Elisa Mazuir

Louis Richevaux

Merie Nassar

Noémie Robil

Pierre De

La Grange

Catherine Lubetzki

Desdemona Fricker
email: desdemona.fricker@parisdescartes.fr

Nathalie Sol-Foulon
email: nathalie.sol-foulon@sorbonne-univer-site.fr

Pierre De La Grange

Oligodendrocyte secreted factors shape hippocampal GABAergic neuron transcriptome and physiology

Keywords: GABAergic neurons, neuro-glia interactions, oligodendrocytes, secreted factors, single-cell RNAseq Cerebral Cortex GABAergic neurons, neuro-glia interactions, oligodendrocytes, secreted factors, single-cell RNAseq

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Communication between oligodendrocytes and neurons is crucial for circuit maturation but still not completely understood. The fast transmission of action potentials relies on insulating properties of myelin sheath which is interrupted at nodes of Ranvier, small axonal domains highly enriched in voltage-gated Na + channels [START_REF] Sherman | Mechanisms of axon ensheathment and myelin growth[END_REF]. The profile of myelination and nodes of Ranvier controls the timing of impulse transmission, critical for coincident arrival of synaptic inputs transmitted by multiple axons in sensory systems [START_REF] Seidl | Regulation of conduction time along axons[END_REF]Freeman et al. 2016;[START_REF] Arancibia-Cárcamo | Node of Ranvier length as a potential regulator of myelinated axon conduction speed[END_REF][START_REF] Monje | Myelin Plasticity and Nervous System Function[END_REF]. Both oligodendrocytes and oligodendrocyte precursor cells (OPCs or NG2 cells) sense neuronal activity, which triggers their differentiation and maturation into myelinating oligodendrocytes [START_REF] Barres | Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons[END_REF][START_REF] Demerens | Induction of myelination in the central nervous system by electrical activity[END_REF]. Adaptive myelination acts to reinforce selected circuits during learning [START_REF] Mckenzie | Motor skill learning requires active central myelination[END_REF]Bechler et al. 2018;[START_REF] Monje | Myelin Plasticity and Nervous System Function[END_REF][START_REF] Stedehouder | Activity-Dependent Myelination of Parvalbumin Interneurons Mediated by Axonal Morphological Plasticity[END_REF]. Oligodendrocytes also release lactate to provide metabolic support to axons [START_REF] Fünfschilling | Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity[END_REF][START_REF] Lee | Oligodendroglia metabolically support axons and contribute to neurodegeneration[END_REF][START_REF] Saab | Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism[END_REF].

Factors secreted by oligodendrocytes induce early formation of node-like clusters, termed prenodes, enriched in Nav channels, Nfasc186 and Ankyrin-G, along the axons of retinal ganglion cells and some hippocampal GABAergic neurons (parvalbumin or somatostatin immunopositive) before myelination [START_REF] Kaplan | Induction of sodium channel clustering by oligodendrocytes[END_REF]Freeman et al. 2015;[START_REF] Bonetto | Selective Axonal Expression of the Kv1 Channel Complex in Pre-myelinated GABAergic Hippocampal Neurons[END_REF]Dubessy et al. 2019). These early clusters are associated with an increased axonal conduction velocity along GABAergic axons (Freeman et al. 2015). In addition, oligodendrocyte lineage cells close to the soma of pyramidal neurons modulate glutamatergic neurotransmission via secreted factors and restrain high-frequency firing through the rapid uptake of K + [START_REF] Sakry | Oligodendrocyte precursor cells modulate the neuronal network by activity-dependent ectodomain cleavage of glial NG2[END_REF][START_REF] Birey | Genetic and Stress-Induced Loss of NG2 Glia Triggers Emergence of Depressive-like Behaviors through Reduced Secretion of FGF2[END_REF][START_REF] Battefeld | Myelinating satellite oligodendrocytes are integrated in a glial syncytium constraining neuronal high-frequency activity[END_REF][START_REF] Jang | Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling in the mouse brainstem[END_REF]Xin et al. 2019). Moreover, oligodendroglial exosomes and OPC secreted protein NG2 mediate glia signaling to neurons F o r P e e r R e v i e w [START_REF] Frühbeis | Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication[END_REF]Sakry et al. 2015). The identity of molecular targets by which oligodendrocyte secreted factors affect GABAergic neuron excitability and synaptic interactions remain unknown.

The present study aimed to identify targets of oligodendrocyte mediated regulation of GABAergic hippocampal neurons. Electrophysiological phenotypes of rat hippocampal neurons were compared in mixed cultures, (with glial cells, CTRL) and purified neuron cultures (without glial cells, PUR). We then tested the effects of adding oligodendrocyte conditioned medium (OCM) to purified cultures. OCM tended to reverse changes in GABAergic neuron physiology and anatomy induced by eliminating glial cells from cultures. Single-cell RNAsequencing of GABAergic neuron cytoplasm collected in patch electrodes let us explore molecular targets of OCM-induced regulation. RNA-seq analysis was validated by the presence of cell-type specific genes, including those for subclasses of GABAergic neurons. Major targets of oligodendrocyte factor signaling to GABAergic neurons included ion channels and transporters contributing to the regulation of membrane potential and action potential generation as well as transmembrane transport of zinc.

MATERIALS AND METHODS

Animals

Care and use of rats in all experiments conformed to institutional policies and guidelines (UPMC, INSERM, and European Community Council Directive 86/609/EEC). The following rat strains were studied: Sprague-Dawley or Wistar rats (Janvier Breeding Center) and VGAT-Venus Wistar rats in which a green fluorescent protein variant is selectively expressed in GA-BAergic cells (Uemastu et al. 2008). We assume that fluorescent cells in cultures correspond to GABAergic neurons.

Culture Media

We used the following culture media. NM, neurobasal medium (21103ium (2Gibco) supplemented with 0.5 mM L-glutamine, B27 (1×; Invitrogen), and penicillin-streptomycin (100 IU/mL). BS, Bottenstein-Sato medium: DMEM Glutamax supplemented with transferrin (50 μg/mL), albumin (50 μg/mL), insulin (5 μg/mL), progesterone (20 nM), putrescine (16 μg/mL), sodium selenite (5 ng/mL), T3 (40 ng/mL), T4 (40 ng/mL) and PDGF (10 ng/ml).

Preparation of oligodendrocyte conditioned medium

Glial cell cultures were prepared from cerebral cortices of P2 Wistar rats as described previously [START_REF] Mazuir | Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments[END_REF]). After meninges were removed, cortices were incubated for 35 min in papain (30 U/mL; Worthington), supplemented with L-cysteine (0.24 mg/mL) and DNase (50 µg/mL) in DMEM at 37°. They were then mechanically homogenized and passed through a 0.70 µm filter. Cells were re-suspended in DMEM with 10% FCS and 1% penicillin-streptomycin. After 7 to 14 days in vitro (DIV), oligodendroglial lineage cells were purified from glial cell cultures which initially contain astrocytes and microglial cells. After cultures were shaken overnight at 230 rpm and 37°C, overlying oligodendroglial and microglial cells could be selectively detached. Microglia were then eliminated by differential adhesion [START_REF] Mccarthy | Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue[END_REF]. Collected cells were incubated in dishes for 15 min. Non-adherent cells were retrieved and centrifuged in DMEM for 5 min at 1500 rpm. They were re-suspended and seeded at a density of 1.5x10 5 /cm 2 on Polyethylene-imine (PEI)-coated dishes with BS medium and 0.5% PDGF. Immunostaining showed that 90 ± 4% of cells were positive for the oligodendroglial marker O4 + , 7.2 ± 2.5% were GFAP + astrocytes and 4.6 ± 0.7% were CD11b + immunopositive microglial cells [START_REF] Mazuir | Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments[END_REF]. Medium from these cultures was collected after 48 hours, filtered (0.22 µm) and stored for use as oligodendrocyte conditioned medium.

