (CE50) under the APAFIS n°1692.

## Plasmids and viral vectors

From the Lentivector F(syn)W-RBN::VGLUT1<sup>venus</sup> previously published (45), we engineered a series of point and deletion mutations of VGLUT1<sup>venus</sup> using conventional site directed mutagenesis protocols (see Table 1). Some experiments were performed using enhanced green fluorescent protein tagged synaptobrevin2 (F(syn)W-RBN::Syb2EGFP; 45). Lentiviral particles were generated by co-transfection of HEK-293T cells with the vector plasmid, a packaging plasmid (CMVD8.9 or CMV-8.74) and an envelope plasmid (CMV-VSVg) using Lipofectamine Plus (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. 48h after transfection, viral particles were harvested in the supernatant, treated with DNaseI and MgCl<sub>2</sub>, passed through a 0.45 µm filter and concentrated by ultracentrifugation (ω<sup>2</sup>t = 3,2 10<sup>10</sup> rad<sup>2</sup>/s) and suspended in a small volume of PBS. Viral stocks were stored in 10µl aliquots at -80° C before use. Viral titres were estimated by quantification of the p24 capsid protein using the HIV-1 p24 antigen immunoassay (ZeptoMetrix Corporation, Buffalo, NY, USA) according to the manufacturer's instructions. All productions were between 100 and 300ng/µl of p24. Alternatively, recombinant adenovirus associated virus vectors (AAV) were engineered with either WT or the transport deficient sVGLUT1 inserts (see Table 1). We tagged these constructs with mCherry-miniSOG (48). Serotype 9 AAV particles were generated by transient transfection of HEK293T cells and viral stocks were tittered by QPCR on the recombinant genome as previously described (49). Both viral vectors control the expression of the inserts through the human synapsin promoter. For competition experiments with endophilinA1 and intersectin1 SH3 domains, we built fusions of the endophilinA1 SH3 domain (aa 290 to aa 352) or the SH3B domain of intersectin1 (aa 903 to 971) with the fluorescent protein mCerulean3 positioned at their N-
358 terminus (50). All three SH3 domains were synthesized and the sequences checked (Eurofins
359 genomics company). These fusions were cloned into the AAV shuttle plasmid.

360 **Hippocampal cell cultures and transgene expression**

361 Hippocampal primary dissociated cultures were prepared from P0 mice. The hippocampi were
362 dissected in ice-cold Leibovitz's L-15 medium (11415064; Gibco), and then incubated in
363 0.05% trypsin-EDTA (25300054, Gibco) for 15 min at 37°C. The tissues were washed with
364 Dulbecco's Modified Eagle's Medium (DMEM, 61965026, Gibco) containing 10% FBS
365 (CVFSVF0001, Eurobio), 1% Penicillin-streptomycin (15140122, Gibco). Cells were
366 mechanically dissociated by pipetting up and down, and plated onto poly-L-lysine (P2636,
367 Sigma) coated coverslips at a density of 20 000 cells/cm². Cells were grown in Neurobasal A
368 medium (12349105, Gibco) containing 2% B27 supplement (17504044, Gibco), 0.5 mM
369 Glutamax (35050038, Gibco), and 0.2% MycoZap plus-PR (VZA2021, Lonza) for 5 days in-
370 vitro (DIV). From DIV 5-6, complete Neurobasal A medium was partially replaced to ½ by
371 BrainPhys medium (51), every two or three days. Imaging of live dissociated neuron cultures
372 was performed at DIV 17-21 in culture medium with Hepes buffer (40 mM). Neurons were
373 transduced at DIV1 or -2 with viral vectors diluted by a factor 1/1000. The viral expression
374 levels were controlled by both western-blot (not shown), and fluorescence intensity to rescue
375 VGLUT1 expression to endogenous VGLUT1 level. The bouton size and fluorescent
376 intensity from the different VGLUT1 mutants were analyzed from the first frames the FRAP
377 sequences. Images were processed by "Find Edges", binary images were created through a
378 threshold setting application to define the boundary of boutons. Area and background
379 subtracted fluorescence intensity were measured. Integrated intensity values were normalized
380 to the value in WT boutons of the same group. The statistical significance of the bouton size
381 and fluorescence intensity between the WT and VGLUT1 mutant rescues were evaluated with
382 unpaired t-tests. For competition experiments with endophilinA1 and intersectin1 SH3
domains, plasmids were delivered by electroporation at DIV0 before plating (Nucleofector, Lonza).

## 385 FRAP imaging

Fluorescence Recovery After Photobleaching (FRAP) experiments were performed to determine the mobility of Syb2 labeled SVs (for silent VGLUT1 mutant rescue experiments) or VGLUT1 labeled SVs (for VGLUT1 C-terminal mutants rescue experiments) at synapses.

