Raphaële Mongrédien

Amaia M Erdozain

Sylvie Dumas

Laura Cutando

Amaia Nuñez

Del Moral

Emma Puighermanal

Sara Rezai Amin

Bruno Giros

Emmanuel Valjent

Javier Javier Meana

Amaia Nuñez Del Moral

Sophie Gautron

Luis F Callado

Véronique Fabre

Vincent Vialou
email: vincent.vialou@inserm.fr

Vincent Vialou

Cartography of hevin-expressing cells in the adult brain reveals prominent expression in astrocytes and parvalbumin neurons

Keywords: Hevin, matricellular protein, parvalbumin neurons, astrocytes, glutamatergic neurons, in situ hybridization lateral part AVM Anteroventral thalamic nucleus, medial part Berg. Bergman glia BLA Basolateral amygdala CA1-3 Cornu ammonis Cb Cerebellum cc Corpus callosum ChP Choroid plexus Cl Claustrum CPu Caudate putamen Cx Cortex DBB

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INTRODUCTION

Hevin, also known as SPARC-like 1 (SPARCL1), ECM2 or Mast9, belongs to the matricellular proteins of the SPARC (secreted protein acidic and rich in cysteine) family, a class of nonstructural extracellular matrix (ECM) molecules that regulate cell morphology, proliferation and migration [START_REF] Girard | Modulation of endothelial cell adhesion by hevin, an acidic protein associated with high endothelial venules[END_REF][START_REF] Gongidi | SPARC-like 1 regulates the terminal phase of radial glia-guided migration in the cerebral cortex[END_REF][START_REF] Sage | Extracellular Proteins That Modulate Cell-Matrix Interactions -Sparc, Tenascin, and Thrombospondin[END_REF][START_REF] Sullivan | Altered tissue repair in hevin-null mice: inhibition of fibroblast migration by a matricellular SPARC homolog[END_REF]). These matricellular proteins include structurally diverse proteins such as thrombospondins 1 and 2, tenascins, SPARC and hevin (Murphy-Ullrich and Sage 2014), which interact with other classes of ECM proteins as well as with cytokines, proteases, neurotrophic factors and cell surface receptors [START_REF] Bornstein | Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1[END_REF]. In the developing central nervous system (CNS), hevin modulates neuronal migration and synaptogenesis. Specifically, hevin helps terminate neuronal migration through its anti-adhesive properties [START_REF] Gongidi | SPARC-like 1 regulates the terminal phase of radial glia-guided migration in the cerebral cortex[END_REF]). At postnatal day (P) 15, a period of intense synaptogenesis, release of hevin promotes the apposition and stabilization of pre-and post-synaptic specialized junction elements by clustering trans-synaptic adhesion molecules, neurexin and neuroligin [START_REF] Risher | Astrocytes refine cortical connectivity at dendritic spines[END_REF][START_REF] Singh | Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1alpha and NL1 via Hevin[END_REF]). Although hevin is strongly expressed in the adult CNS [START_REF] Eroglu | The role of astrocyte-secreted matricellular proteins in central nervous system development and function[END_REF][START_REF] Lloyd-Burton | SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS[END_REF][START_REF] Mendis | SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain[END_REF][START_REF] Mothe | Effect of hyperthermia on the transport of mRNA encoding the extracellular matrix glycoprotein SC1 into Bergmann glial cell processes[END_REF][START_REF] Weaver | Proteolysis of the matricellular protein hevin by matrix metalloproteinase-3 produces a SPARC-like fragment (SLF) associated with neovasculature in a murine glioma model[END_REF], its impact on adult brain function is currently unknown. In animal models of epilepsy, ischemia and injury, hevin is induced in reactive astrocytes, suggesting that it might participate in neuronal remodeling in pathological conditions in the adult stage (Lively and Brown 2008a;Lively and Brown 2008b;[START_REF] Lively | SC1/hevin and reactive gliosis after transient ischemic stroke in young and aged rats[END_REF][START_REF] Mendis | Induction of SC1 mRNA encoding a brain extracellular matrix glycoprotein related to SPARC following lesioning of the adult rat forebrain[END_REF].