Neuronal cultures

Experiments were performed in three different culture conditions (Fig. 1A). Control (CTRL) was mixed hippocampal cultures containing neurons, astrocytes, and oligodendrocyte lineage cells (Sup. Fig1A). They were prepared from E18 rat embryos and were seeded on polyethyleneimine precoated glass coverslips at a density of 50,000 cells/35 mm2 (Freeman et al. 2015). Purified neuron cultures (PUR) were prepared by adding anti-mitotic agents (FdU and U 5µM) for 12 hours, starting at 24 hours after dissection. Immunostaining showed these cultures contained less than 5% astrocytes and virtually no oligodendrocytes. In OCM cultures oligodendrocyte conditioned medium was added to purified neuron cultures. Conditioned medium (500 μl/well) was added at 3 days in vitro (DIV). One-third of the medium was replaced with neurobasal medium (NM) at 7 DIV, and then twice a week. Axon initial segments were visualized by 20 min exposure to an anti-Nfasc antibody (clone A12/18, Antibodies Incorporated) coupled to Alexa 594 (using Apex antibody labeling kit, ref A10474, Thermofisher) before recordings (Sup. Fig1B).

Patch-clamp electrophysiological recording and analysis

Electrophysiological recordings were made from cultures at 17 DIV. Dishes were transferred to a recording chamber mounted on a BX51WI microscope (Olympus, France) and superfused with ACSF containing (in mM): 124 NaCl, 2.5 KCl, 26 NaHCO3, 1 NaH2PO4, 2 CaCl2, 2 MgCl2, and 11 glucose, bubbled with 5% CO2 in O2 (pH 7.3, 305-315 mOsm/L). Temperature was kept at 34° C. Recordings were made with glass pipettes pulled using a Brown-Flaming electrode puller (Sutter Instruments) from borosilicate glass of external diameter 1.5 mm (Clark Capillary Glass, Harvard Apparatus). Pipette resistance was 6 MΩ when filled with a solution containing (in mM): 135 K-gluconate, 1.2 KCl, 10 HEPES, 0.2 ethylene glycol tetraacetic acid (EGTA), 2 MgCl2, 4 MgATP, 0.4 Tris-GTP, 10 Na2-phosphocreatine and 2.7 biocytin. RNase inhibitor was added (40U/µl, Thermo Fischer Scientific; 5µl in 1ml) when harvesting cell contents. Pipette solution pH was adjusted to 7.3 with KOH and the osmolarity was 290 mOsm.

Whole-cell current-clamp recordings were made using a MultiClamp 700B amplifier and pCLAMP software (Molecular Devices). Potential signals were filtered at 6 kHz and digitized at 20-50 kHz.

Recordings in the whole-cell current clamp configuration were made from fluorescent GA-BAergic neurons and non-fluorescent pyramidal shaped neurons in cultures prepared from VGAT-Venus Wistar rats. Excitatory postsynaptic potential (EPSP) and action potential (AP) frequencies, membrane potential and input resistance were measured at resting potential. Responses to families of hyperpolarizing and depolarizing current steps of duration 800 ms were recorded from holding potentials near -60 mV. Current intensities were manually adjusted to induce a maximal hyperpolarization close to -100 mV. Incremental positive steps of +1/10 of that value were then applied until the cell was depolarized above rheobase several times. After recording electrical data for ~10 min, neuronal contents were aspirating into the glass electrode tip. They were extracted into a tube containing 3.5 µl of lysis buffer with RNase inhibitor as a first step to prepare a library of neuronal total RNA [START_REF] Qiu | Singleneuron RNA-Seq: technical feasibility and reproducibility[END_REF]. Electrophysiological signals were analyzed with AxographX and routines written in MATLAB (The Mathwork; [START_REF] Huang | Laminar Localization and Projection-Specific Properties of Presubicular Neurons Targeting the Lateral Mammillary Nucleus, Thalamus, or Medial Entorhinal Cortex[END_REF]. EPSP and AP frequencies were measured from baseline records of duration at least 3000 ms. Sup. Fig2 shows procedures used to measure active and passive membrane parameters.

Reconstruction of neuronal morphology

Pipettes used for patch-clamp recordings included 2.7 mM biocytin. Cultures containing filled cells were fixed at 17 DIV with PFA4% (diluted in PBS 1X; pH 7.2) for 10 min at room temperature (RT). Coverslips were washed three times with PBS 1X and blocked with 5% normal Images of stained cells were acquired on a upright spinning disk microscope (Intelligent Imaging Innovations, Inc) using a 20x glycerol immersion objective (NA 1.0), a CSU-W1 spinning disk head (Yokogawa) and a sCMOS ORCA-Flash4.0 camera (Hamamatsu). Multiple tile regions each with Z step series of separation 1.1 µm were acquired for each cell. Tile scans were stitched using Fiji software with BigStitcher plugin. Neuronal arborizations were drawn with the semi-automatic filament tracer tool of IMARIS software (Bitplane). The axon was identified from Neurofascin immunostaining of its initial segment. Axonal and dendritic lengths and data for Sholl analyses were derived by the IMARIS software.

Statistical analysis of electrophysiological properties and morphology

Statistical analyses were performed using GraphPad Prism version 7.0. Experiments for each condition were carried out in at least 3 independent cultures from at least 3 different litters.

Electrophysiological parameters in Figs. 1 and2 were compared using the Mann-Whitney test (for PYR CTRL vs. GABA CTRL) and using Kruskal Wallis and Dunn's multiple comparison post hoc test (for GABA CTRL vs. GABA PUR, GABA PUR vs. GABA OCM and GABA CTRL vs. GABA OCM). P-values are given in Table 1.

Axonal and dendritic lengths were compared (Fig. 3 and GABA CTRL vs. GABA OCM). Dendritic arborizations were assessed with Sholl analysis which measures the number of dendrites which intersect circles of increasing distance from the neuronal soma (20 µm increments were used). They were analyzed using a linear mixed-effects model (LMM) with culture condition and radial distance as fixed effects, and the cell identifier number as a random effect to account for the successive measurements over the concentric rings (Wilson et al. 2017). Significance for the main effects of condition, distance and their interaction was then evaluated using ANOVA Type II Wald chi-square tests. Analyses were made with R (R Development Core Team, ver 3.5.1, 2019) and plots were generated with the ggplot2 package [START_REF] Wickham | Ggplot2: Elegant Graphics for Data Analysis[END_REF]. LMM was fitted with the function lmer in the lme4 package [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF]. When a factor had a significant effect or when a significant interaction was found between condition and distance, post-hoc pairwise comparisons were completed with Tukey's method. Data were square root transformed before modeling to improve model assumptions of linearity, normality and constant variance of residuals, Data are given as mean ± SD (i.e. standard deviation) in Table 1 and in the text. The level of statistical significance was set at p < 0.05 for all tests. Significance is represented as numbers of asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 cDNA synthesis, library preparation and sequencing mRNA capture, reverse transcription and amplification was achieved using the SMART-Seq v4 ultra low input RNA Kit (Takara,634891). This kit improves synthesis of the full-length cDNA via a template switching mechanism for synthesis of the second strand cDNA. 5µl of sample was used for hybridization with the 3'smart-seq primer, then poly{T}-primed mRNA was converted to cDNA by reverse transcriptase and PCR amplification was done, according to the kit instructions. Full-length cDNA was then processed with a Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096). This kit aims to fragment and add adapter sequences onto template DNA with a single tube Nextera XT tagmentation reaction and so generate multiplex sequencing libraries. The resulting indexed paired-end libraries were sequenced by nextgeneration sequencing (NGS), using NextSeq500 2x75pb, 33 million of reads per sample (Illumina NextSeq 500 platform) (Sup. Fig3A).

RNA-seq data analysis

RNA-Seq data analysis was performed by GenoSplice technology (www.genosplice.com). Sequencing, data quality, read distribution (to check for ribosomal contamination for instance), and insert size estimation were done with FastQC, Picard-Tools, Samtools and rseqc. Reads were mapped using STARv2.4.0 (Dobin et al. 2013) on the rn6 Rat genome assembly. The regulation of gene expression was studied as in [START_REF] Noli | Discordant Growth of Monozygotic Twins Starts at the Blastocyst Stage: A Case Study[END_REF]. For each gene of the Rat FAST DB v2016_1 annotations, reads aligning on constitutive regions (not prone to alternative splicing) were counted. Normalization and differential gene expression was assessed from these reads using DESeq2 running on R (v.3.2.5, [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. FastQC was used for quality control, sequencing quality per base and sequence, per base sequence and GC content, N content, overrepresented sequences, sequence lengths. Sequence coverage of introns and exons was used to ensure that the sequences derived from mRNA rather than genomic DNA.

