Pierre-Eric Lutz

Daniel Almeida

Dominique Filliol

Fabrice Jollant

Brigitte L Kieffer

MD Gustavo Turecki
email: gustavo.turecki@mcgill.ca

Increased functional coupling of the mu opioid receptor in the anterior insula of depressed individuals

The mu opioid receptor (MOR) is a G protein-coupled receptor that plays an essential role in reward and hedonic processes, and that has been implicated in disorders such as depression and addiction. Over the last decade, several brain imaging studies in depressed patients have consistently found that dysregulation of MOR function occurs in particular in the anterior insular cortex, an important brain site for the perception of internal states and emotional regulation. To investigate molecular mechanisms that may underlie these effects, here we assessed genetic polymorphisms, expression, and functional G-protein coupling of MOR in a large post-mortem cohort (N=95) composed of depressed individuals who died by suicide, and healthy controls.

Results indicated that depression, but not comorbid substance use disorder or acute opiate consumption, was associated with increased MOR activity. This effect was partly explained by a specific increase in expression of the inhibitory alpha G-protein subunit GNAI2. Consistent with previous neuroimaging studies, our findings support the notion that enhanced endogenous opioidergic tone in the anterior insula may buffer negative affective states in depressed individuals, a mechanism that could potentially contribute to the antidepressant efficacy of emerging opioidbased medications.

Introduction

Depression is a recurring and often chronic brain disorder that stems from maladaptive processes affecting multiple neurotransmitter systems. While the monoaminergic theory of depression received considerable attention, and fuelled intense research efforts over the last 50 years, another theory, dating back to the early 1900s, posits the implication of the opioid system in regulating mood [START_REF] Lutz | Opioid receptors: distinct roles in mood disorders[END_REF]. Euphoric properties of opiates acting at the mu opioid receptor (MOR), such as morphine and heroin, have been recreationally exploited for centuries, and were once considered a potential 'opioid cure' for depression.

Over the last two decades, both clinical and animal research have further strengthened the notion that adaptations at the level of MOR signalling may contribute to depression pathophysiology [START_REF] Lutz | Opioid receptors: distinct roles in mood disorders[END_REF]. Recently, functional neuroimaging has allowed studies of MOR in patients experiencing various mood states [START_REF] Nummenmaa | Lowered endogenous mu-opioid receptor availability in subclinical depression and anxiety[END_REF]. Positron emission tomography (PET) with the MOR selective radiotracer [ 11 C]-carfentanil found that the induction of a sadness state, or of sustained pain, was associated in both healthy [START_REF] Zubieta | Regional mu opioid receptor regulation of sensory and affective dimensions of pain[END_REF][START_REF] Zubieta | Regulation of human affective responses by anterior cingulate and limbic mu-opioid neurotransmission[END_REF] and depressed [START_REF] Kennedy | Dysregulation of endogenous opioid emotion regulation circuitry in major depression in women[END_REF] women with changes in MOR bioavailability across several brain regions. Among these regions, the insular cortex [START_REF] Namkung | The Insula: An Underestimated Brain Area in Clinical Neuroscience, Psychiatry, and Neurology[END_REF] appears as a major site where MOR modulates affective states, in particular in relation to social experiences, such as social acceptance and rejection, in both healthy volunteers [START_REF] Hsu | Response of the mu-opioid system to social rejection and acceptance[END_REF] and depressed [START_REF] Hsu | It still hurts: altered endogenous opioid activity in the brain during social rejection and acceptance in major depressive disorder[END_REF] subjects.

While PET imaging allows studying MOR availability in living individuals, this approach does not differentiate changes in occupancy of the receptor by endogenous opioid peptides from changes in receptor abundance, complicating interpretation of findings. Since MOR belongs to the large family of inhibitory G protein-coupled receptors (GPCR), radioligand binding assays in postmortem cohorts offer a complementary approach to assess receptor level and signalling. The five previous studies available for MOR in relation to depression were conducted between 1990 to 2012 [START_REF] Zalsman | Postmortem mu-opioid receptor binding in suicide victims and controls[END_REF][START_REF] Meana | mu-opioid receptor and alpha2-adrenoceptor agonist stimulation of [35S]GTPgammaS binding to G-proteins in postmortem brains of opioid addicts[END_REF][START_REF] Gross-Isseroff | Regionally selective increases in mu opioid receptor density in the brains of suicide victims[END_REF][START_REF] Gabilondo | Increased density of mu-opioid receptors in the postmortem brain of suicide victims[END_REF][START_REF] Scarr | Mu opioid receptor availability in people with psychiatric disorders who died by suicide: a case control study[END_REF], focused on relatively small cohorts, and investigated the temporal and frontal cortices, caudate nucleus, and thalamus. Importantly, no investigation was conducted on the cortical structure most consistently identified by the aforementioned brain imaging studies, the insular cortex.

In this context, the goal of the present work was to test the hypothesis that major depressive disorder (MDD) may associate with dysregulated MOR function in the anterior insular cortex (AI). Using post-mortem samples from a large and well-characterized cohort (N=95 total), we compared subjects diagnosed with MDD (N=55), and healthy controls (N=40). Expression of MOR was measured using quantitative PCR, while MOR coupling was assessed using agoniststimulated [ 35 S]-GTPγS binding, and the expression of Gi proteins measured using Nanostring. In addition, a series of single nucleotide polymorphisms located along the MOR gene and potentially contributing to variability in receptor expression or function were characterized. Overall, our results indicate that MDD associates in the anterior insula with increased MOR coupling, which appears partly mediated by increased expression of the alpha(i2) G-protein subunit, suggesting a molecular substrate that may contribute to disease pathophysiology.

Material and methods

Cohort

This study was approved by the Research Ethics Board at the Douglas Mental Health University Institute, Verdun, Quebec. Brain tissue was obtained from the Douglas-Bell Canada Brain Bank (http://douglasbrainbank.ca/). We compared two groups of subjects matched for sex, age, postmortem interval (PMI) and pH (see Table 1): 1) One group of individuals who died by suicide during a major depressive episode (MDD group), and 2) A second group of psychiatrically healthy individuals who died suddenly from accidental causes, and who constituted the control group (C).

Information on psychopathology, socio-demographic variables and development was obtained by way of psychological autopsies performed by trained clinicians, with the informants best acquainted with the deceased, as described elsewhere [START_REF] Dumais | Risk factors for suicide completion in major depression: a case-control study of impulsive and aggressive behaviors in men[END_REF]. The process employed by our group in the collection of information used in psychological autopsies has been extensively investigated and found to produce valid information, especially in the context of observable behaviours and major life events [START_REF] Brent | The validity of diagnoses obtained through the psychological autopsy procedure in adolescent suicide victims: use of family history[END_REF][START_REF] Conner | The validity of proxy-based data in suicide research: a study of patients 50 years of age and older who attempted suicide. II. Life events, social support and suicidal behavior[END_REF][START_REF] Kelly | Validity of DSM-III-R diagnosis by psychological autopsy: a comparison with clinician ante-mortem diagnosis[END_REF]. Both cases and controls were characterized by the same psychological autopsy methods, avoiding the occurrence of systematic biases. Psychopathology diagnoses were obtained using DSM-IV criteria by means of SCID-I interviews adapted for psychological autopsies. Brain samples were dissected from each subject from 0.5 cm-thick coronal brain sections by expert brain bank staff following standard dissection procedures, with the aid of a human brain atlas (see [START_REF] Mai | Atlas of the Human Brain[END_REF] and thehumanbrain.info/brain/bn_brain_atlas/brain.html). Samples were carefully dissected at 4°C after having been flash-frozen in isopentene at -80°C. AI tissue was dissected from the superior part of the insula, just beneath the overlying opercula in the brain section containing the striatum, at the level of nucleus accumbens.

Relationship to previous studies

Our previous study on a partly overlapping but smaller cohort (N=59) focused on the kappa opioid receptor [START_REF] Lutz | Epigenetic Regulation of the Kappa Opioid Receptor by Child Abuse[END_REF] and its epigenetic regulation, while also reporting mRNA expression for the MOR.

The present study investigated an enlarged cohort (N=95), and comprehensively characterized MOR for gene expression, genetic variation, and signalling efficiency.

Quantitative polymerase chain reaction (qPCR)

RNA was extracted from homogenized brain tissue samples using the RNeasy Lipid Tissue Mini Kit (Qiagen). RNA quantity and quality were measured by Nanodrop® and Agilent 2100 Bioanalyzer technologies, and only samples with an RNA integrity number (RIN) ≥ 5 were used (resulting in N=40 C and N=55 MDD subjects). Extracted RNA was reverse-transcribed using M-MLV reverse transcriptase (Invitrogen™). Messenger RNA (mRNA) levels were quantified by real time polymerase chain reaction (RT-PCR) using SYBR® Green DNA intercalating dye and master mix (Bio-Rad) and the ABI 7900HT PCR machine. Primers for targeted genes were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and validated by gel migration and dissociation curves. Complementary DNAs (cDNA) from every subject were pooled and used to prepare calibration curves, from which cDNA quantities of target genes were calculated in each sample according to the measured RT-PCR threshold cycle (Ct value), in quintuplicates. Relative expression levels for each gene of interest were calculated by dividing the cDNA quantity for the gene of interest by the arithmetic mean of cDNA quantities for two reference housekeeping genes, GAPDH and β-actin. MOR, forward primer: TTC CGT ACT CCC CGA AAT GC; reverse primer: ACA ATC TAT GGA ACC TTG CCT G. GAPDH, forward primer: TTG TCA AGC TCA TTT CCT GG; reverse primer: TGT GAG GAG GGG AGA TTC AG. β-actin, forward primer: AAG ACC TGT ACG CCA ACA CA; reverse primer: GCA GTG ATC TCC TTC TGC ATC.

Genotyping

Genomic DNA extracted from brain tissue was first amplified, via PCR, using a Platinum Taq DNA Polymerase (Invitrogen®). Forward and reverse primers were designed using NCBI's Primer-BLAST tool and synthesized by Integrated DNA Technologies (IDT). Final reactions contained 1 µL of gDNA, 1 µL of 10x PCR buffer, 0.2µL of 10mM dNTP mix, 0.6µL of 50mM MgCl2, 0.1µL of Q-solution, 1µL of 10nM forward and reverse primers, 0.1µL of DNA Polymerase, and 6µL of H2O. PCR reactions were carried out using the following conditions: 95°C for 5 min, then 35 cycles of [95°C for 30s, 60°C for 30s, 72°C for 40s], final extension at 72°C for 7 min. Individual amplicons were then purified using a 0.7x AMPure bead mix. Purified amplicons were sent for Sanger sequencing, on a 3730xl DNA Analyzer (Applied Biosystems), at the McGill University and Genome Quebec Innovation Center. Sequence trace files in ABI format, as well as reference sequences in FASTA format, were loaded into the novoSNP 3.0.1 program for Windows users [START_REF] Weckx | novoSNP, a novel computational tool for sequence variation discovery[END_REF]). Variation at each location was determined by following the procedures outlined in Weckx et al [START_REF] Weckx | novoSNP, a novel computational tool for sequence variation discovery[END_REF].