The mobile fraction of Syb2/VGLUT1 labeled SVs is the proportion of fluorescent material that can be replenished after photo bleaching. FRAP was performed using a spinning-disk confocal head Yokogawa CSU-X1 (Yokogawa Electric Corporation, Tokyo, Japan) mounted on an inverted Leica DMI 6000 microscope (Leica Microsystems, Wetzlar, Germany) and equipped with a sensitive EM-CCD QuantEM camera (Photometrics, Tucson, USA), and a FRAP scanner system (Roper Scientific, Evry, France). Surrounding the setup, a thermal incubator was set to 37°C (Life Imaging Services, Switzerland). Z-stacks of 4.8 µm thickness were obtained with a piezo P721.LLQ (Physik Instrumente, Karlsruhe, Germany) at randomly selected fields from hippocampal cell culture with a 63×/1.4 numerical aperture oil-immersion objective. For each stack, five fluorescent boutons, distant from each other, were selected for bleaching. Three passes of the 491 nm laser (40 mW) for Syb2<sup>EGFP</sup> or two laser passes using the 491 nm laser (30 mW) and the 405 nm laser (10 mW) for VGLUT1<sup>venus</sup>, were applied on the mid-plane of the stack and resulted in an average bleaching of 50% of the initial fluorescence intensity at boutons. The bleaching protocol for Venus/EYFP prevents the spontaneous recovery of fluorescence from a dark reversible photochemical state as previously reported (8,52).

Fluorescence recovery was monitored every 30 s during the first 3 min and then every 5 min during the next 70 min. The entire FRAP procedure was controlled by MetaMorph (Molecular Devices, Sunnyvale, USA). Image processing was automated using ImageJ macro
408 commands (53) (available at https://github.com/fabricecordelieres/IJ-Macro_FRAP-MM).

409 Sum projections of the individual stacks, assembly and x-y realignment were applied,
410 resulting in 32 bits/pixel sequences. Integrated fluorescence intensities of the five bleached
411 boutons, and the cells in the field, as well as one background area were extracted. The
412 background signal was subtracted, and data were normalized to the average baseline before
413 bleaching (100%) and corrected for photobleaching against the cells. Experiments were
414 discarded if photobleaching exceeded 60% (risk of phototoxicity). Fluorescence intensity of
415 boutons was normalized to 1 before bleaching, and 0 right after bleaching. A double
416 exponential function was used to fit the average of all normalized FRAP traces and the extra
417 sum-of-squares F test was applied to compare the different best fits. Unpaired t-test was
418 applied to analysis of bouton fluorescence intensity between different mutants and the
419 corresponding WT data sets.

420

421 Live Cell imaging

422 Time-lapse experiments using the spinning disk confocal microscope were performed to
423 quantify the SVs moving along the inter-synaptic axonal segments. Images were sampled at 5
424 frames/s for 30 seconds with 200 ms exposure time (151 frames in total). Synaptic boutons
425 were saturated in order to allow better visualization of the dimmer fluorescent material
426 moving along the axons. Quantification of the speed of moving clusters was performed with
427 the KymoToolbox plugin in ImageJ (Figure 4; available at
428 https://github.com/fabricecordelieres/IJ-Plugin_KymoToolBox) (54). In each sequence, 8
429 axons segments of 10-15 µm were selected for particle tracking. Furthermore, the traffic at
430 inter-synaptic segments was quantified by drawing line ROIs perpendicularly to the axon, and
431 cumulating the integrated density values in each of the 151 images. Background was
432 subtracted, and all values were divided by the average of 10 lowest values of the sequence to
433 normalize for the differences of fluorescence intensity between different sets of experiments.

434 All normalized values from each line selection were summed to evaluate the total amount of
435 material going through the given cross-section. The statistical significance of the differences
436 in cumulative traffic between the WT and VGLUT1 mutants (S540A and P554A) were
437 evaluated with unpaired t-test.

438 Electrophysiology

439 Dual whole-cell patch-clamp recordings were performed from DIV17 to DIV21. Patch
440 pipettes (2-4MΩ) were filled with the following intracellular solution (in mM): 125 CsMeSO₃,
441 2 MgCl₂, 1 CaCl₂, 4 Na₂ATP, 10 EGTA, 10 HEPES, 0.4 NaGTP and 5 QX-314-Cl, pH was
442 adjusted to 7.3 with CsOH. Extracellular solution was a standard ACSF containing the
443 following components (in mM): 124 NaCl, 1.25 NaH₂PO₄, 1.3 MgCl₂, 2.7 KCl, 26 NaHCO₃,
444 2 CaCl₂, 18.6 Glucose and 2.25 Ascorbic acid. To record excitatory and inhibitory miniature
445 currents (mEPSC and mIPSC), Tetrodotoxin (TTX) was added at 1μM into an aliquot of the
446 standard ACSF (Alomone labs). Cultures were perfused at 35°C with an ACSF perfusion
447 speed of 0.02mL/min and equilibrated with 95% O₂/5% CO₂. Signals were recorded at
448 different membrane potentials under voltage clamp conditions for about 2 min (0mV for
449 inhibitory events and -70mV for excitatory events) using a MultiClamp 700B amplifier
450 (Molecular Devices, Foster City, CA) and Clampfit software. Recording at 0mV in voltage
451 clamp allowed us to confirm the effect of TTX on network activity. The recording of
452 miniature events began 2min after adding TTX and the extinction of synchronized IPSCs.
453 Additional recordings were performed at membrane potentials of -20mV, -40mV, -60mV and
454 -80mV. For drug treatment, CNQX was used at 50μM and PTX at 100μM and 2minutes after
455 drug addition, the condition was considered as stable.