From 64 samples, 21 passed these control quality steps. Others were discarded due to sample quality (n=32) or contamination detected from marker expression (n=11) based on the following GFAP, Aquaporin 4,Slc1a2,PDGFRa,MOG,Itgam (Sup. Fig3 B,D,E).

Genes were considered as expressed if their FPKM (i.e. Fragments Per Kilobase Million) value was greater than 98% of the background FPKM value from intergenic regions (Sup. Fig4 Clustering and heatmap analyses were performed using dist" and "hclust" functions in R, with

Euclidean distance and Ward agglomeration method.

Genes were considered to be differentially expressed when the uncorrected p-value ≤ 0.05 and fold-change ≥ 1.5.

Enriched gene ontology (GO) terms were assessed, from the identity of differentially expressed genes, with the DAVID Functional Annotation Tool (v6.8), [START_REF] Huang | The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists[END_REF]). GO terms were considered enriched when fold enrichment ≥ 2.0 and uncorrected p-value ≤ 0.05 (1.3 value in GO term graph matches with a p value of 0.05), and at least 2 regulated genes in the pathway/term.

A Pearson correlation-based approach was used to compare single-cell RNA values with electrophysiological parameters for neurons (Fig. 6). The procedure was restricted to genes expressed in at least five samples, with coefficients greater than 0.6 and significant p-values (p<0.05).

RESULTS

Oligodendrocyte secreted factors control the electrophysiological properties of hippocampal GABAergic neurons

We first asked how the presence of glial cells or oligodendrocyte secreted factors affected GABAergic neuron phenotype. Hippocampal neuron cultures were prepared from VGAT-Venus rat embryos so that GABAergic interneurons could be identified. Spontaneous activity and active and passive membrane properties were recorded in the current clamp mode from fluorescent GABAergic neurons (n=36). These properties were compared with those of unlabeled pyramidal cells (n=11). We further compared the physiology of GABAergic neurons in control cultures (CTRL) with those in purified cultures lacking glial cells (PUR) and with

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those in purified cultures supplemented with oligodendrocyte conditioned medium (OCM) (Fig. 1A,B).

Spontaneous synaptic events and spiking activity of recorded cells was quantified at resting potential with no injected current (Fig. 1C-E). In pyramidal cells (PYR), frequencies of excitatory postsynaptic potentials (EPSP) and action potentials (AP) were low in CTRL cultures (PYR CTRL, EPSPs, 7±2 Hz; APs, 0.03±0.01 Hz; n=11). Mean resting potential was -63±9

mV and mean input resistance was 330±218 MΩ (Fig. 1F,G; Table 1). EPSP frequency and AP discharge rate were both higher in GABAergic neurons from the same cultures. Mean EPSP frequency was 70±13 Hz and AP discharge frequency was 0.6±0.5 Hz (GABA CTRL, n=11).

In PUR neuronal cultures, both EPSP frequency and AP discharge by GABAergic neurons were reduced (GABA PUR, EPSPs, 6±2 Hz, n=10; APs, 0.03±0.03 Hz, n=11). This despite a more depolarized mean resting membrane potential (GABA PUR, -49±11 mV vs GABA CTRL, -55±5 mV) and a significantly higher input resistance than in control cultures (GABA PUR, 144±46 MΩ vs GABA CTRL, 74±53 MΩ). Supplementing PUR cultures with oligodendrocyte conditioned medium tended to increase EPSP and AP frequencies towards CTRL levels (GABA OCM,EPSPs,19±4 Hz,n=13;APs,0.4±0.03 Hz,n=12). Mean membrane potential and input resistance also reverted towards values in CTRL mixed cultures (GABA OCM, -54±7 mV and 110±104 MΩ). These data suggest that oligodendrocyte secreted factors influence active and passive aspects of the physiological phenotype of GABAergic neurons. We next asked whether the properties of fluorescent and non-fluorescent cells were consistent with those of GABAergic neurons and pyramidal cells respectively [START_REF] Pelkey | Hippocampal GABAergic Inhibitory Interneurons[END_REF].

In control cultures, the mean resting potential of fluorescent neurons was significantly more depolarized (GABA CTRL, -55±5 mV vs. PYR CTRL, -63±9 mV; Fig. 1F), and their input resistance was significantly lower than that of non-fluorescent cells (74±53 MΩ vs. 330±218 MΩ; Fig. 1G). Comparing neuronal responses to depolarizing and hyperpolarizing step current injections (Sup. Fig. 2, Fig. 2) revealed significant differences in the membrane time constant tau (Fig. 2C, GABA CTRL, 12±10 ms vs. PYR CTRL, 31±16 ms), sag ratio, which is linked to the presence of an h-current, (Fig. 2D, 1.27±0.10 vs 1.10±0.10), rheobase current (Fig. 2E, 427±284 pA vs 121±83 pA) and firing rate induced by a 200 pA step current injection (Fig. 2F, 3±7 Hz vs 26±19 Hz). Input-output plots of the number of APs against the injected current had a mean initial slope of 99±48 Hz/pA in fluorescent cells, significantly lower than 224±150

Hz/pA for non-fluorescent cells (Fig. 2G). Action potential width in fluorescent neurons was short, 0.50±0.20 ms (Fig. 2I), as is characteristic of some interneurons, compared to an AP width of 1.51±0.8 ms in non-fluorescent neurons. AP thresholds were similar: -32.2±3.5 mV in fluorescent cells (Fig. 2H) and -30.5±3.3 mV in non-fluorescent cells. AP rising amplitude (Fig. 2K) was 61±9 mV in fluorescent cells, significantly lower than a value of 78±7 mV in nonfluorescent cells. Maximum depolarization and repolarization rates were 294±68 V/s and -164±82 V/s respectively in fluorescent cells, compared to 325±212 V/s (Fig. 2L) and -61±28

V/s (Fig. 2M) in non-fluorescent neurons. Action potential after hyper-polarizations (AHP)

were larger in fluorescent cells at -27.6±3.3 mV compared to -19.5±4.4 mV (Fig. 2J). The latency to the first AP at rheobase was significantly shorter, 167±172 ms compared to 349±251 ms (Fig. 2N). Overall these data confirm that fluorescent neurons from VGAT-Venus animals Our next objective was to compare electrophysiological phenotypes for GABAergic neurons in control conditions (CTRL), in purified cultures (PUR) with no glial cells and in PUR cultures supplemented with oligodendrocyte conditioned medium (OCM). We found nodal proteins were clustered on GABAergic axons in CTRL but not in PUR cultures (Sup. Fig1D).

Adding OCM to PUR cultures induced prenode formation as previously shown (Sup. Fig1D;Freeman et al. 2015;Dubessy et al. 2019). We also found some electrophysiological parameters of GABAergic neurons changed when glial cells were absent, and were partially restored by OCM treatment (Fig. 1, 2 and Table 1). The mean resting membrane potential of GABAergic neurons did not change significantly (Fig. 1F, CTRL, -55±5 mV; PUR, -49±11 mV; OCM, -54±7 mV). Input resistance increased in PUR cultures (Fig. These data show that factors of the OCM have significant effects on the input resistance, rheobase current, and AP and AHP amplitudes of GABAergic neurons. These effects partially reverse changes induced by switching from a mixed culture of neurons, oligodendrocytes, and other glial cells, to a purified neuronal culture.

Impact of OCM on hippocampal GABAergic neuron morphology

We next asked whether factors secreted by oligodendrocyte exert trophic effects on GA-BAergic neuron anatomy. Biocytin-filled GABAergic neurons from CTRL, PUR, and OCM cultures were reconstructed and axonal and dendritic lengths were measured (Fig. 3A-C). The absence of glial cells had no significant effect on total axonal length (CTRL, 1179 ± 427 µm, n=6;PUR, 1929PUR, ± 1305 µm, n=5 µm, n=5; Fig. 3B) or total dendritic length (CTRL, 4405 ± 983 µm, n=6; PUR, 4794 ± 1621 µm, n=5; Fig. 3C). Scholl analysis of dendritic arbors revealed slight differences (Fig. 3D). However, in OCM cultures, total axonal length (OCM, 6451 ± 4793 µm, n=8) was significantly increased compared to CTRL (p=0.0148) and mid-dendritic arbors were significantly more complex at distances of 260-700 µm from the soma compared to CTRL and PUR (Fig. 3B,D). Thus, oligodendrocyte derived factors exert effects on axons and dendrites of GABAergic neurons, which were not apparent on switching from CTRL to PUR cultures.