Neuroanatomical studies have identified the cellular phenotype of hevin-expressing cells only during development. From embryonic day (E) 10.5 to P1, hevin is expressed in radial glial cells and vascular endothelial cells and is absent from neuroblasts [START_REF] Lloyd-Burton | SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS[END_REF][START_REF] Mckinnon | Extracellular matrix-associated protein Sc1 is not essential for mouse development[END_REF]. After the first postnatal week, in which the vast majority of radial glial cells undergo differentiation into astrocytes, hevin is expressed in astrocytes while progressively disappearing from blood vessels, and is absent from microglia [START_REF] Lively | SC1/hevin and reactive gliosis after transient ischemic stroke in young and aged rats[END_REF][START_REF] Lloyd-Burton | SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS[END_REF][START_REF] Risher | Astrocytes refine cortical connectivity at dendritic spines[END_REF]. Hevin starts to be expressed in subsets of neurons at P1 and remains expressed in adult neurons [START_REF] Kucukdereli | Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC[END_REF]Lively and Brown 2008c;[START_REF] Lively | SC1/hevin and reactive gliosis after transient ischemic stroke in young and aged rats[END_REF][START_REF] Lloyd-Burton | SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS[END_REF][START_REF] Mckinnon | Extracellular matrix-associated protein Sc1 is not essential for mouse development[END_REF][START_REF] Mendis | SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain[END_REF][START_REF] Risher | Astrocytes refine cortical connectivity at dendritic spines[END_REF]. The spatio-temporal expression profile of hevin is strikingly different from other matricellular proteins such as SPARC, its closest homologue, whose expression decreases throughout development and is mostly absent from neurons [START_REF] Eroglu | The role of astrocyte-secreted matricellular proteins in central nervous system development and function[END_REF][START_REF] Jones | Astrocytes control glutamate receptor levels at developing synapses through SPARC-beta-integrin interactions[END_REF][START_REF] Kucukdereli | Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC[END_REF]. However, studies describing the precise distribution of hevin in the entire adult brain are lacking, and the phenotype of the neurons expressing hevin is unknown.

Here, we determined the regional distribution of hevin throughout the adult mouse brain using isotopic in situ hybridization (ISH). For comparison purposes, we also determined the distribution of SPARC mRNA. In order to determine the cellular phenotype of hevinexpressing cells, we performed double fluorescent in situ hybridization (FISH) with probes targeting hevin and markers of astrocytes, glutamatergic neurons, gamma-aminobutyric acid (GABA) neuron subtypes, monoaminergic and cholinergic neurons. In addition, we characterized the phenotype of hevin mRNA-expressing cells in two human brain regions, the prefrontal cortex and the caudate nucleus.

MATERIALS AND METHODS

Animals

All experiments were performed in conformity with the European Union laws and policies for the use of animals in neuroscience research (European Committee Council Directive 2010/63/EU), and were approved by the local animal research committee. Nine and twelve week-old male and female C57BL6J mice were housed in groups under standard conditions at 22±1°C and a 12 h light/dark cycle, with food and water provided ad libitum.

Preparation of mouse brain sections

Brains were quickly removed after decapitation and frozen at -30°C in 2-methylbutane. Coronal frozen sections (16 µm) were prepared with a cryostat at -20°C, thaw-mounted onto poly-L-lysine-coated glass slides (Superfrost Plus, Menzel Glaser, Braunschweig, Germany) and stored at -80°C until usage. The entire brain was cut and sections were serially mounted with a step of 144 µm between two consecutive sections.

Human brain samples

Human brain samples from twelve subjects who died from sudden and violent causes were obtained at autopsy in the Basque Institute of Legal Medicine in Bilbao, Spain. All subjects were free of psychiatric or neurological disorders based on medical history, postmortem tissue examinations and a toxicological screening on blood at autopsy (detection of antidepressants, antipsychotics, other psychotropic drugs, and ethanol). Samples from the prefrontal cortex and caudate nucleus (n=8) were dissected at the time of autopsy and immediately stored at -70°C until assayed. These two specific areas were chosen, as they are representative of the diversity of hevin expressing cell types in the mouse brain, i.e. astrocytes, parvalbumin neurons and glutamatergic neurons. The demographic and brain sample characteristics of the subjects are shown in Table 1. The study was performed in compliance with legal policy and ethical review boards for postmortem brain studies. Frozen sections (16 µm) were prepared with a cryostat at -20°C, thaw-mounted onto poly-L-lysine-coated glass slides (Superfrost Plus, Menzel Glaser, Braunschweig, Germany) and stored at -80°C until usage.