Agonist-stimulated [ 35 S]-GTPgS binding activity of MOR

The functional activity of MOR was assessed by the [ 35 S]-GTPγS binding assay, as described previously [START_REF] Gonzalez-Maeso | Neurotransmitter receptor-mediated activation of G-proteins in brains of suicide victims with mood disorders: selective supersensitivity of alpha(2A)-adrenoceptors[END_REF]. The method is based on the ability of the radiolabeled GTP analog, [ 35 S]-GTPγS, to specifically bind to G-proteins. Here, 100mg of AI tissue was used for each subject for membrane preparation. Membranes were prepared by homogenizing the tissue in ice-cold 0.25 M sucrose solution 10 vol (ml/g wet weight of tissue). Samples were then centrifuged at 1100 g for 10 min. Supernatants were collected and diluted 5 times in buffer containing 50 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and were then centrifuged at 35,000 g for 30 min. The pellets were homogenized in 2 ml of ice-cold sucrose solution (0.32 M), aliquoted and kept at -80°C until further use. For each [ 35 S]-GTPγS binding assay, 2.5 μg of membrane proteins were used per well.

Samples were incubated with the MOR agonist DAMGO (using 12 increasing concentrations, from 10 -4 to 10 -10 M) for 1 h at 25 °C in assay buffer 50 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 100 mM NaCl, 0.2 mM EGTA containing 30 μM GDP and 0.1 nM [ 35 S]-GTPγS. Incubation was terminated by rapid filtration and washing in ice-cold buffer (50 mM Tris-HCl, 5 mM MgCl2, 50 mM NaCl, pH 7.4). Bound radioactivity was quantified using a liquid scintillation counter (Perkin Elmer Top Count). Non-specific binding was defined as binding in the presence of 10 μM GTPγS, and basal binding was assessed in the absence of DAMGO. Assays were performed in triplicates in different membrane preparations. Non-linear regressions were performed on individual concentrationresponse curves of DAMGO-induced stimulations (Prism, GraphPad Software, San Diego, CA, USA). Data are expressed as the maximal net stimulation (Emax, stimulated minus basal levels) expressed in fmol.mg -1 protein. The concentration that elicited half-maximal effect (EC50) was obtained and normalized as log EC50 values.

Quantification of inhibitory G-proteins alpha subunits expression using Nanostring

Nanostring experiments were performed as described previously [START_REF] Lutz | Association of a History of Child Abuse With Impaired Myelination in the Anterior Cingulate Cortex: Convergent Epigenetic, Transcriptional, and Morphological Evidence[END_REF] at the Jewish General Hospital Molecular Pathology Centre (Montréal, QC, Canada) using Nanostring nCounter targeted gene expression profiling [START_REF] Geiss | Direct multiplexed measurement of gene expression with color-coded probe pairs[END_REF]. Briefly, 5 µl of total RNA (20 ng/µl) was hybridized with the reporter and capture probes at 65 °C in a thermocycler for 19-20 hours. Probes were designed against 4 genes of interest (GNAI1, GNAI2, GNAO1 and GNAZ) and against 4 housekeeping control genes highly expressed in the AI: GAPDH, β-Actin, Tubulin and β-2-Globulin. The samples were processed with the nCounter Prep Station to purify the hybridized targets and affix them to the cartridge. After transfer to the nCounter Digital Analyzer, barcodes were counted and tabulated for each target molecule. The data were analysed using the nSolver version 2.6.43 following the manufacturer's recommendations.

Statistical Analysis

Statistical analyses were carried out using R and Prism v8.4.1. Frequencies of SNPs were analysed using χ 2 test to test for Hardy-Weinberg equilibrium. For MOR expression and [ 35 S]-GTPγS a general linear model (GLM) was used to analyse group differences. Potential correlations between variables that commonly act as confounders (pH, PMI, RIN, and age) and gene expression were assessed separately, and only those variables showing a significant Pearson correlation were included in each final GLM model. Statistical significance was set at p<0.05.

Results

MOR gene expression in the anterior insula.

We first used qPCR to quantify MOR mRNA levels in the two groups of C and MDD subjects. Groups did not differ with respect to the subjects' sex (Fisher's exact test, p>0.99) nor RNA Integrity Numbers (RIN; t=1.25, p=0.21; see Table1). MOR expression was similar across men and women (t=1.30, p=0.20) and, as expected, significantly correlated with RIN values (Fig. S1A). Interestingly, it also increased with age (Fig. 1A; r=0.37, N=90 p=3E-04), in contrast with previous studies that reported a significant decrease in the mediodorsal thalamus [START_REF] Lutz | Epigenetic Regulation of the Kappa Opioid Receptor by Child Abuse[END_REF], with no changes in three other structures (the caudal part of the anterior cingulate cortex, cACC [START_REF] Lutz | Epigenetic Regulation of the Kappa Opioid Receptor by Child Abuse[END_REF], the ventral posterior part of the lateral thalamus [START_REF] Oertel | Geneticepigenetic interaction modulates mu-opioid receptor regulation[END_REF], and the secondary somato-sensory cortex [START_REF] Oertel | Geneticepigenetic interaction modulates mu-opioid receptor regulation[END_REF]). This could indicate brain region-specific patterns of MOR regulation during aging. Importantly, a GLM comparing our two main groups, C and MDD, and considering age and RIN as significant covariates, did not reveal a difference between groups (Fig. 1B,F(1,86)=1.11, p=0.27). Further, while we previously reported that a history of child abuse, a major distal risk factor for MDD, associated in the AI with decreased expression of the kappa opioid receptor [START_REF] Lutz | Epigenetic Regulation of the Kappa Opioid Receptor by Child Abuse[END_REF], no such effect was found for MOR (t=0.83, p=0.41).

All drugs of abuse, including opiates, trigger rewarding effects at least in part by recruiting the MOR [START_REF] Lutz | The multiple facets of opioid receptor function: implications for addiction[END_REF], which has been conceptualized as a molecular gateway to addiction [START_REF] Contet | Mu opioid receptor: a gateway to drug addiction[END_REF]. Animal studies have also reported changes in MOR expression following acute or chronic activation of this receptor. We therefore explored whether MOR expression varied as a function of comorbid substance use disorder (SUD), or ante-mortem acute opiate consumption. Results showed that the SUD diagnosis, present at similar frequency in C and MDD groups (Fisher's exact test, p>0.99; Table 1), had no significant effect (Fig. S1B, t=0.32, p=0.75). Toxicological screenings (conducted on blood, urine, and ocular liquid samples) indicated that all drugs of abuse considered together (opiates, cocaine, amphetamines, ethanol, and tetrahydrocannabinol), or opiates only, were detected in 35 and 8 subjects, respectively, and significantly associated with SUD diagnosis, as expected (p<0.05). However, positive toxicological screening did not associate with any change in MOR expression (p>0.05; Fig. S1C). Altogether, MOR expression in the AI was not modulated by MDD, SUD, recent opiate consumption, or history of child abuse.

MOR gene expression and single nucleotide polymorphisms (SNP).

We investigated a series of SNP in the MOR gene (see observed frequencies in Table 2). These were chosen based on previous literature [START_REF] Oertel | Geneticepigenetic interaction modulates mu-opioid receptor regulation[END_REF][START_REF] Bilbao | A pharmacogenetic determinant of mu-opioid receptor antagonist effects on alcohol reward and consumption: evidence from humanized mice[END_REF][START_REF] Belzeaux | GPR56/ADGRG1 is associated with response to antidepressant treatment[END_REF], and notably included the A118G (rs1799971), a well-studied SNP in relation to MOR function and SUD [START_REF] Mague | OPRM1 SNP (A118G): involvement in disease development, treatment response, and animal models[END_REF]. The distribution of the A and G alleles for A118G was in Hardy-Weinberg equilibrium (HWE), as assessed using a χ 2 test (χ 2 =0.52, p=0.47), with a low frequency of the G allele (approx. 13.6%), as expected in a Quebec population of European descent, and similar to previous studies [START_REF] Cimino | The μ-opioid receptor gene A118G polymorphism is associated with insecure attachment in children with disruptive mood regulation disorder and their mothers[END_REF][START_REF] Troisi | Variation in the mu-opioid receptor gene (OPRM1) moderates the influence of early maternal care on fearful attachment[END_REF]. When pooling results from the present work with those previously obtained in two other brain regions (the caudal part of the anterior cingulate cortex and medio-dorsal thalamus [START_REF] Lutz | Epigenetic Regulation of the Kappa Opioid Receptor by Child Abuse[END_REF]), the A118G genotype was found to have a moderately significant effect on MOR mRNA expression, with higher expression in carriers of the minor G allele (Fig. S2; 2-way ANOVA: genotype effect F(1,184)=6.30, p=0.013; brain region effect F(2,184)=1.53, p=0.22). Previous studies that investigated this same SNP reported no correlation with MOR expression, neither in the thalamus nor the somato-sensory [START_REF] Oertel | Geneticepigenetic interaction modulates mu-opioid receptor regulation[END_REF] or dorsolateral prefrontal cortices [START_REF] Hancock | Cis-Expression Quantitative Trait Loci Mapping Reveals Replicable Associations with Heroin Addiction in OPRM1[END_REF], suggesting a brain region-specific impact of this SNP. For the 15 other SNPs that we investigated, no relationship with MOR expression was observed (p>0.05 for all).

MOR signaling efficiency. We next used [ 35 S]-GTPγS to determine MOR G-protein coupling.

Using DAMGO as a well-validated agonist, we generated dose-response curves for each subject, which were then used to compute maximal receptor stimulation (Emax) and the agonist concentration eliciting half-maximal effect (log EC50).

First, we found a significant correlation between inter-individual variability of MOR mRNA (qPCR) and protein (MOR Emax) levels (r=0.28, p=0.028; Fig. 2A). This suggests that signaling of the receptor in the AI is at least partly explained by its expression from local neurons, and provides a validation of our [ 35 S]-GTPγS procedure. Second, we found that basal [ 35 S]-GTPγS binding (defined as the constitutive activity of all GPCRs, measured in the absence of DAMGO), was slowly decreasing as a function of age (r=(-0.23), p=0.028; Fig. S3A), consistent with previous reports [START_REF] Gonzalez-Maeso | Effects of age, postmortem delay and storage time on receptor-mediated activation of G-proteins in human brain[END_REF]. MOR Emax was not affected by either sex (p=0.42) or age (p=0.31), but significantly correlated with brain tissue pH (r=0.34, p=7.2E-05; Fig. S3B). Finally, we observed that MOR affinity for DAMGO, measured as the log EC50, showed a significant negative correlation with age (r=-0.31, p=0.0023; Fig. 2B), corresponding to a leftward shift of concentration-response curves, and indicating enhanced DAMGO efficacy with aging. This is consistent with results from a previous study conducted in the prefrontal cortex (Broadmann Area 9, see [START_REF] Gonzalez-Maeso | Effects of age, postmortem delay and storage time on receptor-mediated activation of G-proteins in human brain[END_REF]), although the biological significance of this effect is elusive.