Single-cell transcriptomic analysis of electrophysiologically characterized neurons

We used single cell RNA-sequencing (scRNA-seq) of the cytoplasmic contents of GA-BAergic neurons to pursue the identity of protein targets underlying changes in their phenotype induced by OCM. When patch-clamp recordings were completed, the same pipette was used to harvest neuronal cytosolic contents (n=64). Sup. Fig. 3A, B illustrates the experimental workflow and bioinformatics pipeline. We noted a strong correlation (R=0.79) between the number of expressed genes and the number of aligned reads suggesting that increasing reads could improve transcript detection (Fig. 4-1C). RNA-seq data was filtered to exclude samples of poor RNA quality where fewer than 2000 transcripts were detected. We also excluded samples which expressed glial cell specific genes, such as Mog or Gfap, as likely contaminated. Samples from 21 neurons passed these two controls.

Principal component analysis (PCA) of transcriptomic profiles captured 22% of explained variance with the first two principal components, PC1 and PC2 (Fig. 4A). Pyramidal cells and GABAergic neurons were clearly segregated in this two-dimensional space. GABAergic neurons from PUR cultures were separated from GABAergic neurons in CTRL cultures.

Transcriptomes of GABAergic neurons from OCM cultures overlapped with those of CTRL cultures.

We investigated transcriptomic differences between hippocampal neuron types and the transcriptional response to glial factors in GABAergic neurons. The heat map of Fig. 4B shows in red up-regulated genes and in blue genes that were down-regulated with respect to mean expression in all three conditions. High, deep sequencing detected up to 5,700 expressed genes in single neurons. Hierarchical clustering of samples from different cells in the heat map segregated 4 out of 5 pyramidal neurons from GABAergic neurons. GABAergic neurons from purified cultures (PUR) were also grouped together in the dendrogram while GABAergic neurons sampled in CTRL and OCM cultures overlapped in a large branch (Fig. 4B). Elfn1, the serotonin receptor Htr3a, KCC2 the potassium chloride cotransporter 2, Slc12a5, the kinase Erbb4 and the protease reelin, Reln. We note that some molecular markers examined here are not entirely specific to GABAergic neurons, and were also detected in samples from 1 or 2 pyramidal cells (Slc12a5, Calb1, Satb1, Reln, Sst, Npy, Elfn1 and Cck.). Furthermore, no samples from either pyramidal or GABAergic neurons expressed Vip, Calb2 or Nos1 (Fig. 4D).

F

Ion channel and transmitter receptor gene expression in single hippocampal neurons

The cellular and synaptic physiology of neurons depends on the expression of genes coding for ion channels, transporters and neurotransmitter receptors. We examined quantitative expression of these genes in samples from each neuron in our RNAseq data set (Fig. 5A,B).

Different neurons expressed different levels of Atp1a, b genes coding for Na + /K + -transporting ATPase subunits and Clcn3-7 genes coding for Cl -/H + exchangers, which contribute to stabilize membrane potential. The depolarization phase of action potentials is due to opening of voltagegated sodium channels, which consist of an α-subunit forming pore (Scn1a-9a) and auxiliary ß subunits (Scn1b-4b). Almost all neurons expressed high levels of Scn2a (encodes Nav1.2). In contrast, only GABAergic neurons in CTRL cultures with glial cells or with oligodendroglial factors (OCM) expressed Scn1a (Nav1.1) and few expressed Scn8a (Nav1.6). The Na + channel modifier 1 (Scnm1), which governs alternative splicing of pre-mRNAs, was detected in samples from some GABAergic neurons but not from pyramidal cells.

Various potassium channels are crucial for action potential repolarization, generate the AHP and contribute to maintenance of membrane potential. Voltage-gated K + channels differ in structure, biophysics and pharmacology from voltage independent, two-pore-domain (K2P) channels which support leak-type K + conductances. We found that distinct neurons express

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specific combinations of K + channel ∝ (Kcn a to v) and auxiliary subunits (Kcnab1-2) as well as K2P channels (Kcnk1-10). We also examined Ca 2+ channels, Ca 2+ -activated K + channels and hyperpolarization-activated, cyclic nucleotide gated, K + /Na + permeable 'h' channels (HCN).

Fig. 5B shows genes encoding ionotropic glutamate receptors, expressed at excitatory synapses, and including AMPA receptors (Gria1-4), NMDA receptors (Grin1-3) and kainate receptors (Grik1-5). Genes encoding GABAA receptors (Gabra-g), which mediate fast inhibitory neurotransmission and are assembled as heteropentameric chloride channels, are also indicated as are detected genes which code for subunits of nicotinic cholinergic receptors (Chrnab), serotonin receptor (Htr3a) and glycine receptor (Glra2). Fig. 5C shows genes coding for Gprotein linked receptors including metabotropic glutamate (Grm2, 5) and GABAB (Gabbr1, 2) receptors.

Correlation-based approach

These scPatch-seq data permit quantitative assays of transcriptomic features. We attempted to relate them to intrinsic neuronal electrophysiology by searching for correlations between values from transcriptomic samples and different electrophysiological parameters. Fig.

6 plots genes coding for ion channels, transporters and synaptic receptors for which a correlation (p-value <0.05) with electrophysiological parameters was detected. The analysis was based on all neurons with complete electrical and transcriptomic datasets and on cells from all culture conditions. Genes were clustered based on their relations with electrophysiological parameters.

One cluster coding for α1, ß1 and α3 subunits of the Na + , K + -ATPase (Atp1a1, Atp1b1 and Atp1a3), was correlated with neuronal input resistance, Atp1b1 was also correlated with time constant, tau and AP width. The Na + channel subunit Scn2a (coding for Nav1.2) was correlated with the time constant tau and with AP threshold, neuronal input resistance and AP width. A larger cluster, consisting of several K-channel linked genes (Kcnc2, coding for Kv3.2; Kcnk3, Task-1; Kcnip1, K + channel modulatory protein), as well as two Zn transporters (Slc30a3, ZnT3; Slc30a4, ZnT4) was strongly correlated with the Sag ratio, the AP threshold and the after-hyperpolarization amplitude (AP AHP). both increase together. Significant positive or negative correlations with coefficient > 0.6 and p-value <0.05 are marked with a yellow star.

Transcriptomic patterns across groups of neurons in relation to biological processes

Gene Ontology (GO) analysis let us estimate biological processes underlying differential expression of groups of genes in different neurons or in different culture conditions. For Many genes were down-regulated in one comparison and up-regulated in the other. GO process terms identified from their identity regulate neuronal action potential, synapse assembly, Ca 2+dependent exocytosis, protein phosphorylation, kinase signaling and cell division (Fig. 7B,C).

GO-analysis further suggested that genes coding for proteins involved in transmembrane transport of K + were up-regulated by OCM treatment (Fig. 7C). Further insights into processes affected by oligodendrocyte factors were obtained by grouping genes with similar profiles of changes. RNA-seq data from GABAergic neurons in different culture conditions was normalized with respect to expression of each gene in pyramidal cells (Fig. 8A, B and Sup. Fig6). A total of 241 genes were identified as differentially expressed between pyramidal and GABAergic neurons in CTRL cultures and were mostly insensitive to PUR or OCM culture conditions (Sup. Fig6).

We found a significant difference in the group of genes that were reduced in PUR cultures and restored in OCM cultures. For one group of these genes, expression in GABAergic and pyramidal neurons in CTRL conditions were comparable (Fig. 8A; n=137). Another second group of genes differed in that expression in GABAergic neurons was systematically higher than in pyramidal cells in CTRL cultures (Fig. 8B; n=120). GO term analysis of the identities of the first group of genes (Fig. 8a') linked them to processes including negative regulation of protein kinase activity and apoptosis signaling pathways. In contrast, the identities of the second group of genes (Fig. 8b') evoked biological processes including the membrane transport of zinc, K + channels, regulation of membrane potential and action potential and G-protein coupled receptor signaling pathways.