456 Electrophysiology Analysis
Analyses were performed using Clampfit (Molecular Device), in which we created one mini-
excitatory (-70mV) and one mini-inhibitory (0mV) template from a representative recording.
Those templates were used for all recordings and analysis was done blind to the experimental
group. We measured the number of mEPSCs at -70mV and mIPSCs at 0mV and their mean
amplitude. Cell properties were monitored to get a homogenous set of cells, i.e. we analyzed
the seal-test recordings of every cell (see supplementary file 2) and calculated the capacitance
from the Tau measured by Clampfit (supplementary file 2). Cells with a leak current over -
200pA, and/or a membrane resistance over -100MOhms were excluded from the analysis.
Statistical analyses were performed using One-way ANOVA or Kruskal-Wallis test (* for p <
0.05, ** for p < 0.01 and *** for p < 0.001).

467 Antisera

The detection of wild type VGLUT1 was performed with rabbit polyclonal antiserum (8,25)
whereas the detection of VGLUT1<sup>venus</sup> was done with a mouse monoclonal anti-GFP antibody
(11814460001, Roche). Anti-VIAAT (131004, SYSY) and Anti-VGLUT2 (AB2251,
Millipore) guinea pig polyclonal antisera were used. For electron microscopy we used a
guinea pig anti-VGLUT1 polyclonal antiserum (AB5905, Merck) and a rabbit polyclonal
antiserum against endophilinA1 (38). Secondary HRP-coupled anti-rabbit, anti-mouse and
anti-guinea pig antibodies were used for western blot detection (711-035-152, 715-035-150,
706-035-148, respectively, Jackson ImmunoResearch).

476 Immunocytochemistry

Neuron cultures were washed with cold 1X PBS and fixed with 4% paraformaldehyde in 1X
PBS for 5min at room temperature. Immunostainings were performed as previously described
(25). Wide-field pictures were acquired using an epifluorescence Nikon Eclipse NIS-element
microscope with the 40x objective. The ImageJ software was used to quantify the
fluorescence intensity. A mean filter, background subtraction, and threshold were applied to
cover the punctate signals and generate a selection mask. The mean density values were extracted. The averages of 5 frames per culture were probed and more than 3 cultures per age were measured.

## Immunohistochemistry and electron microscopy

VGLUT1 and EndophilinA1 were simultaneously detected by combination of immunoperoxidase and immunogold methods, respectively, on brain sections at the ultrastructural level.

*Animals and tissue preparation* The mice were deeply anesthetized with sodium chloral hydrate and then perfused transcardially with 0.9% NaCl, followed by fixative consisting of 2% paraformaldehyde (PFA) with 0.2% glutaraldehyde in 0.1M phosphate buffer (PB), pH 7.4, at 4°C. The brain was quickly removed and left overnight in 2% PFA at 4°C. Sections from cerebellum and caudate putamen were cut on a vibrating microtome at 70mm and collected in PBS (0.01M phosphate, pH 7.4). To enhance the penetration of the immunoreagents in the preembedding procedures, the sections were equilibrated in a cryoprotectant solution (0.05MPB, pH 7.4, containing 25% sucrose and 10% glycerol) and freeze-thawed by freezing in isopentane cooled in liquid nitrogen and thawed in PBS. The sections were then preincubated in 4% normal goat serum (NGS) in PBS.

*Double detection of VGLUT1 and EndophilinA1 by combined preembedding immunoperoxidase and immunogold methods.* The brain sections were incubated in 4% NGS for 30 min and then in a mixture of VGLUT1 (1:5000) and EndophilinA1 (1:500) antibodies, supplemented with 1% NGS overnight at RT. The sections were then incubated with goat anti-guinea-pig IgGs conjugated to biotin (1:200), washed in PBS and incubated in a mixture of avidin-biotin-peroxidase complex (ABC), (1:100; Vector Laboratories, Burlingame, CA) and goat anti-rabbit IgGs conjugated to gold particles (1.4 nm diameter; Nanoprobes, Stony Brook, NY; 1:100 in PBS/BSA-C) for 2 hr in PBS/BSA-
C. The sections were then washed in PBS and post-fixed in 1% glutaraldehyde in PBS.
507

After washing (PBS; sodium acetate buffer, 0.1M, pH 7.0), the diameter of the gold
508
immunoparticles was increased using a silver enhancement kit (HQ silver; Nanoprobes)
509
for 5min at RT in the dark. After washing (2xPBS, 1xTB 0.05M, pH 7.6), the
510
immunoreactive sites VGLUT1 were revealed using DAB. The sections were then stored
511
in PB and processed for electronmicroscopy.
512

Preparation for electron microscopy The sections were post-fixed in osmium tetroxide
513
(1% in PB, 0.1M,pH7.4) for 10 min at RT. After washing, they were dehydrated in an
514
ascending series of dilutions of ethanol that included 1% uranyl acetate in 70% ethanol.
515
They were then treated with propylene oxide and equilibrated in resin overnight
516
(Durcupan AC M; Fluka, Buchs, Switzerland), mounted on glass slides, and cured at 60°C
517
for 48 hr. The immunoreactive areas identified on thick sections were cut in semithin
518
sections (1-mm-thick), then in ultrathin sections on a Reichert Ultracut S.
519
Ultrathinsections were collected on pioloform-coated single slot copper grids. The
520
sections were stained with lead citrate and examined in a Philips C M10 electron
521
microscope.
522