We next wondered whether MOR coupling was modulated by genetic variation, as per our SNP data. We used dominant negative models and linear regression to investigate relationships between MOR Emax and observed allelic frequencies. Results showed a nominally significant correlation for rs1074287, a SNP located upstream the canonical MOR transcription start site and within the first intron of rare transcript variants (see Fig. S4A). Accordingly, a higher potency of the DAMGO agonist was observed in carriers of the minor G allele (p=0.017; Fig. S4B), suggesting higher MOR protein abundance in these subjects. This effect, however, did not stand multiple testing correction (n=16 SNPs; Bonferroni-corrected p-value=0.27), and will require replication in a larger cohort. In addition, no significant effect of the minor G allele was observed on MOR expression (p=0.88; Fig. S4C). For other SNPs investigated, no significant correlation was observed (p>0.05; data not shown), including for the A118G SNP (Fig. 2C). Regarding the later, to our knowledge, two previous studies similarly compared MOR coupling across A and G carriers [START_REF] Oertel | Geneticepigenetic interaction modulates mu-opioid receptor regulation[END_REF][START_REF] Oertel | A common human micro-opioid receptor genetic variant diminishes the receptor signaling efficacy in brain regions processing the sensory information of pain[END_REF], both using brain tissue dissected from the secondary somatosensory cortex (SII) and ventral posterior part of the lateral thalamus. The first study [START_REF] Oertel | A common human micro-opioid receptor genetic variant diminishes the receptor signaling efficacy in brain regions processing the sensory information of pain[END_REF] (total sample size N=33) found lower DAMGO efficacy in G carriers in the SII, but not in the thalamus, while the second study [START_REF] Oertel | Geneticepigenetic interaction modulates mu-opioid receptor regulation[END_REF] (total sample size N=60), conducted in another cohort, found lower efficacy in G carriers in both brain regions. Combined with our findings, this suggests that the A118G might exert a brain-region specific effect on MOR coupling. This hypothesis is further supported by results from an animal study conducted in mice genetically modified to model the human polymorphism, which reported similar brain-region specificity (with no significant effect of genotype in the AI, see [START_REF] Wang | Brain region-and sex-specific alterations in DAMGO-stimulated [(35) S]GTPgammaS binding in mice with Oprm1 A112G[END_REF]).

Finally, we investigated MOR coupling in relation to psychopathology. Similar to aforementioned analysis of MOR mRNA expression, we first wondered whether the [ 35 S]-GTPγS assay was impacted by ante-mortem consumption of drugs of abuse, or SUD diagnosis. Results showed that none of these factors had any effect on MOR Emax (SUD: p=0.55, FigS3C; acute opiate: p=0.74, FigS3D). We then built a GLM model to search for an association with MDD, taking into account the effect of pH as covariate (see above and FigS3B). Results indicated that depressed subjects showed significantly higher Emax (F(1,94)=4.97, p=0.028; Fig2D, an effect that remained significant even when correcting for the effect of rs1074287; p=0.0447). This was not accompanied by any change in logEC50 as a function of MDD (p=0.58), indicating that MOR protein signaling, but not affinity, is affected by MDD status. Overall, these results show that coupling of the MOR to inhibitory G-proteins is potentiated in individuals diagnosed of MDD, independently of comorbid SUD.

Expression of inhibitory alpha G-protein subunits. MOR belongs to the family of inhibitory G-

protein coupled receptors that activate Gi alpha-subunits, thereby inhibiting adenylate cyclase activity and lowering cAMP levels. To determine whether changes in expression of these proteins might contribute to altered MOR coupling in MDD subjects, we used Nanostring to quantify the expression of the four major corresponding genes: GNAI1, GNAI2, GNAO1, and GNAZ. We first looked at potential effects of clinical and technical covariates, and found none for sex or SUD diagnosis (p>0.05), while RIN affected GNAI1 only (p=0.029). Of note, aging associated with a significant decrease in GNAZ expression (p=0.016; FigS5), an effect that may contribute to the decrease in basal binding (i.e. in the absence of MOR agonist) previously observed in [ 35 S]-GTPγS experiments (see above, and FigS4A). More importantly, we investigated potential association with MDD, and observed a selective increase in the expression of GNAI2 in depressed individuals (GLM, F(1,89)=7.42, p=0.0077; Fig3), which remained significant even when controlling for the number of genes investigated by Nanostring (Bonferroni-corrected p-value=0.031). Therefore, our results suggest that increased expression of the GNAI2 alpha subunit is a molecular adaptation that may contribute to enhanced MOR coupling in subjects with MDD.

Discussion

Inspired by recent imaging studies reporting convergent evidence for MOR dysfunction in the AI of individuals with MDD, the present post-mortem study investigated its molecular expression and function at this brain site.

We first quantified MOR gene expression using qPCR, and found no significant change as a function of MDD. To our knowledge, only two previous studies quantified MOR mRNA in depressed individuals compared to healthy controls, and reported increased levels in the prefrontal cortex (BA9, see [START_REF] Escriba | Increased mRNA expression of alpha2Aadrenoceptors, serotonin receptors and mu-opioid receptors in the brains of suicide victims[END_REF]), or no change in the mediodorsal thalamus or anterior cingulate cortex (BA24/32, [START_REF] Lutz | Epigenetic Regulation of the Kappa Opioid Receptor by Child Abuse[END_REF]), altogether suggesting that MDD-related changes in MOR expression may be subtle, and specific to particular brain regions.

We then used [ 35 S]-GTPγS to assess MOR signalling. In the past, four human studies have investigated MOR density using the radiolabelled agonist [ 3 H]-DAMGO [START_REF] Zalsman | Postmortem mu-opioid receptor binding in suicide victims and controls[END_REF][START_REF] Gross-Isseroff | Regionally selective increases in mu opioid receptor density in the brains of suicide victims[END_REF][START_REF] Gabilondo | Increased density of mu-opioid receptors in the postmortem brain of suicide victims[END_REF][START_REF] Scarr | Mu opioid receptor availability in people with psychiatric disorders who died by suicide: a case control study[END_REF], while only one study used agonist-stimulated [ 35 S]-GTPγS to measure both receptor affinity and density [START_REF] Gonzalez-Maeso | Neurotransmitter receptor-mediated activation of G-proteins in brains of suicide victims with mood disorders: selective supersensitivity of alpha(2A)-adrenoceptors[END_REF]. The two oldest studies, although conducted in small cohorts ( [START_REF] Gross-Isseroff | Regionally selective increases in mu opioid receptor density in the brains of suicide victims[END_REF], N=2-7 subjects/group; [START_REF] Gabilondo | Increased density of mu-opioid receptors in the postmortem brain of suicide victims[END_REF], N=15/group), provided preliminary evidence for increased MOR abundance in the frontal and temporal cortices, and the caudate nucleus. However, the three subsequent studies, performed on frontal cortex tissue (BA9 and BA24) in larger cohorts ( [START_REF] Zalsman | Postmortem mu-opioid receptor binding in suicide victims and controls[END_REF], N=9-10/group; [START_REF] Gonzalez-Maeso | Neurotransmitter receptor-mediated activation of G-proteins in brains of suicide victims with mood disorders: selective supersensitivity of alpha(2A)-adrenoceptors[END_REF], N=28/group; [START_REF] Scarr | Mu opioid receptor availability in people with psychiatric disorders who died by suicide: a case control study[END_REF], N=20-51/group), failed to replicate these findings, as they all reported no significant differences between subjects who died by suicide (mostly diagnosed with mood disorders) and controls. In the present study, we investigated a larger cohort (N=40-55/group) and focused instead on the AI, a brain region previously not investigated using such assays. Our findings indicate that MDD was associated in the AI with a significant increase in MOR activity, an effect that occurred in the absence of any change in receptor affinity, and that was not accounted for by other confounders such as opiate consumption, SUD, or clinical covariates. This suggests, therefore, that the AI is a prominent brain site where MDD-related changes affecting MOR may contribute to disease pathophysiology.

To understand molecular adaptations potentially underlying enhanced MOR coupling, and because MOR is an inhibitory G-protein-coupled receptor, we next used Nanostring to quantify expression of the 4 major inhibitory alpha G-protein subunits. Results showed a specific increase in the expression of GNAI2 in depressed individuals. Because [ 35 S]-GTPγS Emax measures at saturating agonist concentration reflect maximal signalling when all receptors are recruited, this increased GNAI2 expression likely explains the increase in Emax detected in the GTPgS assay.

Importantly, these findings appear consistent with imaging studies published over the last 15 years (see [START_REF] Lutz | The opioid system and the social brain: implications for depression and suicide[END_REF] for review), mostly conducted using positron emission tomography (PET) and the MOR selective radiotracer [ 11 C]-Carfentanil. These studies first indicated that acute emotionally salient stimuli (whether the recall of sad autobiographical events [START_REF] Zubieta | Regulation of human affective responses by anterior cingulate and limbic mu-opioid neurotransmission[END_REF], exposure to social rejection [START_REF] Hsu | Response of the mu-opioid system to social rejection and acceptance[END_REF], or viewing pictures of appetizing food [START_REF] Nummenmaa | mu-opioid receptor system mediates reward processing in humans[END_REF]) led in heathy volunteers and depressed individuals [START_REF] Hsu | It still hurts: altered endogenous opioid activity in the brain during social rejection and acceptance in major depressive disorder[END_REF] to rapid changes in MOR availability, notably at the level of the AI, reflecting the well-characterized notion that MOR activity modulates hedonic tone.

Beyond these fluctuations, prolonged low mood in MDD also seems to affect MOR function. Accordingly, while an initial PET study reported lower MOR availability in depressed women, which reached significance in the posterior thalamus only (possibly due to modest statistical power, N=14/group) [START_REF] Kennedy | Dysregulation of endogenous opioid emotion regulation circuitry in major depression in women[END_REF], a more recent study found convergent results by investigating a large cohort characterized for subclinical depressive symptoms (N=135, with 113 males; see [START_REF] Nummenmaa | Lowered endogenous mu-opioid receptor availability in subclinical depression and anxiety[END_REF]). Widespread reductions in MOR availability were observed as a function of symptom severity across numerous cortical regions, with the insula, precuneus, and temporoparietal regions most strongly affected. Importantly, such reduced availability can be interpreted in PET experiments as reflecting either increased occupancy of the MOR by endogenous opioid peptides or, alternatively, lower receptor density or affinity. Results from the present post-mortem study argue in favor of the former possibility. Because increased MOR signaling was found in our study, we hypothesize that depression associates in the AI with higher MOR coupling efficiency (Emax) that at least partly stem from higher GNAI2 expression. In addition, PET studies suggest enhanced endogenous release of opioid peptides (lower MOR availability). Therefore, these 2 distinct mechanisms seem to converge to potentiate the insular opioidergic tone, and likely reflect a compensatory mechanism that counteracts low mood.

While MOR-mediated regulation of reward and mood has been primarily investigated in animal models in brain structures associated with the mesolimbic dopaminergic system, such as the ventral tegmental area and basal ganglia [START_REF] Lutz | Opioid receptors: distinct roles in mood disorders[END_REF][START_REF] Russo | The brain reward circuitry in mood disorders[END_REF], it has been left relatively unexplored in cortical structures. Studies in human, however, clearly indicate that MOR signalling in the AI contributes to important physiological function, in particular the processing of emotional aspects of pain that may arise from either somatosensorial stimuli (physical pain), or from situations that threatens the organism's survival, internal homeostasis, or social position (i.e. psychological or social pain) [START_REF] Eisenberger | Social ties and health: a social neuroscience perspective[END_REF][START_REF] Pecina | Endogenous opioid system dysregulation in depression: implications for new therapeutic approaches[END_REF]. Our findings are consistent with this hypothesis, and suggest that opiate medications that recruit the MOR (e.g. buprenorphine, frequently combined with samidorphan), and are currently under intense examination for the management of depression and suicidal ideas [START_REF] Fava | Opioid system modulation with buprenorphine/samidorphan combination for major depressive disorder: two randomized controlled studies[END_REF][START_REF] Yovell | Ultra-Low-Dose Buprenorphine as a Time-Limited Treatment for Severe Suicidal Ideation: A Randomized Controlled Trial[END_REF], may act at least in part by potentiating in the AI the beneficial impact of MOR on social and emotional distress.