Finally, we present target genes whose expression was affected by the absence of glial cells in These target transcripts code for ion channels, transporters, synaptic markers, vesicle trafficking, cytoskeleton remodeling, cell adhesion molecules, growth factors and signaling (Fig. 8C)

and were mostly up-regulated in CTRL and OCM neurons. They included Na + channels, Scn1a (Nav1.1) andScn1b (ß1Nav) andK + channels, Kcna1 (Kv1.1), Kcnab1 (ß1Kv), Kcnk3 (Task-1), Kcnip1 (KChip-1, a K + channel modulatory protein). Genes encoding zinc transporters (Slc39a11, Slc30a4 and Slc30a3), were upregulated by OCM, while a Na + /K + /Ca 2+ exchanger (Slc24a2) was one of the few transcripts downregulated by OCM. Among genes coding for signaling molecules, growth factors and receptors, the kinases Nek7, Pak1 and Akt1, and growth factors Vegfb, Pdfga, Fgf9 and Rara were up-regulated in OCM culture conditions. Only the protein kinase C binding protein Nell2 and the neuropeptide hormone Proenkephalin (Penk)

were downregulated by OCM. To address possible coordinated changes, we also analyzed pathways of regulated genes between OCM and PUR, or CTRL and PUR. We found that the MAPK, AMPc and PI3K-Akt signal transduction pathways are significantly up-regulated by glial cells and OCM, respectively (Fig. 8D,E). showed (Fig. 2) GABAergic neurons in culture conserved properties, including a relatively depolarized resting potential, and action potentials of short duration followed by a prominent AHP, which distinguish them from hippocampal pyramidal cells [START_REF] Spruston | Perforated patch-clamp analysis of the passive membrane properties of three classes of hippocampal neurons[END_REF]Fricker et al. 1999;[START_REF] Staff | Resting and active properties of pyramidal neurons in subiculum and CA1 of rat hippocampus[END_REF][START_REF] Gu | Interneurons. Fast-spiking, parvalbumin + GABAergic interneurons: from cellular design to microcircuit function[END_REF][START_REF] Prestigio | Spike-related electrophysiological identification of cultured hippocampal excitatory and inhibitory neurons[END_REF].

The frequency of excitatory synaptic events impinging on hippocampal GABAergic neurons was strongly reduced in the absence of glial cells. This is consistent with data showing that glutamatergic synaptic transmission is enhanced by factors secreted by glia [START_REF] Turko | Differential Dependence of GABAergic and Glutamatergic Neurons on Glia for the Establishment of Synaptic Transmission[END_REF]) including astrocytes [START_REF] Baldwin | Molecular mechanisms of astrocyte-induced synaptogenesis[END_REF]. Here we demonstrate that oligodendrocytes regulate synaptic excitation of GABAergic interneurons. Adding OCM to PUR cultures partly restored EPSC frequencies. Additional effects of OPC or astrocyte -released factors, as well as cell-cell contacts in CTRL culture may explain differences with OCM effect.

Anatomical analysis revealed that oligodendrocyte secreted factors (OCM) enhanced the complexity of dendritic arbors, although dendritic length differed little between cultures that did (CTRL) or did not (PUR) contain glial cells. Novel synapses formed on more complex dendrites in OCM cultures may contribute to the increased EPSC frequency in these conditions.

We found similar electrophysiological properties for GABAergic neurons grown in CTRL and OCM cultures, notably a low input resistance and high rheobase. Rheobase was reduced and AP firing frequency significantly depressed in PUR cultures. The PUR condition may be similar to a lack of maturation in some respect [START_REF] Okaty | Transcriptional and electrophysiological maturation of neocortical fast-spiking GABAergic interneurons[END_REF] and our study may suggest that the maturation process depend on oligodendrocyte secreted factors. The phenotype of low intrinsic excitability corresponds to mature neurons expressing a large array of voltage dependent or independent ion channels. We show (see Figure 8C-E [START_REF] Wilkins | Oligodendrocytes promote neuronal survival and axonal length by distinct intracellular mechanisms: a novel role for oligodendrocyte-derived glial cell line-derived neurotrophic factor[END_REF]. We previously showed that Contactin-1 together with Phosphacan or Tenascin-R secreted by oligodendrocytes mediate early formation of prenodal clusters along hippocampal GABAergic axons likely through a direct clustering effect (Dubessy et al., 2019).

In addition to its role in prenodal clustering, Contactin-1 also plays important role in the hippocampus, in synaptic plasticity, neurogenesis, and memory in adult mice [START_REF] Murai | Contactin supports synaptic plasticity associated with hippocampal long-term depression but not potentiation[END_REF][START_REF] Puzzo | F3/Contactin promotes hippocampal neurogenesis, synaptic plasticity, and memory in adult mice[END_REF]

Technical points

This work combined patch clamp recordings with single neuron RNA-seq analysis. Relatively few studies have demonstrated strong correlations between single-neuron transcriptomic profiles and electrophysiological phenotypes [START_REF] Cadwell | Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq[END_REF]Földy et al. 2016;[START_REF] Fuzik | Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes[END_REF][START_REF] Muñoz-Manchado | Diversity of Interneurons in the Dorsal Striatum Revealed by Single-Cell RNA Sequencing and PatchSeq[END_REF][START_REF] Scala | Layer 4 of mouse neocortex differs in cell types and circuit organization between sensory areas[END_REF]. We took several steps to ensure and verify the validity of our results. The duration of patch electrode recordings was deliberately limited to ~10 min in order to minimize perturbation of the transcriptome [START_REF] Fuzik | Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes[END_REF]). When possible, patch electrodes targeted isolated neuronal somata to reduce possible mRNA contamination from adjacent cells [START_REF] Tripathy | Transcriptomic correlates of neuron electrophysiological diversity[END_REF].

As for previously published single-cell RNAseq datasets, the number of sequenced reads per cell was found to be positively correlated with detected transcript counts and did not reach a plateau [START_REF] Cadwell | Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq[END_REF]Tasic et al. 2016;[START_REF] Tripathy | Transcriptomic correlates of neuron electrophysiological diversity[END_REF]. We attempted to validate our data by cross-correlating transcripts detected for recorded pyramidal and inhibitory neurons with expected profiles for these cell types. Most GABAergic neurons expressed molecular markers, including peptides and Ca-binding proteins, specific to known subclasses of these cells (Zeisel et al. 2015;[START_REF] Gouwens | Integrated morphoelectric and transcriptomic classification of cortical GABAergic cells[END_REF]. Larger numbers of sequenced cells would have permitted enhanced statistics to assure data quality even if links between specific transcripts and identified cell types tends to support our approach.

Single neuron transcriptomes obtained in this way helped us define a global view of processes initiated by oligodendrocyte conditioned medium. They showed glial factors modify the transcriptome of GABAergic neurons to change intrinsic electrophysiological properties, AP generation, EPSC frequencies and dendritic anatomy. A minority of GABAergic neurons expressed genes for Reelin and NPY as do neurogliaform cells [START_REF] Pelkey | Hippocampal GABAergic Inhibitory Interneurons[END_REF].

GABAergic neuron data revealed genes coding for proteins relevant to specific aspects of inhibitory cell physiology. They included Nav1.1 (Scn1a), Kv3.2 (Kcnc2) and Task-1(Kcnk3) in neurons with PV and/or SST genes [START_REF] Chow | K(+) channel expression distinguishes subpopulations of parvalbumin-and somatostatin-containing neocortical interneurons[END_REF][START_REF] Torborg | TASK-like conductances are present within hippocampal CA1 stratum oriens interneuron subpopulations[END_REF][START_REF] Lorincz | Cell-type-dependent molecular composition of the axon initial segment[END_REF]. Kv3 channels with fast kinetics curtail action potentials permitting sustained firing at high frequencies [START_REF] Rudy | Kv3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing[END_REF][START_REF] Gu | Interneurons. Fast-spiking, parvalbumin + GABAergic interneurons: from cellular design to microcircuit function[END_REF][START_REF] Hu | Complementary Tuning of Na+ and K+ Channel Gating Underlies Fast and Energy-Efficient Action Potentials in GABAergic Interneuron Axons[END_REF]. Task-1 forms K + permeable leak channels which contribute to resting potential and membrane resistance [START_REF] Okaty | Transcriptional and electrophysiological maturation of neocortical fast-spiking GABAergic interneurons[END_REF]). We found high levels of genes for the zinc transporters, ZnT3