523 Biochemistry

All steps were performed at 4°C or on ice. Brains of wild type adult mice were dissected for
524
the collection of brain regions. The samples were treated with homogenization buffer (0.32 M
525
sucrose, 4 mM HEPES pH 7.4). Cultures expressing the different VGLUT1 mutants were
526
collected on DIV 17 with 1× PBS. Both buffers were supplemented with protease inhibitor
527
cocktail (539134, Millipore) and Halt™ phosphatase Inhibitor Cocktail (78420, Thermo
528
Fisher Scientific). When necessary, protein samples were treated with alkaline phosphatase
529
prior to the biochemical analysis. FastAP Thermosensitive Alkaline Phosphatase (1 unit/µl)
530
and FastAP 10× buffer (EF0654, Thermo Scientific) were added to samples and incubated for
531
1 h at 37°C. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
phosphate affinity SDS-PAGE (55) were conducted according to standard methods. For
phosphate affinity SDS-PAGE (Mn²⁺-Phos-tag SDS-PAGE), 25 µM Phostag (AAL-107,
Wako) and 0.1 mM MnCl₂ were added to the resolving gel before polymerization. Western
blotting was performed according to standard procedures using HRP-coupled secondary
antibodies for qualitative detection. Chemi-luminescence signals were visualized with
ChemiDoc MP System (Bio-Rad) using SuperSignal™ West Dura Extended Duration
Substrate (34075, Thermo Scientific).

540
541 SOURCE DATA FILES

542 Source data used to build figures are available in the following separate supplementary files:
543 Figure 1-source data 1.xlsx ; Figure 1-source data 2.xlsx ; Figure 1-figure supplement1 source
544 data 1.xlsx ; Figure 2-source data 1.xlsx ; Figure 3-source data 1.xlsx ; Figure 3-source data
545 2.xlsx ; Figure 3-figure supplement 1 source data.xlsx ; Figure 4-source data 1.xlsx ; Figure 4-
546 source data 2.xlsx ; Figure 4-source data 3.xlsx ; Figure 4-source data 4.xlsx ; Figure 4-source
547 data 5.xlsx ; Figure 4-source data 6.xlsx ; Figure 4-source data 7.xlsx ; Figure 4-source data
548 8.xlsx ; Figure 4-source data 9.xlsx ; Figure 4-figure supplement 2 source data.xlsx ; Figure 5-
549 source data 1.xlsx

550 ACKNOWLEDGEMENTS

551 Elisa Luquet, Sally Wenger, and Charlène Josephine for excellent technical support. Nils
552 Brose for a strong support throughout the project. Serge Marty and Martin Oheim for fruitful
553 discussions. Christian Rosenmund, Prof Roger Tsien, Prof Mark A Rizzo, for sharing
554 reagents. Several experiments required the use of Bordeaux University/CNRS/INSERM core
555 facilities: Bordeaux Imaging Center (member of France BioImaging supported by the French
556 National Research Agency; ANR-10-INBS-04); Vect'UB viral vector facility; Biochemistry
557 and Biophysics of Proteins core facility; Mouse breeding facility. X.M.Z. was supported by
558 the Erasmus Mundus ENC program and the labex BRAIN extension grant (ANR-10-LABX-
559 43 BRAIN). Funding from the Agence Nationale de la Recherche (ANR-12-JSV4-0005-
560 01 VGLUT-IQ ; ANR-10-LABX-43 BRAIN; ANR-10-IDEX-03-02 PEPS SV-PIT to E.H.
561 and PSYVGLUT 09-MNPS-033 to S.E.M.). K.S. and M.F.A. were supported by the
562 Fondation pour la Recherche Médicale (FDT20120925288 and ING20150532192
563 respectively).

564 CONFLICT OF INTEREST

565 The authors declare no conflict of interest.
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TABLES

<table><thead><tr><th></th><th>Mutation</th><th>Domain/Motif</th><th>Name</th><th>putative function</th></tr></thead><tbody><tr><td rowspan="12">VGLUT1</td><td>R80Q/R176K/R314Q</td><td>TM1;TM4;TM7</td><td>sVGLUT1</td><td>Glutamate transport</td></tr><tr><td>Δ504-560</td><td>Whole C-terminus</td><td>ΔC-term</td><td>SV/VGLUT Trafficking</td></tr><tr><td>DQL514AQA</td><td>PDZ type 3 binding</td><td>DQL514AQA</td><td>Unknown</td></tr><tr><td>Δ530-560</td><td>Proline Rich Domains 1+2</td><td>ΔPRD1+2</td><td>SH3 domain binding</td></tr><tr><td>PP534AA</td><td>Proline Rich Domain1</td><td>PP534AA</td><td>SH3 domain binding</td></tr><tr><td>S540A</td><td>SYGAT</td><td>S540A</td><td>Unknown</td></tr><tr><td>T544A</td><td>SYGAT</td><td>T544A</td><td>Unknown</td></tr><tr><td>Δ550-560</td><td>Proline Rich Domain 2</td><td>ΔPRD2</td><td>Endophilin binding</td></tr><tr><td>P554A</td><td>Proline Rich Domain 2</td><td>P554A</td><td>Endophilin binding</td></tr><tr><td>EndoA1 290-352</td><td>SH3</td><td>SH3</td><td>Binds PRD and ITSN1 SH3B</td></tr><tr><td>EndoA1 290-352<sup>E329K,S336K</sup></td><td>SH3</td><td>SH3<sup>E329K,S336K</sup></td><td>Binds PRD and ITSN1 SH3B</td></tr><tr><td>ITSN1 903-971</td><td>SH3B</td><td>SH3B</td><td>Endo SH3 binding through E329 and S336</td></tr></tbody></table>

781

782

Table 1: List of mutant constructs tested.