In conclusion, the present work brings new knowledge that, in combination with imaging studies, indicates that the AI is a critical brain site where increased MOR signalling occurs in the context of MDD and suicide. Future studies will be necessary to determine whether similar adaptations may affect other brain regions where MOR contributes to reward and affective regulation.

Tables and Figures legends

Table 1. Sample Demographics. Healthy controls (C) and subjects who died by suicide during a major depressive episode (MDD) were matched for sex, age, post-mortem interval (PMI), and pH. There were no differences, between groups, in the number of male and female subjects, the presence of alcohol or drug abuse, or RIN values (see main text). Age, RIN, PMI and pH values are shown as mean ± sem.

Table 2. Single nucleotide polymorphisms (SNP) profiled in this study in the OPRM1 gene

encoding the mu opioid receptor. SNPs were determined using Sanger sequencing (see methods). Genomic position (in the Human GRCh37/hg19 reference genome), gene feature (promoter/exon/intron), observed counts and percentages of major and minor alleles in our cohort, and expected percentages (according to the 1000 Genomes Project) are shown.

Funding

  and disclosures. The authors reported no biomedical financial interests or potential conflicts of interest. PEL was supported by fellowships from the Fondation Fyssen, Fondation Bettencourt-Schueller, Canadian Institutes of Health Research (201311MFE-320636-218885), American Foundation for Suicide Prevention (PDF-0-081-13), and Fondation pour la Recherche Médicale (ARF20160936006). This work was also supported by the Agence Nationale de la Recherche (ANR grant JCJC SignOp, PEL). Contributions. PEL, FJ, BLK and GT conceived the study. PEL performed RNA extractions, qPCR, and Nanostring experiments. DA conducted SNP genotyping. DF, PEL and BLK conceived and performed [ 35 S]-GTPγS experiments. PEL prepared the manuscript, and all authors approved its final version.

Figure 1 .

 1 Figure 1. Relative real-time quantitative PCR (RT-PCR) expression of the mu opioid receptor (MOR) in the anterior insula by age and clinical group. A. MOR expression was quantified by RT-PCR in quintuplicates, using calibration curves and two reference housekeeping genes: GAPDH and β-actin (see methods for details). The scatter plot displays a significant positive linear correlation between MOR expression and age, in all subjects, independent of clinical group (r=0.37, p=3E-04). B. Expression of MOR in healthy controls and subjects who died during a major depressive episode (MDD). There was no significant difference between the two clinical groups (F(1,86)=1.11, p=0.27). Mean ± sem, as well as individual expression values, are shown.

Figure 2 .

 2 Figure 2. Mu opioid receptor (MOR) DAMGO agonist-stimulated [ 35 S]-GTPγS binding in the anterior insular cortex of healthy controls and subjects with major depressive disorder (MDD). Coupling efficiency of MOR was evaluated using [ 35 S]-GTPγS binding and 12 increasing concentrations of the potent and specific agonist DAMGO, followed by non-linear regression of concentration-response curves, to compute Emax (maximal net stimulation of MOR; [ 35 S]-GTPγS net stimulation over basal binding) and logEC50 (agonist concentration for half-maximal effect, log transformed) values. A. The scatter plot displays a significant positive linear correlation (r=0.28, p=0.028) between MOR mRNA levels (measured by RT-PCR) and protein signaling (Emax). B. Scatter plot displaying a significant negative linear correlation (r=(-0.31), p=0.0023) between age and MOR affinity for DAMGO (logEC50). (C-D) Concentration-response curves of [ 35 S]-GTPγS stimulation, by log10[DAMGO], in the anterior insular cortex. C. Dose-response curve in non-

Figure 3 .

 3 Figure 3. Relative expression of major inhibitory alpha G-protein subunits in the anterior insula of healthy controls and subjects with major depressive disorder (MDD). NanoString was used to quantify the mRNA relative expression of 4 inhibitory alpha G-protein subunits. No difference was found between controls and depressed individuals for 3 subunits: GNAI1, GNAO1 and GNAZ. In contrast, subjects with MDD showed a significant increase in the expression of the GNAI2 subunit, compared to controls (F(1,89)=7.42, p=0.0077). Mean +/-SE, as well as, individual expression values are shown, * p <0.05.


Ph.D Arnaud Tanti

M.D. Ph.D Pierre-Eric Lutz

B.Sc John Kim

Liam O'leary

M.D. Ph.D Gustavo Turecki

Ph.D Naguib Mechawar
email: naguib.mechawar@mcgill.ca

Evidence of decreased gap junction coupling between astrocytes and oligodendrocytes in the anterior cingulate cortex of depressed suicides

Glial dysfunction is a major feature in the pathophysiology of mood disorders. While altered astrocyte (AS) and oligodendrocyte-lineage (OL) cell functions have been associated with depression, the crosstalk between these two major glial cell types has never been assessed in that context. AS are potent regulators of OL cells and myelination, in part through gap junction-mediated intercellular communication made possible by the heterotypic coupling of AS-specific (Cx30 and Cx43) and OL-specific (Cx32 and Cx47) connexins, allowing cytosolic transport and metabolic support to OL cells. Because changes in the expression of AS-specific connexins have been previously reported in the brain of depressed individuals, this study aimed at addressing the integrity of AS-OL coupling in the anterior cingulate cortex (ACC) of depressed suicides. Using immunofluorescence and confocal imaging, we characterized the distribution of the AS-specific Cx30 in the ACC, and mapped its expression onto oligodendrocyte somas and myelinated axons as well as brain vasculature in post-mortem brain samples from depressed suicides (N=48) and matched controls (N=23). The differential gene expression of key components of the gap junction nexus was also screened through RNA-sequencing dataset previously generated by our group, and validated by quantitative real-time PCR. Our results indicate that Cx30 expression mapping to OL cells is selectively decreased in depressed suicides, an effect that was associated with decreased expression of OL-specific connexins Cx32 and Cx47, as well as the downregulation of major connexin-interacting proteins essential for the scaffolding, trafficking and function of gap junction channels. These results provide a first evidence of impaired gap junction mediated communication between astrocytes and oligodendrocytes in the ACC of individuals with mood disorders. These changes in glial coupling are likely to have significant impact on brain function, and may contribute to the altered OL function previously reported in this brain region.

INTRODUCTION

Glial dysfunction has emerged as an important pathophysiological hallmark of depression and suicide. In particular, post-mortem studies have consistently shown changes in astrocyte numbers and morphology, as well as in the expression of several astrocytic markers in fronto-limbic regions of depressed individuals [START_REF] Baucom | Glial fibrillary acidic protein immunoreactivity in the prefrontal cortex distinguishes younger from older adults in major depressive disorder[END_REF][START_REF] Si | Age-Dependent Reductions in the Level of Glial Fibrillary Acidic Protein in the Prefrontal Cortex in Major Depression[END_REF][START_REF] Rajkowska | Coverage of Blood Vessels by Astrocytic Endfeet Is Reduced in Major Depressive Disorder[END_REF][START_REF] Gittins | A morphometric study of glia and neurons in the anterior cingulate cortex in mood disorder[END_REF][START_REF] Torres-Platas | Astrocytic hypertrophy in anterior cingulate white matter of depressed suicides[END_REF][START_REF] Torres-Platas | Glial fibrillary acidic protein is differentially expressed across cortical and subcortical regions in healthy brains and downregulated in the thalamus and caudate nucleus of depressed suicides[END_REF][START_REF] Nagy | Repression of Astrocytic Connexins in Cortical and Subcortical Brain Regions and Prefrontal Enrichment of H3K9me3 in Depression and Suicide[END_REF][START_REF] Nagy | Astrocytic abnormalities and global DNA methylation patterns in depression and suicide[END_REF][START_REF] Rajkowska | Astrocyte pathology in the ventral prefrontal white matter in depression[END_REF][START_REF] Czéh | Clinical Findings Documenting Cellular and Molecular Abnormalities of Glia in Depressive Disorders[END_REF] , while pharmacological or genetic disruption of astrocyte function in rodents has been associated with depressive-like behaviour [START_REF] Leng | Menin Deficiency Leads to Depressivelike Behaviors in Mice by Modulating Astrocyte-Mediated Neuroinflammation[END_REF][START_REF] Banasr | Glial Loss in the Prefrontal Cortex Is Sufficient to Induce Depressive-like Behaviors[END_REF] . Recently, oligodendrocyte-lineage (OL) cells have also been associated with depression and suicide. These cells, which allow for a formidable form of brain plasticity by responding to environmental cues and experiences in an activity-dependent manner [START_REF] Almeida | On Myelinated Axon Plasticity and Neuronal Circuit Formation and Function[END_REF][START_REF] Gibson | Bad wrap: Myelin and myelin plasticity in health and disease[END_REF][START_REF] Forbes | All Wrapped Up: Environmental Effects on Myelination[END_REF] , were found to display signs of functional impairments in both post-mortem brain samples from depressed individuals [START_REF] Uranova | Oligodendroglial density in the prefrontal cortex in schizophrenia and mood disorders: a study from the Stanley Neuropathology Consortium[END_REF][START_REF] Uranova | Electron microscopy of oligodendroglia in severe mental illness[END_REF][18][START_REF] Tanti | Child abuse associates with an imbalance of oligodendrocyte-lineage cells in ventromedial prefrontal white matter[END_REF][START_REF] Hayashi | A novel, rapid, quantitative cell-counting method reveals oligodendroglial reduction in the frontopolar cortex in major depressive disorder[END_REF][START_REF] Rajkowska | Oligodendrocyte morphometry and expression of myelin -Related mRNA in ventral prefrontal white matter in major depressive disorder[END_REF][START_REF] Edgar | A putative functional role for oligodendrocytes in mood regulation[END_REF][START_REF] Aston | Transcriptional profiling reveals evidence for signaling and oligodendroglial abnormalities in the temporal cortex from patients with major depressive disorder[END_REF][START_REF] Kim | Correlation analysis between genome-wide expression profiles and cytoarchitectural abnormalities in the prefrontal cortex of psychiatric disorders[END_REF] and stress-induced animal models of depression [START_REF] Laine | Genetic Control of Myelin Plasticity after Chronic Psychosocial Stress[END_REF][26][START_REF] Miyata | Association between chronic stress-induced structural abnormalities in Ranvier nodes and reduced oligodendrocyte activity in major depression[END_REF] . These findings could represent a neurobiological substrate to the altered connectivity and white matter integrity that have been reported in imaging studies [START_REF] Tham | White matter abnormalities in major depression: evidence from post-mortem, neuroimaging and genetic studies[END_REF][START_REF] Kim | White matter alterations are associated with suicide attempt in patients with panic disorder[END_REF][START_REF] Peng | White-matter density abnormalities in depressive patients with and without childhood neglect: A voxel-based morphometry (VBM) analysis[END_REF][START_REF] Drevets | Brain structural and functional abnormalities in mood disorders: implications for neurocircuitry models of depression[END_REF][START_REF] Johansen-Berg | Anatomical connectivity of the subgenual cingulate region targeted with deep brain stimulation for treatment-resistant depression[END_REF][START_REF] Steffens | Structural Integrity of the Uncinate Fasciculus and Resting State Functional Connectivity of the Ventral Prefrontal Cortex in Late Life Depression[END_REF][START_REF] Birn | Evolutionarily conserved prefrontal-amygdalar dysfunction in early-life anxiety[END_REF][START_REF] Koolschijn | Brain volume abnormalities in major depressive disorder: A meta-analysis of magnetic resonance imaging studies[END_REF] , highlighting that myelin plasticity in discrete cortico-limbic areas could mediate some of the behavioral changes characterizing depression or modulate individual vulnerability to this mood disorder.