Correlation between gene expression and electrophysiological parameters

We attempted to link expression of genes for ion channels and neurotransmitter receptors with elements of electrophysiological phenotypes using a correlation-based analysis on data from different cell types and culture conditions. The analysis suggests genes for α1, ß1 and α3 subunits of the Na + , K + -ATPase, Atp1a1, Atp1b1 and Atp1a3, are linked to neuronal input resistance. The Na + /K + -ATPase maintains transmembrane ionic gradients and resting membrane thus affecting neuronal excitability [START_REF] Larsen | Managing Brain Extracellular K(+) during Neuronal Activity: The Physiological Role of the Na(+)/K(+)-ATPase Subunit Isoforms[END_REF]). The Na-channel subunit Scn2a (Nav1.2) was found to be correlated with action potential width, neuronal input resistance, and the membrane time constant, tau. A cluster including several K + channels (Kcnc2, Kcnk3 and Kcnip-1) and two Zinc transporters (Slc30a3 and Slc30a), was expressed selectively in GA-BAergic neurons, and correlated with AP threshold, sag ratio and the after-hyperpolarization amplitude. We should note that these correlations do not imply causality and caution that classdriven correlations are an important confound in our dataset. Some within-cell-type correlations may have been missed [START_REF] Bomkamp | Transcriptomic correlates of electrophysiological and morphological diversity within and across excitatory and inhibitory neuron classes[END_REF].

Biological processes affected by glial cells and oligodendroglial secreted factors

Clustering genes with similar patterns of altered expression revealed GO process terms regulated by factors in OCM. Processes identified in this way matched quite efficiently with changes in GABAergic neuron phenotype inferred from electrophysiological and anatomical observations. Enriched processes included synapse assembly, action potential generation, transmembrane transport of ions specifically zinc, and kinase signaling. They derived from differential expression of K + channel genes, including Kcna1 (Kv1.1), Kcnab1 (Kv ß1chain), Kcnip1

(KChIP) and Kcnk3 (Task-1), and Na + channel genes, including Scn1a (Nav1.1) and Scn1b

(Nav ß1chain). We found two kinases which were upregulated in OCM. Nek7 is involved in microtubule polymerization during the formation of PV+ interneuron connections (Hinojosa et

  0.1% Triton for 15 min at RT. Biocytin-filled cells and their axon initial segments were visualized. Cultures were incubated with anti-Neurofascin (1:100, ab31457, Abcam) for 2 hours at RT. After three PBS rinses, they were incubated with Streptavidine-Alexa 488 (ThermoFisher Scientific) to visualize biocytin-filled neurons and anti-rabbit-Alexa 594 (1:1000, ThermoFisher Scientific) for Neurofascin for 1 hour at RT. Stained cultures were mounted with Fluoromount-G.

  ) using Kruskal Wallis and Dunn's multiple comparison post hoc test (for GABA CTRL vs. GABA PUR and GABA PUR vs. GABA OCM

Figure 1 :

 1 Figure 1: Patch clamp recording and cytosol harvesting of hippocampal neurons. (A) Sche-

  neurons, and non-fluorescent cells to pyramidal cells.

Figure 3 :

 3 Figure 3: Axo-dendritic morphologies of biocytin filled GABAergic neurons. (A) Reconstruc-

Figure 4 :

 4 Figure 4: Gene expression analysis of the content of pyramidal or GABAergic neuron cyto-

Figure 5 :

 5 Figure 5: Cell-type specific expression of mRNAs coding for ion channel and receptors. (A)

Figure 6 :

 6 Figure 6: Pearson correlation between scRNASeq data and electrophysiological parameters.

  genes were differentially expressed in GABAergic neurons and pyramidal neurons in CTRL cultures (Fig.7A, Sup. Fig5). GO process terms derived from the identity of the genes were related to synaptic transmission, organization and transmitter transport as well as cortical development. Comparing GABAergic neurons in CTRL and PUR cultures we found 219 genes were differentially expressed, mostly down-regulated in PUR conditions (Fig.7B, Sup. Fig5). Inversely, comparing GABAergic neurons in PUR and OCM cultures, 192 genes were differentially expressed, mostly up-regulated in OCM conditions (Fig.7C, Sup. Fig5).

Figure 7 :

 7 Figure 7: Heatmap of genes expressed differentially between conditions. (A) Differences be-

  inversely by OCM and which are linked to GABAergic cell physiology.

Figure 8 :

 8 Figure 8: Gene ontology analysis of regulated genes. (A, B) Expression within clusters with a

  Gene expression in hippocampal neuron typesTranscriptomic data are defining the classification of cortical neurons(Zeisel et al. 2015;[START_REF] Cembrowski | Hipposeq: a comprehensive RNA-seq database of gene expression in hippocampal principal neurons[END_REF][START_REF] Harris | Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics[END_REF][START_REF] Sugino | Adult mouse cortical cell taxonomy revealed by single cell transcriptomics[END_REF] Yuste et al. 2020). A recent study based on Patch-seq data from several 1000s of cells may offer the best current correspondence between transcriptomic, anatomical and electrophysiological data for GABAergic mouse cortical neurons[START_REF] Gouwens | Integrated morphoelectric and transcriptomic classification of cortical GABAergic cells[END_REF]. Our data can be interpreted in the light of those studies. Most GABAergic neurons studied in the hippocampal cultures studied here expressed Somatostatin (SST) associated with other peptide markers. Several subtypes of hippocampal inhibitory cells express SST including long-range inhibitory neurons which possess myelinated axons (and also express Calbindin and Npy), oriens-lacunosum moleculare (O-LM) interneurons (also Elfn1 and Pnoc), or oriens-bistratified neurons (also Tac1, Npy, Satb1 and Erbb4)[START_REF] Somogyi | Defined types of cortical interneurone structure space and spike timing in the hippocampus[END_REF][START_REF] Jinno | Structural organization of long-range GABAergic projection system of the hippocampus[END_REF][START_REF] Harris | Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics[END_REF]. We found some cells expressed genes for Sst, Pnoc and Pvalb[START_REF] Jinno | Colocalization of parvalbumin and somatostatin-like immunoreactivity in the mouse hippocampus: Quantitative analysis with optical disector[END_REF][START_REF] Jinno | Structural organization of long-range GABAergic projection system of the hippocampus[END_REF][START_REF] Harris | Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics[END_REF]).

(

  Slc30a3) and ZnT4 (Slc30a4), which are found in SST-containing interneurons[START_REF] Paul | Transcriptional Architecture of Synaptic Communication Delineates GABAergic Neuron Identity[END_REF]. ZnT3 is a vesicular transporter which may contribute to the co-release of zinc in synaptic vesicles with GABA (McAllister and Dyck 2017).