783
784
785
786 FIGURE LEGENDS
787
788 **Figure 1:** Dose dependent regulation of SV super-pool size by VGLUT1.
789
790 A: Expression of Synaptobrevin2 fused to Enhanced Green Fluorescent Protein (Syb2<sup>EGFP</sup>) in hippocampal neurons at 18 days in culture. B: examples of FRAP sequences from both Slc17a7 (VGLUT1) +/- and -/- genotypes. Boutons are imaged for 3 min, bleached, and recovery is recorded for 73 min. C: average FRAP kinetics. 27 synapses from +/- and 31 synapses from -/- were measured by FRAP and the average traces are displayed here (for +/- N = 8 cultures; for -/- N = 11 cultures). The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by 2 divergent models (F ratio = 19.32; P<0.0001). Fast FRAP recovery was monitored every 5 s in an independent set of experiments (inset). D: Expression of transduced VGLUT1<sup>venus</sup> in hippocampal neurons. E: Average FRAP recovery of VGLUT1<sup>venus</sup> at 68 minutes post-bleach in rescue or over-expression. Over-expression reduces the mobility of SVs (Unpaired t test, P = 0.0385, t = 2.176. For overexpression: N = 4 cultures, n = 14 synapses; for rescue: N = 3 cultures, n = 15 synapses). scale bar: 2µm in A and D, 1 µm in B.
800
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803

804 **Figure 2:** Structure of VGLUT1 and expression of mutants in neurons.

805 A: schematic of VGLUT1 structure with 560 amino acids, 12 transmembrane domains, N- and C-termini facing the cytoplasmic side, and N-glycosylation at the first luminal loop. In 806 blue the 3 residues mutated to silence VGLUT1 transport (sVGLUT1 see Figure3). Red bars 807 mark the 3 deletions used (ΔC-term, ΔPRD1+2, ΔPRD2). Red residues were mutated to 808 alanine in order to test their role in the super-pool regulation supported by VGLUT1. All 809 mutations carried a venus tag at the C-terminus. B: examples of expression patterns obtained 810 with VGLUT1 WT and mutants upon transduction in hippocampal neurons matching 811 endogenous expression levels. Note the dense punctate expression and low somatic signal 812 typical of VGLUT1 distribution. Scale bar 10µm. C: Measurement of bouton area. None of 813 the mutants displayed a shift in bouton size compared to WT controls. D: Measurement of 814 fluorescence intensity. None of the mutants displayed a significant shift in fluorescence 815 intensity compared to WT controls.
816
817
818
819

**Figure 3:** Glutamate transport and SV tonicity are not involved in the reduction of SV super-pool size.

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A: Expression of VGLUT1<sup>mCherry</sup>, sVGLUT1<sup>mCherry</sup> and Syb2<sup>EGFP</sup> in hippocampal neurons. FRAP was performed on Syb2<sup>EGFP</sup> at axons, both on those with, and those without mCherry signal. Scale bar 5µm. B: Average FRAP kinetics from knock-out cells rescued by VGLUT1<sup>mCherry</sup>, sVGLUT1<sup>mCherry</sup>, and VGLUT1 KO synapses not rescued. Synapses from each genotype were measured by FRAP and the average traces are displayed (N = 7 independent cultures, 16 synapses for -/-; N = 5 cultures, 23 synapses for WT rescue, and N = 10 cultures, 36 synapses for sVGLUT1 rescue). The 3 traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by 2 divergent models (one for -/- synapses and the other one for both rescues, F ratio = 30.25; P<0.0001). FRAP kinetics for the 2 types of rescued synapses are best fitted by one convergent model (F ratio = 1.235; P = 0.294). C: Spontaneous excitatory activity in VGLUT1 KO rescued neurons. Example traces of mEPSC activity in wild type and sVGLUT1 rescue conditions. D: Comparison of the amplitude of mEPSC events in wild type and sVGLUT1 rescue conditions (N=3 independent cultures, n=31 cells, mean amplitude=15.3 pA ± 1.15 SEM for wild type rescue; N = 3 independent cultures, n=56 cells, mean amplitude = 13.12 pA ± 0.71SEM for sVGLUT1 rescue; unpaired t-test P= 0.011). E: Comparison of the frequency of mEPSC events in wild type and sVGLUT1 rescue conditions. (N = 3 independent cultures, n=31 cells, mean frequency= 3.51 Hz ± 0.6 SEM for wild type rescue; N = 3 independent cultures, n=56 cells, mean frequency = 2.51 Hz ± 0.58 SEM for sVGLUT1 rescue; unpaired t-test P= 0.008). Note that sVGLUT1 conditions are both significantly smaller than wild type rescue conditions.