While astrocytes and oligodendrocytes have been independently implicated in the pathophysiology of depression, the interplay between these cell types has never been examined in this context. Astrocytes are known potent regulators of OL cell proliferation, survival and maturation as well as myelination. This multi-faceted influence of astrocytes is exerted either indirectly through their implication in neurotransmission, or directly through the release of various molecules affecting the physiology of oligodendrocytes and myelin maintenance [START_REF] Domingues | Oligodendrocyte, Astrocyte, and Microglia Crosstalk in Myelin Development, Damage, and Repair[END_REF][START_REF] Dutta | Regulation of myelin structure and conduction velocity by perinodal astrocytes[END_REF][START_REF] Kıray | The multifaceted role of astrocytes in regulating myelination[END_REF][START_REF] Li | Astrocytes in Oligodendrocyte Lineage Development and White Matter Pathology[END_REF] .

Part of the astrocytic control over OL cells and myelination is permitted through cytosolic exchanges allowed by gap junction coupling between astrocytes and oligodendrocytes [START_REF] Domingues | Oligodendrocyte, Astrocyte, and Microglia Crosstalk in Myelin Development, Damage, and Repair[END_REF][START_REF] Kıray | The multifaceted role of astrocytes in regulating myelination[END_REF][START_REF] Nualart-Marti | Gap junction communication in myelinating glia[END_REF][START_REF] Jl | Gap Junctions Couple Astrocytes and Oligodendrocytes[END_REF] . Gap junction coupling, which can also be homotypic, thus gives rise to extended networks of interconnected glial cells in the brain. The docking of two connexin hexamers (hemichannel, or connexon) from two adjacent cells forms a channel allowing the exchange of a wide range of small molecules such as ions, metabolites, second messengers and neurotransmitters, referred to as gliotransmitters [START_REF] Evans | Gap junctions: structure and function (Review)[END_REF] . Astrocytes mainly express Cx30 and Cx43, while oligodendrocytes mainly express Connexin 32 (Cx32) and Cx47. Functional channels are formed between astrocytes and oligodendrocytes (A:O) through the heterotypic coupling of Cx30-Cx32 and Cx43-Cx47 [START_REF] Jl | Gap Junctions Couple Astrocytes and Oligodendrocytes[END_REF][START_REF] Magnotti | Functional heterotypic interactions between astrocyte and oligodendrocyte connexins[END_REF][START_REF] Maglione | Oligodendrocytes in mouse corpus callosum are coupled via gap junction channels formed by connexin47 and connexin32[END_REF][START_REF] Nagy | Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS[END_REF][START_REF] Rash | Identification of cells expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in gap junctions of rat brain and spinal cord[END_REF][START_REF] Orthmann-Murphy | Two Distinct Heterotypic Channels Mediate Gap Junction Coupling between Astrocyte and Oligodendrocyte Connexins[END_REF] .

Glial gap junction coupling has been shown to be of critical importance for oligodendrocyte function, including normal myelination. In double knockout mouse models, loss of oligodendrocyte-expressed Cx32 and Cx47 induces important abnormalities in the central nervous system [START_REF] Menichella | Connexins are critical for normal myelination in the CNS[END_REF] , including thinner or absent myelin sheaths, oligodendrocyte cell death and axonal loss. Interestingly, the double deletion of both astrocyte-expressed Cx30 and Cx43 also leads, albeit to a lesser extent, to myelination impairments [START_REF] Lutz | Deletion of Astrocyte Connexins 43 and 30 Leads to a Dysmyelinating Phenotype and Hippocampal CA1 Vacuolation[END_REF] . Moreover, mice with concomitant deletions of an oligodendrocyte connexin and an astrocyte connexin, for example Cx43/Cx32 or Cx30/Cx47 double knock-out mice, show impaired astrocyte-oligodendrocyte coupling, severe myelin pathology such as myelin loss, and decreased numbers of OL cells [START_REF] May | Connexin47 protein phosphorylation and stability in oligodendrocytes depend on expression of Connexin43 protein in astrocytes[END_REF][START_REF] Tress | Panglial Gap Junctional Communication is Essential for Maintenance of Myelin in the CNS[END_REF] . Overall, these studies demonstrate that astrocyte-oligodendrocyte gap junction coupling is particularly important for oligodendrocyte function.

Our group and others recently reported that the expression of both astrocytic Cx30 and Cx43 is significantly decreased in multiple areas of the depressed suicide brain, including the prefrontal cortex, orbitofrontal cortex, mediodorsal thalamus, and caudate nucleus [START_REF] Nagy | Repression of Astrocytic Connexins in Cortical and Subcortical Brain Regions and Prefrontal Enrichment of H3K9me3 in Depression and Suicide[END_REF][START_REF] Wilson | Reduced connexin 43 immunolabeling in the orbitofrontal cortex in alcohol dependence and depression[END_REF] . These findings have been replicated in chronic stress mouse models of depression [START_REF] Moulana | Chronic Unpredictable Stress Reduces Immunostaining for Connexins 43 and 30 and Myelin Basic Protein in the Rat Prelimbic and Orbitofrontal Cortices[END_REF][START_REF] Sun | Gap junction dysfunction in the prefrontal cortex induces depressive-like behaviors in rats[END_REF] , with decreased diffusion of gap junction permeable dyes concomitant to changes in Cx43 expression [START_REF] Sun | Gap junction dysfunction in the prefrontal cortex induces depressive-like behaviors in rats[END_REF] . The underlying mechanisms and functional consequences of such coupling changes are unclear, but given that astroglial networks are involved, notably, in regulating neuronal activity and plasticity, metabolic and osmotic support, blood-brain barrier integrity, and myelination [START_REF] Kıray | The multifaceted role of astrocytes in regulating myelination[END_REF][START_REF] Pannasch | Emerging role for astroglial networks in information processing: from synapse to behavior[END_REF][START_REF] Dossi | Human astrocytes in the diseased brain[END_REF][START_REF] Rial | Depression as a Glial-Based Synaptic Dysfunction[END_REF] , alterations in glial coupling are likely to have significant consequences on brain function. Importantly, it remains to be determined whether decreased expression of astrocytic connexins may reflect changes in gap junction coupling of astrocytes among themselves, and/or with other cell types.

We examined this question in the present study by focusing on astrocyte-oligodendrocyte coupling in post-mortem anterior cingulate cortex (ACC) samples from depressed suicides and matched sudden-death controls. This cortical region was selected because we previously found it to display convergent molecular and cellular abnormalities of myelination and oligodendrocyte function in depressed suicides 18 , particularly in individuals with a history of child abuse (CA). Based on these previous results and evidence suggesting that such individuals may represent a distinct clinical and neurobiological subtype of depression [START_REF] Teicher | Childhood maltreatment and psychopathology: A case for ecophenotypic variants as clinically and neurobiologically distinct subtypes[END_REF] , we also examined samples from a group of depressed suicides with a history of CA. Our experimental strategy was to combine double-labeling immunofluorescence and confocal microscopy to map the expression of the astrocyte connexin Cx30 onto oligodendrocyte somas and myelinated fibers.

We also mapped Cx30 expression onto cerebral vasculature as a way to address the cell-type specificity of the potential changes observed in Cx30 mapping. We also screened for the expression of genes known to be important for the scaffolding and function of gap junction channels (GJCs), as well as genes coding for the main astrocyte-and oligodendrocyte-specific connexins. These experiments provided evidence of a significant decrease of Cx30-immunoreactivity (-IR) specifically on oligodendrocyte-lineage cells and a significant downregulation of oligodendrocyte-specific connexins, as well as key associated molecules in the ACC of depressed suicides, altogether suggesting impaired AS-OL gap junction communication in the brain of depressed suicides.

MATERIAL AND METHODS

Human post-mortem samples

This project was approved by the Douglas Hospital Research Ethics Board. Brain tissues were obtained from the Suicide section of the Douglas-Bell Canada Brain Bank (Douglas Hospital Research Centre, Montreal, Canada) in collaboration with the Quebec Coroner's Office and with informed consent from next of kin. Samples were obtained from individuals having died by suicide in the context of a depressive episode (Depressed Suicides -DS; N=48) and from healthy controls (Controls -CTRL; N= 23) having died suddenly without psychiatric or neurological illness. Groups were matched for age, post-mortem interval (PMI), tissue pH and RNA Integrity Number (RIN) (Table 1). Cases and controls were defined with the support of medical charts and Coroner records, and based on previously described and validated psychological autopsies [START_REF] Dumais | Risk factors for suicide completion in major depression: a case-control study of impulsive and aggressive behaviors in men[END_REF] . In brief, the Structured Clinical Interview for DSM-IV Psychiatric Disorders (SCID-I) was conducted by a trained interviewer with one or more informants best acquainted with the deceased and reviewed by a panel of clinicians to reach consensual diagnosis. Cases with a history of CA all scored highest (1 or 2) in the rating scale of sexual and physical abuse from an adapted version of the Childhood Experience of Care and Abuse (CECA) [START_REF] Bifulco | Childhood Experience of Care and Abuse (CECA): a retrospective interview measure[END_REF][START_REF] Bifulco | Memories of childhood neglect and abuse: corroboration in a series of sisters[END_REF] . Histological experiments for Cx30 quantification and colocalization were performed on a subset of matched controls (N=8-11) and depressed suicides (N= 12-17).

Tissue dissections

As previously described 18 , ACC samples were dissected by expert brain bank staff from frozen coronal sections with the guidance of a human brain atlas [START_REF] Mai | Atlas of the human brain[END_REF] . Histology blocks (~1cm 3 ) were collected in sections equivalent to plate 6 of this atlas (-30mm from the center of the anterior commissure) immediately rostral to the genu of the corpus callosum, using the cingulate and the callosal sulci as the rostral and caudal landmark limits, respectively. Frozen blocks were fixed overnight in 10% formalin, cryoprotected in a 20% sucrose/PBS solution, and cut serially in 50µm-thick sections with a cryostat. Sections were kept in cryoprotectant (30% glycerol, 30% ethylene glycol, PBS) until processed for immunofluorescence.

Immunohistochemistry

Free-floating sections were rinsed in PBS and then incubated overnight and at room temperature in primary antibodies diluted in a solution of PBS-Tx-100/2% normal donkey serum. Following washes in PBS, sections were incubated for 2 hours at room temperature with Alexa-488, Cy3 or Cy5-conjugated secondary antibodies produced in donkey (Jackson ImmunoResearch) diluted 1:500 in PBS-Tx-100/2% normal donkey serum. Sections were then mounted on slides, coverslipped (Vectashield mounting medium with DAPI, Vector Laboratories) and kept at 4°C until imaging. The primary antibodies used in this study along with their respective dilutions are listed in Table 2.