  ). In addition, we found that the PI3K-Akt, MAPK and AMPc signal transduction pathways are significantly up-regulated by OCM and glial cells. These signaling pathways control gene expression by phosphorylating a number of transcription factors and thus regulating their transcriptional activity. Previous studies have shown that these pathways are activated by oligodendrocytes secreted factors to exert their neuromodulatory functions on cultured neurons[START_REF] Meyer-Franke | Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture[END_REF][START_REF] Wilkins | Oligodendrocytes promote neuronal survival and axonal length by distinct intracellular mechanisms: a novel role for oligodendrocyte-derived glial cell line-derived neurotrophic factor[END_REF]. PIP3-Akt1-mTOR activation is critical for neuronal development, and notably for hippocampal function[START_REF] Wang | Control of synaptic strength, a novel function of Akt[END_REF][START_REF] Lai | Akt1 deficiency affects neuronal morphology and predisposes to abnormalities in prefrontal cortex functioning[END_REF][START_REF] Goebbels | A neuronal PI(3,4,5)P3-dependent program of oligodendrocyte precursor recruitment and myelination[END_REF][START_REF] Balu | Akt1 deficiency in schizophrenia and impairment of hippocampal plasticity and function[END_REF]. It increases the caliber of axons and the expression of numerous genes encoding regulatory proteins, which might be sufficient to trigger all steps of myelination[START_REF] Goebbels | A neuronal PI(3,4,5)P3-dependent program of oligodendrocyte precursor recruitment and myelination[END_REF].In conclusion, our study provides new insights into communication between glial cells and neurons showing that factors secreted by oligodendrocytes induce transcriptomic changes which may modulate the physiology and anatomy of hippocampal GABAergic neurons. Further study analyzing OCM effect on purified GABAergic neurons would allow to discriminate indirect effect via glutamatergic neurotransmission and cell-autonomous effect. In addition, it is now established that PV + neurons as well as some Sst + neurons with long range projections through the hippocampus and more distant regions are frequently myelinated(Jinno et al. 2007;[START_REF] Micheva | A large fraction of neocortical myelin ensheathes axons of local inhibitory neurons[END_REF][START_REF] Stedehouder | Fast-spiking parvalbumin interneurons are frequently myelinated in the cerebral cortex of mice and humans[END_REF]. It is fundamental to understand how OPCs and oligodendrocytes feedback to neurons and contribute to the assembly, maturation and myelination of cortical circuits during post-natal development. Moreover, CNS demyelination likely results in altered neuron-oligodendrocyte interactions, and this disruption might influence neuronal function and myelin repair capacity in demyelinating diseases such as Desai NJ, Rutherford LC and Turrigiano GG. 1999. Plasticity in the intrinsic excitability of cortical pyramidal neurons. Nat Neurosci. 2(6):515-520. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 29:15-21. Dubessy A-L, Mazuir E, Rappeneau Q, Ou S, Abi Ghanem C, Piquand K, Aigrot M-S, Thétiot M, Desmazières A, Chan E, Fitzgibbon M, Fleming M, Krauss R, Zalc B, Ranscht B, Lubetzki C, Sol-Foulon N. 2019. Role of a Contactin multi-molecular complex secreted by oligodendrocytes in nodal protein clustering in the CNS. Glia. 67:2248-2263. Favuzzi E, Marques-Smith A, Deogracias R, Winterflood CM, Sanchez-Aguilera A, Mantoan L, Maeso P, Fernandes C, Ewers H, Rico B. 2017. Activity-dependent gating of parvalbumin interneuron function by the perineuronal net protein brevican. Neuron. 95(3):639-655. Fawcett JW, Oohashi T and Pizzorusso T 2019. The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function. Nat Rev Neurosci. 20: 451-465. Földy C, Darmanis S, Aoto J, Malenka RC, Quake SR, Südhof TC. 2016. Single-cell RNAseq reveals cell adhesion molecule profiles in electrophysiologically defined neurons. Proc Natl Acad Sci USA. 113:E5222-5231. Freeman SA, Desmazières A, Fricker D, Lubetzki C, Sol-Foulon N. 2016. Mechanisms of sodium channel clustering and its influence on axonal impulse conduction. Cell Mol Life Sci. 73:723-735. Freeman SA, Desmazières A, Simonnet J, Gatta M, Pfeiffer F, Aigrot MS, Rappeneau Q, Guerreiro S, Michel PP, Yanagawa Y, Barbin G, Brophy PJ, Fricker D, Lubetzki C, Sol-Foulon N. 2015. Acceleration of conduction velocity linked to clustering of nodal components precedes myelination. Proc Natl Acad Sci USA. 112:E321-328.

F

  Quantification of oligodendroglial cell phenotype in CTRL hippocampal cultures depending on positivity of PDGFR, O4, PLP and MBP. n=3 different cultures; mean ± SEM; between 1,500 to 2,000 cells were counted on acquired images for each staining and in each experiment. (B) Image from OCM culture, incubated with Nfasc Ab directly coupled with Alexa594, before patch-clamp recordings. Fluorescent GABAergic neuron (VGAT-venus) with Nfasc staining showing axon initial segment and prenodal clusters along axon. (C) Nfasc staining (in red) on fixed OCM culture, corresponding to reconstruction of neuronal morphology of a biocytin injected neuron (in green). Scale bar: 100 µm (D) Immunostainings of mixed hippocampal neurons (CTRL) and purified neurons in the absence (PUR) or presence of OCM (OCM), at 17 DIV. Na v is in green and GAD67 in red. Nav clusters (indicated by formed along hippocampal GABAergic axons (identified by the presence of the axon initial segment (AIS)) in CTRL and OCM cultures. Scale bar: 20 µm Sup. Figure 2: (A) Voltage response of an inhibitory cell to rheobase injected current, the minimal intensity needed to initiate an action potential (AP). In this example, rheobase is 320 pA. The AP voltage threshold is defined as the point at the foot of the first AP where dV/dt exceeds 30 mV/ms, indicated by a red dot. (B) The first AP at rheobase (dashed grey area from A). The following measures of active neuronal properties presented in Fig. 1 and 2 are derived from analysis of this waveform: Onset latency (purple line), AP threshold (red dot), AP rise amplitude (green line), AP width (dark blue line), AP afterhyperpolarization (AHP, light blue dot and line). Green point represents AP peak. (C) The membrane time constant (Tau) is the shorter time constant determined by fitting a double exponential function to a membrane response (-10 mV frombaseline) to a small hyperpolarizing current. (D) Sag ratio is related to the I h current. Measures of the ratio between ΔVmin and ΔVmean were made on three consecutive traces where the steady state voltage during the second half of the hyperpolarizing to -100mV. ΔVmin is the difference between the minimal potential reached during of the first part of the hyperpolarizing step and the baseline (∆Vmin). ∆Vmean is the difference between the mean potential during the second part of the step and the baseline (∆Vmean). (E) Phase plot representation of the first action potential at rheobase, as the first derivative of membrane potential against membrane potential. Dots indicate membrane potential parameters as in (B): the derivative increases from the threshold (red dot) to maximum depolarization rate (maximum slope of AP depolarization, green line) and then decreases to the AP peak (green dot). After peak, the derivative decreases to reach the maximum repolarization rate (maximum slope of AP repolarization) and then further to AP AHP (blue dot), the most negative voltage point of the AP waveform.

6 :

 6 Overview of transcript library preparation. Cytosol content was extracted from hippocampal neurons by a patch pipette. SMART-Seq v4 technology was used for mRNA capture, reverse transcription and cDNA amplification. Full-length cDNA was processed with the Nextera XT DNA Library Preparation Kit from Illumina to generate multiplex sequencing libraries. Next-generation sequencing was used to produce libraries with the Illumina NextSeq500 after a 2x75bp paired end sequencing. (B) Bioinformatic pipeline of scRNASeq data treatment. Each step is shown (left column) together with the tool used (right column). (C) Number of expressed genes plotted against the number of uniquely mapped reads. (R 2 =0.79 all samples, R 2 = 0.57, selected samples). Black symbols represent cells that passed quality control and grey symbols cells which failed quality control. Circles represent pyramidal neurons under CTRL conditions, squares GABAergic neurons under CTRL conditions. GABAergic neurons in purified neuron cultures are shown in red and in purified neuron cultures treated with OCM in green. (D) Mapping statistics of reads for each sample (top, quality control passed, bottom, quality control failed). (E) Distribution of reads of transcripts with different origins for each sample (top, quality control passed, bottom, quality control failed).Sup. Figure4: Comparison of distributions for genes expressed (representative example, sample 34, PYR) within noncoding regions (intergenic, black) and coding regions. Genes were not detected when their expression fell within 98% of intergenic regions. In this case, Scn1b and Kcnc1 were considered to be expressed while Bsn was not.Sup. Figure5: Lists of regulated genes in the different comparisons, PYR vs GABA CTRL (1), GABA PUR vs GABA CTRL (2), GABA PUR vs GABA OCM (3), GABA OCM vs GABA CTRL (4) Expression of genes in a cluster with similar expression pattern in different culture conditions. Gene expression was normalized to that of pyramidal cells in control conditions. The number of genes for each cluster is indicated. The continuous black line shows the mean gene expression in different culture conditions.