843
844

**Figure 4:** The VGLUT1 PRD2 domain mediates SV super-pool size and mEPSC frequency reductions

845
846
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848
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A: Comparison of VGLUT1<sup>venus</sup> and VGLUT1<sup>P554A-venus</sup> rescues of VGLUT1 KO on SV exchange rates at synapses. 21 (WT) and 22 (P554A) synapses from each rescue were measured by FRAP and the average traces are displayed (N = 5 cultures for WT and N = 5 cultures for P554A). The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by 2 divergent models (F ratio = 19.64; P < 0.0001). B: Similar experiments where performed for ΔPRD1+2, ΔPRD2,
DQL514AQA, PP534AA, S540A and T544A. The results are displayed here as a comparison of fluorescence recovery to the corresponding WT control 68 min after bleaching. Only mutants affecting PRD2 display a lack of reduction to WT levels and a significantly higher SV exchange rate (t test, WT vs. ΔPRD1+2: *P* = 0.0005, *t* = 3.790; vs. ΔPRD2: *P* < 0.0001, *t* = 4.369; vs. P554A: *P* = 0.0293, *t* = 2.266). C: Time lapse imaging of SV axonal transport. VGLUT1<sup>venus</sup>, or VGLUT1<sup>P554A-venus</sup>, or VGLUT1<sup>S540A-venus</sup> were expressed in VGLUT1 KO neurons. Example sequence extracted from the boxed fiber (left) sampled every 200ms over 30s (middle). Vertical arrowhead points to a venus fluorescent dot traveling along the axon. Kymograph of fluorescence movements within the example fiber (boxed in middle panel). Vertical arrowhead points to the same traced event shown in the middle panel. Scale bar: left 5µm, middle 2µm. D: Cumulative axonal fluorescence traffic measured over time-lapse sequences. A significant increase in VGLUT1<sup>venus</sup> traffic is seen when P554A is expressed compared to WT and S540A mutant (unpaired t test, WT vs. P554A: *P* = 0.0092, *t* = 3.564; vs. S540A: *P* = 0.8963, *t* = 0.1351. *N* = 5 cultures for WT, and *N* = 4 cultures for both P554A and S540A.). E: Average speed of VGLUT1<sup>venus</sup> dots was extracted from kymographs. No significant changes in speed was seen for mutants tested compared to WT (One-way ANOVA, *P* = 0.0634, *F* ratio = 2.431. Speed for each mutant, WT: 1.564 ± 0.02692 µm/s; P554A: 1.536 ± 0.02860 µm/s; S540A: 1.460 ± 0.02942 µm/s). F: Spontaneous excitatory activity in VGLUT1 KO neurons rescued by wild type and P554A VGLUT1 constructs. Example traces of mEPSC activity in sham controls, wild type and VGLUT1<sup>P554A</sup> rescue conditions. G: Comparison of the amplitude of mEPSC events in wild type and VGLUT1<sup>P554A</sup> rescue conditions (*N* = 3 independent cultures, *n*=53 cells, mean amplitude=12.73 pA ± 0.79 SEM for sham controls; *N* = 3 independent cultures, *n*=61 cells, mean amplitude=16.05 pA ± 1.05 SEM for wild type rescue; *N* = 3 independent cultures, *n*=53 cells, mean amplitude = 15.59 pA ± 1.02 SEM for vglut1<sup>P554A</sup> rescue; Mann-Whitney test of wild type versus VGLUT1<sup>P554A</sup>, *P*=0.485). Note that both wild type and VGLUT1<sup>P554A</sup> rescue mEPSC amplitudes significantly and to the same extent compared to sham controls. H: Comparison of the frequency of mEPSC events in wild type and VGLUT1<sup>P554A</sup> rescue conditions (*N* = 3 independent cultures, *n*=53 cells, mean frequency= 2.14 Hz ± 0.37 SEM for sham controls; *N* = 3 independent cultures, *n*=61 cells, mean frequency= 5.53 Hz ± 0.87 SEM for wild type rescue; *N* = 3 independent cultures, *n*=53 cells, mean frequency = 7.59 Hz ± 0.97 SEM for VGLUT1<sup>P554A</sup> rescue; Mann-Whitney test of wild type versus VGLUT1<sup>P554A</sup>, *P*=0.031). Note that VGLUT1<sup>P554A</sup> mEPSC events are significantly more frequent than wild type rescue conditions. I: Comparison of the frequency of mIPSC events in wild type and VGLUT1<sup>P554A</sup> rescue
conditions (N = 3 independent cultures, n=53 cells, mean frequency=6.21Hz ± 0.69 SEM for sham controls; N = 3 independent cultures, n=61 cells, mean frequency=7.28 Hz ± 0.69 SEM for wild type rescue; N = 3 independent cultures, n=53 cells, mean frequency = 6.79 Hz ± 0.63 SEM for VGLUT1<sup>P554A</sup> rescue; Mann-Whitney test of wild type versus VGLUT1<sup>P554A</sup>, P=0.781). Note that all groups are equivalent regarding mIPSC amplitudes (Figure 4-figure supplement 2 displays the full IPSC data set). J: Post-hoc analysis of VGLUT1<sup>venus</sup> average fluorescence integrated intensity at boutons in cultures monitored in electrophysiology. (N = 2 independent cultures, mean integrated intensity of punctate VGLUT1<sup>venus</sup> signal= 3.28 AU ± 0.15 SEM for wild type rescue; N = 2 independent cultures, mean integrated intensity of punctate VGLUT1<sup>venus</sup> signal = 3.11 AU ± 0.31 SEM for VGLUT1<sup>P554A</sup> rescue; T test, P=0.348).