Confocal imaging and analysis

Image acquisitions was performed on an Olympus laser scanning confocal microscope (FV1200) equipped with a motorized stage. For the analysis of Cx30 density and colocalization experiments, stack images were taken with a x60 objective (NA = 1.42) and 3.5 digital zoom, with XY pixel width of 0.06µm and z spacing of 0.8µm. This spacing was chosen to prevent any dot from being counted twice in two successive z slices. Parameters were kept consistent across all acquisitions. For Cx30 total density measurements, 4 random stacks with an average of 15 z-sections per stack were randomly acquired within layer 5/6 of the ACC. For each z-section, the number of Cx30 puncta was quantified by the find maxima function of ImageJ with a noise tolerance set at 350, and divided by the image area to calculate a density per mm 2 . We used this approach as it gave the most accurate estimate of puncta number compared to manual counting on a small set of captured images. For each stack the z-sections densities were then averaged, and stack averages computed per subject to yield final Cx30 densities. For Cx30 mapping onto Nogo-A positive cells, an average of 20 cells were manually segmented within 3 images stacks acquired within layer 5/6 of the ACC and using previously described acquisition parameters. For each z-section, cell selection was enlarged by 0.5um, and number of Cx30 puncta quantified as described above, using the find maxima function with a noise tolerance of 350. Total Cx30 puncta numbers per cell were averaged to yield the average number of Cx30 mapping to Nogo-A+ cells per subject. Cx30 mapping to myelinated fibers and blood vessels was performed similarly in 4 random stacks taken within layer 5/6 of the ACC and using the same acquisition parameters. For each z-section of the stacks, PLP or Laminin IR were subjected to auto-thresholding and converted to mask. A median filter with a 3 pixels radius was applied, followed by a round of dilation and erosion. For each z-section the number of Cx30 puncta within PLP or Laminin masks was then quantified using the find maxima function (noise tolerance of 350), and the density of Cx30 calculated by dividing the number of Cx30 puncta by PLP or Laminin area respectively. Densities per stack were then averaged to yield the final density of Cx30 mapping to PLP or laminin per subject.

RNA-sequencing and RT-PCR validation

The RNA sequencing dataset, which was generated with a cohort including all of the subjects from the present study, was thoroughly described in a report we recently published 18 . In brief, total RNA was extracted from frozen ACC samples using the RNeasy Lipid Tissue Mini Kit (Qiagen) and a 2100

Bioanalyzer (Agilent) used to measure RNA concentration and RIN. Libraries preparation was performed at the Genome Quebec Innovation Center with the TrueSeq Stranded Total RNA Sample Preparation and Ribo-Zero Gold kits (Illumina), and quantified using PCR (KAPA Library Quantification). Sequencing was done on an Illumina HiSeq 2000 (100bp paired-end), with ~62 million reads/library. After adapter trimming, alignment and gene quantification using FASTX-Toolkit, Trimmomatic, TopHat v2.1.0 and HTSeq-count respectively, differential expression between groups was analyzed with DESeq2 using RIN, age and gender as covariates. Differential expression was screened in a panel of genes built based on the literature and the evidence of their role in gap junction signaling and cell-cell junctions (Figure 4).

Validation of differential gene expression was performed by quantitative RT-PCR for the 8 following genes: GJB6, GJA1, GJB1, GJC2, CAV1, CAV2, OCLN and DBN1 (Table 3). Starting from 50mg of frozen tissue, total RNA was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen) with DNase digestion. RNA quantity and RIN were measured with the 2200 TapeStation (Agilent), and only samples with RIN>5.5 were used for analysis. One µg of RNA was used to synthetize cDNA using M-MLV reverse transcriptase (Invitrogen) with oligo-dT and random hexamers. Quantitative PCR reactions were performed using SYBR Green intercalating dye and master mix (Biorad) on a QuantStudio 6 Flex Real-

RESULTS

General distribution and validation of Cx30 immunostaining

Using immunofluorescence, we first sought to characterize the general distribution of Cx30 in the ACC and validate antibody specificity. As expected [START_REF] Kunzelmann | Late onset and increasing expression of the gap junction protein connexin30 in adult murine brain and long-term cultured astrocytes[END_REF][START_REF] Nagy | Connexin30 in rodent, cat and human brain: selective expression in gray matter astrocytes, co-localization with connexin43 at gap junctions and late developmental appearance[END_REF] , Cx30-IR took the form of a very dense and clear punctate labeling which was highly enriched in cortical grey matter neuropil (Fig. 1A) while virtually absent in adjacent white matter. Since Cx30 is known to be strongly associated with cerebral vasculature, where astrocytes establish numerous gap junctions at the perivascular endfeet [START_REF] Nagy | Connexin30 in rodent, cat and human brain: selective expression in gray matter astrocytes, co-localization with connexin43 at gap junctions and late developmental appearance[END_REF][START_REF] Ezan | Deletion of Astroglial Connexins Weakens the Blood-Brain Barrier[END_REF][START_REF] Bock | A new angle on blood-CNS interfaces: A role for connexins?[END_REF][START_REF] Rouach | Astroglial Metabolic Networks Sustain Hippocampal Synaptic Transmission[END_REF] , we combined immunostainings for Cx30, vimentin, and GFAP to confirm that we could observe this distributional pattern. Vimentin was chosen because it is an intermediate filament expressed by cells in blood vessels walls as well as by a subpopulation of astrocytes [START_REF] O'leary | Program No. 042.01. Regional characterization of vimentin-immunoreactive astrocytes in the human brain[END_REF] . The distribution of Cx30 was homogenous throughout cortical layers, and as illustrated in Fig. 1B, Cx30 expression was particularly enriched along astrocyte endfeet lining blood vessels in highly vascularized areas. We further addressed the specificity of this distribution by immunostaining for GFAP together with both astrocyte-specific connexins, Cx30 and Cx43. As shown in Fig. 1C and previously reported [START_REF] Nagy | Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS[END_REF][START_REF] Ezan | Deletion of Astroglial Connexins Weakens the Blood-Brain Barrier[END_REF][START_REF] Rouach | Astroglial Metabolic Networks Sustain Hippocampal Synaptic Transmission[END_REF] , Cx30 and Cx43 highly colocalized at the level of GFAP+ endfeet along blood vessels, indicating that our Cx30 staining was reliable. We then quantified the overall density of Cx30 puncta in the ACC of controls and depressed suicides, segregating cases based on their known history of CA (Fig. 2A). Surprisingly, although based on the literature we expected that Cx30 puncta density may be decreased in depressed suicides, we found no difference between groups (Kruskal-Wallis ANOVA: H(2,23) = 0.3268, P =0.84), regardless of the history of CA, as both depressed suicides with (DS-CA) and without (DS-nCA) history of CA showed Cx30 densities similar than controls.

This was unlikely to be attributable to cofounding factors, as the density of Cx30 puncta was not correlated with age nor PMI (PMI vs Cx30 density: R 2 = 0.01, P = 0.63; Age vs Cx30 density: R 2 = 0.05, P = 0.33; Fig. 2D andE). Because previous studies have linked antidepressant medication and alcohol abuse to changes in the expression of astrocytic connexins [START_REF] Wilson | Reduced connexin 43 immunolabeling in the orbitofrontal cortex in alcohol dependence and depression[END_REF][START_REF] Jeanson | Antidepressants Impact Connexin 43 Channel Functions in Astrocytes[END_REF] , we compared Cx30 densities in cases with or without known history of substance abuse (Fig. 2B), as well as cases with or without known antidepressant prescription during the last 3 months before death (Figure 2C). No significant effect of substance abuse (Kruskal-Wallis ANOVA: H(2,23) = 0.02247, P=0.99) or medication (Kruskal-Wallis ANOVA: H(2,23) = 0.3308, P=0.85) was found, supporting the notion that, at least in the ACC, overall Cx30 density is not altered as a function of psychopathology, medication or comorbid substance abuse.

Decreased mapping of Cx30 onto oligodendrocyte cell bodies and myelinated fibers in depressed suicides

Because of the very high level of Cx30 expression and puncta labeling, small differences in Cx30 associated with specific cell-cell interactions could go unnoticed while assessing overall Cx30 distribution and density in the tissue. Since Cx30 is involved in astrocyte-oligodendrocyte gap junction coupling both at the soma and onto myelinated fibers [START_REF] Jl | Gap Junctions Couple Astrocytes and Oligodendrocytes[END_REF] , we mapped Cx30-IR to oligodendrocytes and myelinated fibers to address possible changes specific to astrocyte-oligodendrocyte coupling (Fig. 3A). Using Nogo-A-IR as a reliable marker of mature oligodendrocytes [START_REF] Tanti | Child abuse associates with an imbalance of oligodendrocyte-lineage cells in ventromedial prefrontal white matter[END_REF][START_REF] Kuhlmann | Nogo-A is a reliable oligodendroglial marker in adult human and mouse CNS and in demyelinated lesions[END_REF] , we first quantified the number of Cx30-IR puncta colocalizing with individual NogoA+ cells in layers 5/6 of the ACC (Fig. 3B). Although the average To address whether changes in Cx30 distribution could also be observed between astrocytes and other cell types, we used a similar approach to assess the Cx30-IR associated with cerebral vasculature immunolabeled for laminin (Fig. 3D). Unlike what was observed on Nogo-A-IR cells and PLP-IR axons, the density of Cx30-IR along laminin-IR blood vessels was similar between groups (Kruskal-Wallis ANOVA: H(2,25)= 0.2, P=0.9). These results highlight that changes in Cx30-mediated gap junction coupling in the ACC of depressed suicides may be specific to astrocyte-oligodendrocyte communication.

Downregulation of oligodendrocyte-specific connexins and scaffolding components of gap junction channels in the ACC of depressed suicides

Changes in gap junction coupling are likely to involve transcriptomic alterations of genes essential for the synthesis of GJC components, their scaffolding, trafficking and function. To complement our histological approach, we screened the gene expression of some key regulators of the gap junction proteome from a previously published RNA-sequencing dataset comparing controls and matched depressed suicides in the ACC 18 . This dataset had allowed to find a global downregulation of oligodendrocyte-related genes, as well as morphometric impairments of myelin in depressed suicides with a history of CA. Here, a nonexhaustive literature-based list of genes was screened that included major connexins and some of their known binding partners, such as cytoskeletal and scaffolding components of the GJC (Fig. 4A andB).

The latter included zona occludens 1 (ZO-1), ZO-2 and ZO-3, spectrin, drebrin and catenins, which together form a network of proteins that bridges connexins to microfilaments and microtubules and interact with occludins, claudins and cadherins to form junctional nexuses [START_REF] Laird | The gap junction proteome and its relationship to disease[END_REF][START_REF] Dianati | Connexins, E-cadherin, Claudin-7 and β-catenin transiently form junctional nexuses during the post-natal mammary gland development[END_REF][START_REF] Dbouk | Connexins: a myriad of functions extending beyond assembly of gap junction channels[END_REF][START_REF] Martins-Marques | Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart[END_REF] .