)

  voltage-gated channel subfamily A member regulatory beta subunit 1 insulin-like growth factor binding protein 4 solute carrier family 38, member 7 apoptosis-associated tyrosine kinase Coenzyme A synthase potassium voltage-gated channel subfamily A member 2 XPC complex subunit, DNA damage recognition and repair factor

2

  nucleotidase, cytosolic IIIB ubiquitin-like domain containing CTD phosphatase 1 cyclin and CBS domain divalent metal cation transport mediator 1 general transcription factor IIF subunit 2 cms1 ribosomal small subunit homolog (yeast) nuclear receptor subfamily 2, group F, member 1 mRNA decay factor RAS-like family 11 member A insulin-like growth factor binding protein 4 nitric oxide synthase interacting protein basic helix-loop-helix domain containing, class B

TATA)

  DNA-damage-inducible, gamma potassium calcium-activated channel subfamily M regulatory beta subunit 2 ubiquitin-like domain containing CTD phosphatase 1 potassium two pore domain channel subfamily K member 3 with sequence similarity 69, member A leucine rich repeat neuronal 3 p21 (RAC1) activated kinase 1 corticotropin releasing hormone binding protein KDEL endoplasmic reticulum protein retention receptor 2 activity and apoptosis inhibitor 1 LSM14A mRNA processing body assembly factor CAP-GLY domain containing linker protein family, member 4 solute carrier family 4 MRP 14 subunit UTP3, small subunit processome component homolog (S. cerevisiae)

F

  alpha-N-acetyl-neuraminide alpha-2, 8-sialyltransferase 3 pelota homolog (Drosophila) o r P e e r R e v i e w SST + /PV -cells Total of SST + cells PV + /SST -cells Total of PV + cells PV + /SST + cells neurons (GAD + ) expressing PV, SST or both in CTRL, PUR and OCM hippocampal cultures. n=3 different cultures; mean ±SEM; about 70 to 100 GAD + neurons were counted on acquired images in each experiment.

  Figure for Reviewer 3. Correlating dendritic length and resistance. (A) I-V curves of

F

  Measure of AIS lengths on plot profile of Na v intensity from acquired images after Na v and GAD immunostainings of mixed hippocampal neurons (CTRL) and purified neurons in the absence (PUR) or presence of OCM (OCM), at 17 DIV. Data represent mean ± SD.

Table 1 :

 1 Electrophysiological properties of pyramidal and GABAergic neurons. Top, mean

	EPSP, PYR CTRL vs. GABA CTRL, p<0.0001 (Mann-Whitney test #); GABA CTRL vs.
	GABA PUR, p < 0.0001; GABA CTRL vs. GABA OCM, p=0.0123. AP, GABA PUR vs.
	GABA OCM, p=0.0126 (Kruskal-Wallis and Dunn's post hoc *). (F, G) Resting membrane
	potential (F) and input resistance (G) of recorded neurons in different conditions. Color symbols
			PYR CTRL			GABA CTRL			GABA PUR			GABA OCM
		Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
	Resting membrane potential (mV)	-63	9	11	-55	5	11	-49	11	9	-54	7	13
	Neuronal Input Resistance (Mohm)	330	218	11	74	53	11	144	46	10	110	104	13
	Tau 1 (ms)	31	16	11	12	10	11	15	7	10	14	6	12
	Sag ratio at -100 mV	1.10	0.10	11	1.27	0.10	11	1.17	0.10	10	1.33	0.20	13
	Rheobase current	121	83	11	427	284	11	169	121	10	347	276	13
	Firing rate at 200 pA (Hz)	26	19	11	3	7	11	21	23	10	7	12	13
	Input-output slope (Hz/nA)	225	150	11	99	48	11	188	139	10	113	115	12
	AP threshold (at 10V/s)	-30.5	3.3	11	-32.2	3.5	11	-31.8	5.7	10	-35.3	2.9	13
	AP width (ms)	1.51	0.80	11	0.50	0.20	11	0.96	0.60	10	0.74	0.50	13
	AP AHP (mV)	-19.5	4.4	11	-27.6	3.3	11	-23.8	3.2	10	-29.4	7.3	13
	AP rise amplitude	78	7	11	61	9	11	56	13	10	68	8	13
	AP maximum depolarization rate (V/s)	325	212	11	294	68	11	192	105	10	327	103	13
	AP maximum repolarization rate (V/s)	-61	28	11	-164	82	11	-91	57	10	-119	51	13
	Onset latency at rheobase	349	251	11	167	172	11	28	24	10	64	86	13
		PYR CTRL vs GABA CTRL	GABA CTRL vs GABA PUR GABA PUR vs GABA OCM GABA CTRL vs GABA OCM
		p value (MW) Significance p value (KW) Significance p value (KW) Significance p value (KW) Significance
	Resting membrane potential (mV)	0.0175	*	0.6894	ns	0.6360	ns	>0.9999	ns
	Neuronal Input Resistance (Mohm)	<0.0001	****	0.0156	*	0.1240	ns	>0.9999	ns
	Tau 1 (ms)	0.0041	**	0.2966	ns	>0.9999	ns	0.4235	ns
	Sag ratio at -100 mV	0.0005	***	0.1387	ns	0.0317	*	>0.9999	ns
	Rheobase current	0.0019	**	0.0464	*	0.1854	ns	>0.9999	ns
	Firing rate at 200 pA (Hz)	0.0008	***	0.0523	ns	0.4131	ns	0.9355	ns
	Input-output slope (Hz/nA)	0.0336	*	0.3019	ns	0.1416	ns	>0.9999	ns
	AP threshold (at 10V/s)	0.1932	ns	0.5216	ns	0.0571	ns	0.9382	ns
	AP width (ms)	<0.0001	****	0.1396	ns	0.5170	ns	>0.9999	ns
	AP AHP (mV)	0.0003	***	0.1268	ns	0.0075	**	>0.9999	ns
	AP rise amplitude	<0.0001	****	>0.9999	ns	0.0480	*	0.2263	ns
	AP maximum depolarization rate (V/s)	0.529	ns	0.1148	ns	0.0151	*	>0.9999	ns
	AP maximum repolarization rate (V/s)	0.0001	***	0.0815	ns	0.5344	ns	>0.9999	ns
	Onset latency at rheobase	0.0281	*	0.1681	ns	>0.9999	ns	0.3630	ns

show cells from which sequence data was derived. Mann-Whitney test for PYR CTRL vs.

GABA CTRL significance levels indicated with #, Kruskal-Wallis and Dunn's post hoc for GABA CTRL vs. PUR vs. OCM indicated by *. P-values are given in Table

1

. Boxplots represent the median (middle line), the mean (+ sign), 25 th and 75 th percentiles (box) and the top and bottom values (whiskers).

values, SD and number of cells for each parameter and culture condition. Bottom, p-values and significance levels from Mann-Whitney (MW) tests (PYR CTRL vs. GABA CTRL) and from

  Table 1 summarizes these physiological data and provides statistical support for comparisons.

	Figure 2: Glial factors affect the electrophysiological properties of GABAergic neurons. (A)
	Voltage responses to depolarizing and hyperpolarizing current steps recorded from representa-
	tive neurons at 17 DIV in the different conditions. Top, action potentials initiated at rheobase;
	bottom, current intensities as shown in insets. (B) Action potential waveforms and phase plots
	(Y axis, dV/dt (V/s); X axis, membrane potential (mV). Green line, maximum depolarization
	rate; blue line, maximum repolarization rate. (C) to (N), Effects of culture conditions (PYR
	CTRL, GABA CRTL, GABA PUR and GABA OCM) on 12 parameters characterizing neu-
	ronal intrinsic properties. Each symbol is one neuron. Color symbols correspond to sequenced
	cells as in Fig. 1 F, G. Significance levels indicated as in Fig. 1, i.e. # for Mann-Whitney test F o r and * for Kruskal-Wallis and Dunn's post hoc test. Parameters measured as shown in Sup. Fig.
	2. P-values given in Table 1. Boxplots represent the median (middle line), the mean (+ sign), P e e 25 th and 75 th percentiles (box) and the top and bottom values (whiskers).
	r
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al. 2019) which modulate glutamatergic neurotransmission on pyramidal cells but their effects on GABAergic neurons are not known. Multiple glial factors could affect optimal survival and maturation of CNS neurons, although the precise identity and combination of factors may differ depending on neuronal type and age

[START_REF] Meyer-Franke | Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture[END_REF][START_REF] Goldberg | The relationship between neuronal survival and regeneration[END_REF]

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Cerebral Cortex

Acknowledgements

We thank Marie-Stéphane Aigrot and Loane Wallon, Claire Lovo from ICM Quant, Yannick Marie and Emeline Mundwiller from iGenSeq, and François-Xavier Lejeune from iCONICS, for technical support. We thank Bernard Zalc, Richard Miles and Anne Desmazieres for discussion and critical reading of the manuscript. This work was supported by the French MS research foundation ARSEP (Aide à la Recherche sur la Sclérose en Plaques), Bouvet-Labruyère prize to NSF, and Biogen funding to EM.