897

Figure 5: A tripartite complex between VGLUT1, endophilinA1 and intersectin1 mediates SV super-pool reduction.

A: Schematic model of the competition experiment designed to test for the effective
recruitment of intersectin1 at the VGLUT1/endophilinA1 complex (61). The three SH3
domains over-expressed in this competition assay disrupt distinct parts of the tripartite
complex. The SH3 domain of endophilinA1 displaces the endogenous endophilinA1 BAR
domain (Endo SH3<sup>WT</sup>). The SH3 domain of endophilinA1 mutated at E329K and S336K
disrupts the interaction of intersectin1 with the VGLUT1/endophilinA1 complex(61). Finally,
the SH3B domain of intersectin1 should displace the endogenous intersectin1 and allows us to
assess whether the full-length intersectin1 is required. B: Over-expression of SH3 domains
fused with mCerulean3 in VGLUT1<sup>venus</sup> neurons. Axons filled with mCerulean3 were
selected for FRAP experiments. Scale bar 2µm. C: FRAP measurement of SV super-pool in
SH3 domains competition experiments. Synapses from each over-expression condition were
measured by FRAP and the average recovery after 68 minutes are displayed (0.5346 ±
0.04128, N=4 and n=20 for sham; 0.6071 ± 0.03705, N=5 and n=25 for Endo-SH3<sup>WT</sup>; 0.7323
± 0.04909, N=4 and n=18 for Endo-SH3<sup>E329K,S336K</sup>; 0.6894 ± 0.04036, N=5 and n=25 for
ITSN1-SH3B). Sham and Endo-SH3<sup>WT</sup> converged to a lower SV exchange recovery (Mann-
Whitney test, P=0.141) while Endo-SH3<sup>E329K,S336K</sup> (Mann-Whitney test, P=0.005 **) and
ITSN1-SH3B (Mann-Whitney test, P=0.014 *) generated higher SV exchange recovery
curves.

900

901

902

903

904

905

906

907

908

909

910

911

912

913

914

915

916

917

918
919 **Supporting Information :**

920 **Figure 1-figure supplement 1:** VGLUT2 does not rescue SV super-pool size in VGLUT1
921 knock out neurons.

922 A: Average FRAP kinetics obtained upon rescue of VGLUT1 KO cells by either
923 VGLUT1<sup>venus</sup> or VGLUT2<sup>venus</sup> lentivirus. 24 synapses from VGLUT1<sup>venus</sup> and 20 synapses
924 from VGLUT2<sup>venus</sup> were measured by FRAP and the average traces are displayed
925 (VGLUT1<sup>venus</sup> rescue N = 7 cultureVGLUT2<sup>venus</sup> N = 7 cultures). The two traces were fitted
926 using double exponential components equations and the convergence of the traces to a
927 common fit was tested using the extra sum of squares F test. The F test indicates that the
928 traces are best fitted by 2 divergent models (F ratio = 19.27 ; P<0.0001).

929

930 **Figure 3-figure supplement 1:** VGLUT2 expression diminishes in hippocampal neurons
931 until DIV17

932 A: Immunofluorescence of VGLUT2 on hippocampal neurons at DIV 6, 12, 17, and 22. Scale
933 bar 40µm. B: Quantification of VGLUT2 expression during hippocampal neuron culture
934 development. Note the 3-fold reduction of VGLUT2 level between DIV 6 and 17, while the
935 expression reached a low plateau between DIVB17 and 22. DIV6, N=4, mean intensity =
936 612.8 ± 53.8; DIV12, N=3, mean intensity = 325.9 ± 39.0; DIV17, N=7, mean intensity =
937 202.8 ± 8.2; DIV22, N=5, mean intensity = 191.2 ± 24.2. One way ANOVA p≤0.001; pair
938 wise comparisons: DIV6 vs. DIV22 p <0,001 ; DIV6 vs. DIV17 p <0,001 ; DIV6 vs. DIV12 p
939 <0,001 ; DIV12 vs. DIV22 p=0,044 ; DIV12 vs. DIV17 p=0,052 ; DIV17 vs. DIV22 p=0,989

940

941 **Figure 3-figure supplement 2:** Vesicular glutamate uptake function does not influence
942 mIPSCs features.

943 A. Spontaneous inhibitory activity in VGLUT1 KO rescued neurons. Example traces of
944 mIPSC activity in wild type and sVGLUT1 rescue conditions. B: Comparison of the
945 frequency of mIPSC events in wild type and sVGLUT1 rescue conditions (N = 3 independent
946 cultures, n=31 cells, mean frequency= 4.08 Hz ± 0.59 SEM for wild type rescue; N = 3
947 independent cultures, n=56 cells, mean frequency = 3.99 Hz ± 0.52 SEM for sVGLUT1
948 rescue; unpaired t-test P= 0.62). C: Comparison of the amplitude of mIPSC events in wild
type and sVGLUT1 rescue conditions (N = 3 independent cultures, n=31 cells, mean
949 amplitude=18.06 pA ± 1.07 SEM for wild type rescue; N = 3 independent cultures, n=56 cells,
950 mean amplitude = 17.69 pA ± 0.73 SEM for sVGLUT1 rescue; unpaired t-test P= 0.70).