Among connexin genes, only those coding for two oligodendrocyte-specific connexins, Cx32 (GJB1) and Cx47 (GJC2), were found to be downregulated in depressed suicides (P<0.05). These connexins are binding partners of the astrocyte-specific connexins Cx30 and Cx43, respectively, which pair together to form functional GJCs between astrocytes and oligodendrocytes [START_REF] Jl | Gap Junctions Couple Astrocytes and Oligodendrocytes[END_REF][START_REF] Magnotti | Functional heterotypic interactions between astrocyte and oligodendrocyte connexins[END_REF] . Moreover, two major connexininteracting proteins, alpha-and beta-catenins [START_REF] Talhouk | Heterocellular interaction enhances recruitment of α and β-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells[END_REF][START_REF] Fujimoto | Dynamics of connexins, E-cadherin and alpha-catenin on cell membranes during gap junction formation[END_REF] , both adherens junction components, were also downregulated in cases versus controls (P<0.05). Several major cytoskeletal components essential for the scaffolding of GJCs and junctional nexus were also found to be differentially expressed. The major tight junction proteins ZO-1 and ZO-2, interacting with each other and with connexins to regulate gap junction plaque size and distribution [START_REF] Laird | The gap junction proteome and its relationship to disease[END_REF][START_REF] Hunter | Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion[END_REF][START_REF] Singh | Connexin 43 Interacts with Zona Occludens-1 and -2 Proteins in a Cell Cycle Stage-specific Manner[END_REF] , were downregulated in samples from depressed suicides (P<0.05 and P<0.01 respectively). We also screened for other core proteins of tight junctions, such as occludin and claudins, which are also part of the junctional nexus, some of which co-immunoprecipitate with the oligodendrocyte-expressed Cx32 [START_REF] Kojima | Induction of Tight Junctions in Human Connexin 32 (hCx32)-Transfected Mouse Hepatocytes: Connexin 32 Interacts with Occludin[END_REF]80 . Again, occludin was significantly downregulated in depressed suicides (P<0.01), while among the claudin family of proteins, the only member found to be differentially expressed between groups was CLDN11 (claudin-11, P<0.05), an essential component of myelin interlamellar tight junctions [START_REF] Denninger | Claudin-11 Tight Junctions in Myelin Are a Barrier to Diffusion and Lack Strong Adhesive Properties[END_REF][START_REF] Gow | CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice[END_REF][START_REF] Morita | Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis[END_REF] , also suggesting that cell-cell junctions involving oligodendrocytes may be particularly impaired in depressed suicides.

Caveolin-1 and Caveolin-2, two major regulators of connexin trafficking and assembly, as well as gap junction mediated intercellular communication [START_REF] Schubert | Connexin family members target to lipid raft domains and interact with caveolin-1[END_REF][START_REF] Langlois | Caveolin-1 and -2 Interact with Connexin43 and Regulate Gap Junctional Intercellular Communication in Keratinocytes[END_REF] , were also found to be downregulated in depressed suicides (P<0.05). Finally, of the key actin-binding proteins important to bridge connexins to actin microfilament cytoskeleton [START_REF] Giepmans | Gap junctions and connexin-interacting proteins[END_REF][START_REF] Butkevich | Drebrin Is a Novel Connexin-43 Binding Partner that Links Gap Junctions to the Submembrane Cytoskeleton[END_REF][START_REF] Ursitti | Role of an alternatively spliced form of alphaII-spectrin in localization of connexin 43 in cardiomyocytes and regulation by stressactivated protein kinase[END_REF][START_REF] Toyofuku | Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes[END_REF][START_REF] Majoul | Many faces of drebrin: from building dendritic spines and stabilizing gap junctions to shaping neurite-like cell processes[END_REF] , DBN1 (developmentally regulated brain protein, drebrin, P<0.05), SPTAN1 (Alpha-II Spectrin, P<0.01), SPTB (beta-I spectrin, P<0.05), SPTBN2 (beta-III spectrin, P<0.05), and SPTBN4 (Beta-IV Spectrin, P<0.01) were all upregulated in depressed suicides compared to controls.

A subset of these differentially expressed genes was selected for validation with quantitative RT-PCR.

We focused on the connexins enriched in astrocytes (Cx30 and cx43) and oligodendrocytes (Cx32 and Cx47) as well as CAV1, CAV2, OCLN and DBN1 (Fig. 4C andD). Importantly, because in our RNAsequencing study all depressed suicides had a history of CA, which may specifically lead to oligodendrocyte abnormalities, qPCR validation was performed in both DS-nCA and DS-CA groups similarly to our histological experiments, to clarify whether observed changes were associated with psychopathology or were specific to history of CA. Overall, regardless of CA, depressed suicides displayed a general downregulation of Cx32, Cx47, CAV1 and CAV2 as well as OCLN expression and an upregulation of DBN1 expression compared to controls (Fig. 4C). These changes were however not as statistically robust as previously observed using RNA-seq. Importantly, because these changes did not seem specific to a history of CA, as no significant difference was found between DS-CA and DS-nCA for any of these genes, we combined both depressed suicides groups (Fig4D) and found significant downregulations of oligodendrocyte connexins Cx32 and Cx47, CAV1 and CAV2 as well as OCLN (CTRL vs Depressed Suicides, P<0.05). The upregulation of DBN1 observed in the RNA-seq dataset was however not validated, although a small trend towards increased expression was detectable (CTRL vs Depressed Suicides, P=0.13).

DISCUSSION

In this study, we aimed to address the hypothesis that cellular communication between astrocytes and oligodendrocytes could potentially be impaired in depressed suicides, as previous evidence had linked changes in the expression of astrocytic connexins as well as myelin and oligodendrocyte function impairments in this population. We characterized the distribution of Cx30 in the human post-mortem ACC by immunolabeling, and found that Cx30-IR is selectively decreased in somas of mature oligodendrocytes and along myelinated fibers in depressed suicides, suggesting that Cx30-mediated gap junction coupling between astrocytes and oligodendrocytes may be disrupted. This was supported by the reduced expression of genes coding for the two major oligodendrocyte-specific connexins, Cx32 and Cx47, which upon pairing with the astrocytic connexins Cx30 and Cx43 allow the formation of functional GJCs between these two glial cell types. Additional key components of the gap junction proteome and cell-cell junctions, in particular components essential for the scaffolding, trafficking and function of GJCs, were also found to be downregulated in depressed suicides. Altogether, these results highlight that glial cell communication through cell-cell junctions may be an important feature of the pathophysiology of depression, possibly bridging the cumulative evidence of astrocytes and oligodendrocytes abnormalities previously observed in this population.

Several post-mortem studies, including from our group, have consistently reported a downregulation in the expression of astrocytic connexins in frontal regions of suicides. Ernst et al. first found both Cx30 and Cx43 to be strongly downregulated in the dorsolateral prefrontal cortex of suicides [START_REF] Ernst | Dysfunction of astrocyte connexins 30 and 43 in dorsal lateral prefrontal cortex of suicide completers[END_REF] . Nagy et al. further

showed that this phenomenon also occurred in other cortical and subcortical brain regions, namely the prefrontal, primary motor and visual cortex, the dorsomedial thalamic nucleus, and the caudate nucleus [START_REF] Nagy | Repression of Astrocytic Connexins in Cortical and Subcortical Brain Regions and Prefrontal Enrichment of H3K9me3 in Depression and Suicide[END_REF] .

Finally, Miguel-Hidalgo and collaborators also reported that Cx43 expression was decreased in the orbitofrontal cortex of depressed individuals versus controls [START_REF] Wilson | Reduced connexin 43 immunolabeling in the orbitofrontal cortex in alcohol dependence and depression[END_REF] . We were therefore surprised that this result was not replicated in our cohort of subjects, perhaps highlighting a regional specificity of the ACC with regards to astrocytic changes associated with psychopathology. This discrepancy was not driven by covariates, history of substance dependence or antidepressant treatment. In agreement with our results for Cx30 and Cx43 in the ACC, other canonical markers of astrocytes previously found to be downregulated in BA8/9 of depressed suicides, alongside astrocytic connexins, were not affected in the ACC. These markers include ALDH1L1, GLUL, and Sox9, an astrocyte-enriched transcription factor previously shown to bind to and potently regulate Cx30 expression [START_REF] Ernst | Dysfunction of astrocyte connexins 30 and 43 in dorsal lateral prefrontal cortex of suicide completers[END_REF] . Considering that astrocyte connexins are engaged in a wide range of GJCs between astrocytes and other cell types, we hypothesize that astrocytes dysfunction may not be a critical pathophysiological feature in the ACC, but that their interaction with other cell types may be still be compromised through changes occurring in some of their interacting partners. In that scenario whole levels of Cx30 expression may not be indicative of discrete changes occurring in other cell types. We were particularly interested in glial communication between astrocytes and oligodendrocytes, as we previously showed that cortical myelination is critically impaired in the ACC of depressed suicides 18 , and that A/O GJCs are essential for the maintenance of myelin and oligodendrocyte function [START_REF] Nualart-Marti | Gap junction communication in myelinating glia[END_REF] .

Our results indicated that indeed, while only representing a small fraction of the Cx30 localization, Cx30 mapping to mature oligodendrocytes and myelinating fibers, but not cerebral vasculature, is selectively decreased in depressed suicides. In accordance with these immunohistological results, we found that the gene expression of the canonical partner of Cx30 on the oligodendrocyte side, Cx32, was also significantly downregulated. Taken together, these observations strengthen our interpretation that A/O gap junction coupling may be impaired in depression, possibly through changes occurring in oligodendrocytes. Interestingly, the other oligodendrocyte-specific connexin, Cx47, which associates with Cx43, was also downregulated in depressed suicides. Although we did not quantify the mapping of Cx43 onto oligodendrocytes and myelinated fibers because of antibody incompatibilities, these results also suggest that A/O gap junction coupling impairment may not be specific to Cx30-mediated GJCs but likely extend to Cx43-Cx47 pairing as well. While we focused on A/O coupling, it would be interesting to use this approach to explore other cell-cell interactions, in particular O/O coupling through homotypic pairing of Cx32 and Cx47, both of which are downregulated in the ACC of depressed suicides.

To our knowledge, mapping connexin expression in situ onto different cell types has never been achieved previously in post-mortem brain tissue, in which immunofluorescence can be challenging to perform.

This strategy has some limitations, however. Given the limited resolution of confocal microscopy, it is impossible to infer whether the decreased mapping of Cx30 to oligodendrocytes and myelinated fibers represents a loss of Cx30 pairing to its partner Cx32 or changes in hemichannel trafficking. While a more direct approach consisting in mapping the colocalization of Cx30 with Cx32 onto oligodendrocytes or their interaction by co-immunoprecipitation would have certainly allowed a more precise assessment of A/O coupling, this proved challenging in our hands because of antibodies incompatibility. Despite this limitation, our immunohistological approach was supported by strong evidence showing impaired expression of key components of the gap junction proteome in the ACC of depressed suicides. Alongside reduced expression of the two oligodendrocyte connexins Cx32 and Cx47, major cytoskeletal scaffolding components and regulators of gap junction trafficking and assembly showed a collective downregulation.

While some of those are major tight and adherens junction proteins, such as occludin, ZO1 and ZO2, or claudin-11, strong evidence highlights their role in supporting the scaffolding and function GJCs [START_REF] Laird | The gap junction proteome and its relationship to disease[END_REF][START_REF] Dbouk | Connexins: a myriad of functions extending beyond assembly of gap junction channels[END_REF][START_REF] Giepmans | Gap junctions and connexin-interacting proteins[END_REF] .