951
952

953 **Figure 4-figure supplement 1:** VGLUT1 is phosphorylated but not at the conserved 540-SYGAT motif
954

955 A: Effect of alkaline phosphatase on the electrophoretic mobility of VGLUT1 from forebrain
956 homogenates (and positive control VIAAT; Vesicular Inhibitory Amino-Acid Transporter).

957 B: regional distribution of VGLUT1 phosphorylation state. Homogenates of several brain
958 regions where probed for VGLUT1 signal as in panel A. OB: olfactory bulb, St: Striatum,
959 Hipp: Hippocampus, BS: Brain Stem, Thal: Thalamus, Cb: Cerebellum, Cx: Cortex. C:
960 PhosTag assay on VGLUT1 KO primary neuron cultures rescued with VGLUT1<sup>venus</sup> WT and
961 C-terminal mutant constructs. Note that PhosTag assays prevent from using a size ladder. All
962 constructs display a phospho-shifted band that disappeared upon desphosphorylation by
963 alkaline phosphatase. PI: phosphatase inhibitors; AP: alkaline phosphatase. Selected lanes
964 blotted on the same membranes were reorganized for display purpose.

965

966 **Figure 4-figure supplement 2:** VGLUT1 PRD2 domain removal does not affect mIPSCs
967 features.

968 A: Spontaneous inhibitory activity in VGLUT1 KO neurons rescued by wild type and
969 VGLUT1 P554A constructs. Example traces of mIPSC activity in sham controls, wild type
970 and VGLUT1<sup>P554A</sup> rescue conditions. B: Comparison of the frequency of mIPSC events in
971 wild type and VGLUT1<sup>P554A</sup> rescue conditions (N = 3 independent cultures, n=53 cells, mean
972 frequency= 6.21 Hz ± 0.69 SEM for sham controls; N = 3 independent cultures, n=61 cells,
973 mean frequency= 7.28 Hz ± 0.69 SEM for wild type rescue; N = 3 independent cultures, n=53
974 cells, mean frequency = 6.79Hz ± 0.63 SEM for VGLUT1<sup>P554A</sup> rescue; Mann-Whitney test of
975 wild type versus VGLUT1<sup>P554A</sup>, P=0.781). C: Comparison of the amplitude of mIPSC events
976 in wild type and VGLUT1<sup>P554A</sup> rescue conditions (N = 3 independent cultures, n=53 cells,
977 mean amplitude=19.29 pA ± 1.34 SEM for sham controls; N = 3 independent cultures, n=61
978 cells, mean amplitude=22.47 pA ± 1.13 SEM for wild type rescue; N = 3 independent cultures,
979 n=53 cells, mean amplitude = 19.24 pA ± 0.78 SEM for VGLUT1<sup>P554A</sup> rescue; Mann-
980 Whitney test of wild type versus VGLUT1<sup>P554A</sup>, P=0.137).

981

982 **Figure 5-figure supplement 1:** EndophilinA1 accumulates at synaptic vesicle clusters of
983 VGLUT1 synapses.
984 Subcellular distribution of EndophilinA1 in VGLUT1 immunopositive terminals in mossy
985 fiber terminals of the cerebellum (A,B) and cortico-striatal projections (C,D). We used pre-
986 embedding immunogold (EndophilinA1) combined with immunoperoxidase (VGLUT1)
987 methods. VGLUT1 immunoreaction product was visible at the level of SV throughout the
988 axoplasm of the terminals. Immunoparticles for EndophilinA1 are detected throughout the
989 axoplasm all over the clusters of VGLUT1 immunopositive vesicles. Scale bar : 500nm

990

991 **Supplementary file 1: Supplementary tables collating statistical analysis.**

992

993 **Supplementary file 2: Seal test recording of every cell in the electrophysiology analysis.**
<figure><img src="image_2.png" /></figure>

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A

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C

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B

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D

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E

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<figure><img src="image_35.png" /></figure>
<figure><img src="image_36.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>B</figcaption></figure>

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<figure><img src="image_41.png" /></figure>

VGLUT1 KO neurons

VGLUT2 rescue

VGLUT1 rescue

***
<figure><img src="image_42.png" /></figure>

DIV6

<figure><img src="image_43.png" /></figure>

DIV12

<figure><img src="image_44.png" /></figure>

DIV17

<figure><img src="image_45.png" /></figure>

DIV22

<figure><img src="image_46.png" /></figure>

Mean Fluorescence Intensity of VGLUT2 puncta (AU)

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>

NS p=0,052

NS
<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>
A

<figure><img src="image_52.png" /></figure>

C

Phos-tag assay

<figure><img src="image_53.png" /></figure>

B

<figure><img src="image_54.png" /></figure>

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Cerebellum granular layer

<figure><img src="image_59.png" /></figure>

Cerebellum granular layer

<figure><img src="image_60.png" /></figure>

Striatum

<figure><img src="image_61.png" /></figure>

Striatum

<figure><img src="image_62.png" /></figure>