Alongside our observed changes in actin-binding proteins bridging connexins to cytoskeletal microfilaments, such as spectrins and catenins, this hints towards a convergent disruption of the structural integrity of cell-cell junctions. In view of our immunohistological results and reduced expression of Cx32 and Cx47, we hypothesize that this may reflect disruptions of gap junction integrity, although it is likely that such global effects may translate in other cell-cell junctions, in particular tight and adherens junctions. Tight junctions make up the radial component of CNS myelin, acting as an interlaminar diffusion barrier and structural support [START_REF] Bronstein | Tight Junctions in CNS Myelin[END_REF] . The fact that claudin-11, the major component of oligodendrocyte tight junctions [START_REF] Gow | CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice[END_REF] , was the only member of the claudin family to be differently expressed in depressed suicides suggests that transcriptional changes associated with psychopathology are particularly prominent in OLs.

What upstream causes and mechanisms could underlie the disrupted gap junction connectivity between astrocytes and oligodendrocytes in the ACC of depressed suicides? One possible answer could be neuroinflammation, which has been increasingly associated with depression and suicide [START_REF] Brites | Neuroinflammation and Depression: Microglia Activation, Extracellular Microvesicles and microRNA Dysregulation[END_REF][START_REF] Torres-Platas | Evidence for increased microglial priming and macrophage recruitment in the dorsal anterior cingulate white matter of depressed suicides[END_REF][START_REF] Devorak | Cellular and Molecular Inflammatory Profile of the Choroid Plexus in Depression and Suicide[END_REF] . Indeed, several pro-inflammatory molecules are thought to alter the composition and structure of tight junctions by modulating some of their key components such as occludin, claudins, and zona occludens proteins [START_REF] Rochfort | Cytokine-mediated dysregulation of zonula occludens-1 properties in human brain microvascular endothelium[END_REF][START_REF] Van Itallie | Occludin is required for cytokine-induced regulation of tight junction barriers[END_REF][START_REF] Capaldo | Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins[END_REF] , thereby affecting their permeability and function. This has been documented in the CNS, and disruption of blood brain barrier integrity through alterations in tight junctions has recently emerged as an interesting pathophysiological feature of depression [START_REF] Menard | Social stress induces neurovascular pathology promoting depression[END_REF] . With regards to connexins and gap junction integrity, chronic inflammation, microglial activation and astrogliosis have also been associated with Cx32 and Cx47 downregulation, and widespread loss of O/O and O/A gap junctions [100][101][102][103][104] . Notably, loss of Cx32 GJs at oligodendrocyte cells bodies and along myelinated fibers in grey matter were previously reported in multiple sclerosis 103 , similar to our observations showing decreased Cx30-IR on oligodendrocyte cell bodies and myelinated axons. Given that increased microglia/macrophage activity has been reported in the ACC of depressed suicides [START_REF] Torres-Platas | Evidence for increased microglial priming and macrophage recruitment in the dorsal anterior cingulate white matter of depressed suicides[END_REF] , the present evidence of cell-cell junctions and altered A/O communication may represent a consequence of depression-associated low-level chronic neuroinflammation.

We recently reported a robust downregulation of the oligodendrocyte transcriptome and decreased myelination of small caliber axons in the ACC of depressed suicides 18 . Remarkably, these effects were associated with a history of severe CA, but were absent in a matched sample of depressed suicides without history of CA. The present study provides evidence that altered A/O communication in the ACC may be a common neurobiological feature of major depression, irrespective of history of early-life adversity, and could possibly be linked to the characteristic changes in ACC volume and function described in depressed patients [START_REF] Drevets | Brain structural and functional abnormalities in mood disorders: implications for neurocircuitry models of depression[END_REF]105 . It can be further hypothesized that the more global impact of CA on oligodendrocytes and myelination may predispose to these changes and mediate specific alterations of inter-regional connectivity found in depressed individuals exposed to early-life adversity 106 .

In conclusion, our study suggests that the astrocyte-oligodendrocyte gap junction connectivity is decreased in the ACC of depressed suicides, possibly through decreased Cx30 coupling to oligodendrocyte-and myelin-specific connexins. This is associated with a convergent downregulation of key structural components and regulators of cell-cell junctions. Because A/O GJCs play an important role in providing metabolic support to oligodendrocyte-lineage cells, regulate precursor cells proliferation and differentiation, and are essential for myelin integrity, we argue that our findings may relate to reported alterations of myelination previously observed in depressed suicides and further support glial dysfunction as a major hallmark of depression. Ultimately, as suggested by recent preclinical studies [START_REF] Sun | Gap junction dysfunction in the prefrontal cortex induces depressive-like behaviors in rats[END_REF][START_REF] Jeanson | Antidepressants Impact Connexin 43 Channel Functions in Astrocytes[END_REF]107 , targeting connexins and cell-cell junctions could prove useful in the search of novel therapeutic approaches for depression.

  mapping of Cx30 onto individual mature oligodendrocytes was relatively low, we found a significant difference between groups (Kruskal-Wallis ANOVA: H(2,23)= 9.09, P<0.05), with decreased Cx30-IR mapping to Nogo-A-IR cells in depressed suicides compared to controls, regardless of history of CA (Dunn's multiple comparisons tests: CTRL vs DS-nCA and CTRL vs DS-CA: P<0.05). We then combined Myelin Proteolipid Protein (PLP) and Cx30 immunofluorescence to quantify the density of Cx30 mapping to myelinated fibers (Fig.3C). In accordance with decreased astrocyte-oligodendrocyte coupling at the soma, we also found differences between groups (Kruskal-Wallis ANOVA: H(2,24) = 7.471, P<0.05), with Cx30-IR decreased along PLP-IR myelinated axons in depressed suicides regardless of history of CA, although differences were more statistically robust in DS-CA (Dunn's multiple comparison test, CTRL vs DS-CA: P<0.05), as only a trend was detectable between DS-nCA and controls (Dunn's multiple comparison test, CTRL vs DS-nCA: P<0.1). Densities of Cx30 on PLP-IR myelinated axons were similar between DS-CA and DS-nCA (Dunn's multiple comparison test, DS-nCA vs DS-CA: P=0.99).

Figure 1 .

 1 Figure 1. Distribution of connexin 30 immunoreactivity in the human anterior cingulate cortex

Figure 2 .

 2 Figure 2. Depressed Suicides show no change in the density of Cx30-IR compared to controls in the

Figure 3 .

 3 Figure 3. Specific decrease in Cx30-IR mapping to oligodendrocytes and myelinated fibers in the

Figure 4 . 7 : 4 .

 474 Figure 4. Genes involved in the scaffolding, trafficking and function of gap junction channels and

  Figure 3.

Figure 4 .

 4 Figure 4.

Table 1 .

 1 Subjects characteristics

		CTRL	DS-nCA	DS-CA
	N	23	23	25
	Axis 1 diagnosis	none	MDD (19); DD-NOS (4)	MDD (21); DD-NOS (4)
	History of CA	none	none	25
	Age (years)	46.2 ± 22.2	49.3 ± 10.3	42.2 ± 14.8
	Sex (M/F)	18/5	20/3	18/7
	PMI (hours)	35.4 ± 21.5	51.7 ± 29.4	42 ±22.5
	pH	6.5 ± 0.3	6.6 ± 0.3	6.6 ± 0.3
	RIN	6.9 ± 0.6	6.5 ± 0.3	6.8 ± 0.8
	Substance dependence	0	9	8
	AD medication	0	9	8
	Data represent mean ± SD. AD: antidepressant; DD-NOS: Depressive Disorder Not Otherwise Specified;
	MDD: Major Depressive Disorder; PMI: post-mortem interval; RIN: RNA Integrity Number.

Table 2 .

 2 Primary antibodies used in this study

	Primary antibody	Species Dilution	Manufacturer	Reference number
	Connexin 30	Mouse	500	Santa Cruz Biotechnology sc-514847
	Connexin 43	Rabbit	500	Cell Signaling	3512
	Myelin Proteolipid	Rabbit	1000	Abcam	ab105784
	Protein (PLP)
	Nogo-A	Rabbit	100	Millipore	AB5888
	Laminin	Rabbit	1000	Sigma-Aldrich	L9393
	Vimentin	Rabbit	1000	Abcam	ab92547
	GFAP	Chicken 1000	Abcam	ab4674

Table 3 .

 3 Primers used for quantitative RT-PCR validation of differential expression

	Gene	Primer sequence (5'3')	Primer length (bp)	Amplicon size (bp)
		Forward:
	GJB6	CGGCCGCAGAGCAGTTAAAA Reverse:	20 20	102
		CCCGTTTGCAAAACACAGGA
	GJA1	Forward: TACCAAACAGCAGCGGAGTT Reverse: TGGGCACCACTCTTTTGCTT	20 20	139
		Forward:
	GJB1	GGCAGTGACAGGGAGGTGT Reverse:	19 20	83
		TAGAATGCCGGTTCACGCCA
	GJC2	Forward: AATTTTCTGGCCTGGAGAAGGACC Reverse: TCAGCACGATGCGGAAGAC	24 19	154

Time PCR system (ThermoFisher Scientific), using four replicates per gene. Primers were designed with Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and validated by dissociation curves and gel migration. Relative expression was calculated on the QuantStudio Real-Time PCR Software version 1.2 (ThermoFisher Scientific) using calibration curves and normalization by the housekeeping gene GAPDH.

Statistics

All statistical analyses were performed on Statistica version 12 (StatSoft) and Prism version 6 (GraphPad Software). Distribution of the data and homogeneity of variances between groups were assessed with Shapiro-Wilk and Levene's tests, respectively. For Cx30 density measurements and mapping experiments comparing Controls (CTRL), Depressed Suicides (DS-nCA) and Depressed Suicides with a history of CA (DS-CA), group effects were detected with Kruskal-Wallis non-parametric one-way ANOVAs, followed by Dunn's multiple comparisons post-hoc test. Effects of a history of substance abuse and medication in Depressed Suicides on Cx30 density were addressed using Kruskal-Wallis one-way non-parametric ANOVA. Linear regressions were used to assess the effects of age and PMI on Cx30 density. Differential expression in the RNA-sequencing dataset was performed using DEseq2, using a linear model controlling for RIN, age and gender. Differential expression in RT-PCR data was assessed using unpaired two-tailed t-tests comparing cases to controls. Nominal p-values are reported with a significance threshold set at 0.05.

ACKNOWLEDGMENTS

This research was funded by grants from CIHR (PJT-156346) and the AFSP (SRG-0-088-15) to N.M. A.T. was supported by a TD postdoctoral fellowship, and currently by an AFSP Young Investigator Innovation Grant (YIG-0-146-17). P-E.L. was supported by scholarships from the American Foundation of Suicide Prevention, the Fondation Deniker, the Fondation pour la Recherche Médicale, and the UNAFAM ('Union nationale de familles et amis de personnes malades et/ou handicapées psychiques').

The authors wish to thank the Douglas-Brain Canada Brain Bank (DBCBB) staff as well as the Douglas Institute's Molecular and Cellular Microscopy Platform coordinator for their precious assistance. Both of these research platforms are funded by a Core Facilities and Technology Development grant from Healthy Brains for Healthy Lives (CFREF). The DBCBB is further supported by the RQSHA (FRQ-S).