Radioactive ISH

We used radioactive ISH on mouse brain slices of the entire brain to determine the distribution of hevin and SPARC mRNA. Antisense oligonucleotide probes (35-mer) were designed to detect specifically mRNAs encoding mouse hevin (SPARCL1) and mouse SPARC (Table 2). All antisense probes were tested for specificity using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In situ hybridization was carried out as described previously [START_REF] Vialou | Neurochemical characterization of pathways expressing plasma membrane monoamine transporter in the rat brain[END_REF]). The probes were 3' end-labeled with [alpha-35 S] dATP (370 gigabecquerel/mmol; Perkin Elmer, Waltham, Massachusetts, USA) using terminal deoxynucleotidyl transferase (Roche Applied Biosystems, Nonnenwald, Germany) at a specific activity of 5.10 8 disintegrations per minute/µg. Sections were fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS) for 1 h, washed in PBSX1, rinsed in water, dehydrated in 70% ethanol and air-dried. Hybridization was carried out at 42°C for 16 h in hybridization medium (Oramacell, Paris, France) containing a mixture of labeled antisense oligonucleotides (4.10 5 counts per minute/100 µl each). Sections were washed to a final stringency of 0.5 SSC (7.5 mM sodium citrate, 75 mM NaCl, pH 7.0) at 53°C, dehydrated in ethanol, air-dried and exposed to Fujifilm BioImaging Analyzer BAS-5000 (Fujifilm Life Science, Stamford, Connecticut, USA) for 15 days. Three different oligo-probes tested separately showed an identical distribution pattern, demonstrating the specificity of the approach (data not shown). The control procedure to confirm the specificity of ISH labeling using sense-radiolabeled probes gave no signal.

ISH image analysis

Digitized autoradiography scans (Fujifilm Life Science) were exported in TIFF format using MCID image analysis software (Imaging Research, St. Catharines, Ontario, Canada). Coronal brain sections from two male and two female C57BL6J mice (12 weeks old) spanning the entire brain (from olfactory bulbs to brainstem) were used. Around 45 sections with a step of 144 µm between two consecutive sections per mice were analyzed. Specific brain regions were identified according to the mouse brain atlas [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]. A qualitative estimate of hevin mRNA intensity was assessed at the macroscopic level on digitized autoradiography scans as highest density (++++), high density (+++), moderate density (++), and low density (+) by considering signal strength. The intensity of the hevin signal was always within the linear range of the 14 C standard exposed together with the hybridized tissue. Representative sections were corrected for contrast and cropped using Photoshop CS6 (Adobe Systems, San Jose, California, United States), and assembled on Illustrator CS6 (Adobe Systems).

Double fluorescent ISH (FISH)

FISH was used to determine the phenotype of hevin-expressing cells in both mouse and human brain using probes of specific cellular markers (Table 2), as described previously [START_REF] Anderson | Astrocyte glutamate transport: review of properties, regulation, and physiological functions[END_REF][START_REF] Chazalon | The GABAergic Gudden's dorsal tegmental nucleus: A new relay for serotonergic regulation of sleep-wake behavior in the mouse[END_REF][START_REF] Erdozain | Structural and Functional Characterization of the Interaction of Snapin with the Dopamine Transporter: Differential Modulation of Psychostimulant Actions[END_REF][START_REF] Schweizer | Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion[END_REF][START_REF] Viereckel | Midbrain Gene Screening Identifies a New Mesoaccumbal Glutamatergic Pathway and a Marker for Dopamine Cells Neuroprotected in Parkinson's Disease[END_REF]. Complementary RNA probes were synthesized with DIG-or fluorescein-labeled ribonucleoside tri-phosphate (Roche Applied Biosystems). The specificity of the probes was verified using NCBI blast. Cryosections were air-dried, fixed in 4% paraformaldehyde and acetylated in 0.25% acetic anhydride/100 mM triethanolamine (pH 8). Sections were hybridized for 18 h at 65°C in 100 µl of formamide-buffer containing 1 µg/ml hevin digoxigenin-labeled riboprobe (DIG) and 1 µg/ml cell-marker fluorescein-labeled riboprobe for mouse sections and with hevin fluorescein-labeled riboprobe and DIG-labeled cell-marker riboprobe for human sections (Table 2). Sections were washed at 65°C with SSC buffers of decreasing strength, and blocked with 20% fetal bovine serum and 1% blocking solution (Roche Applied Biosystems). Fluorescein epitopes were detected with horseradish peroxidase (HRP) conjugated anti-fluorescein antibody at 1:1000 and revealed with TSA kit (Perkin Elmer) using biotin-tyramide at 1:75 followed by incubation with Neutravidin Oregon Green conjugate at 1:750. HRP-activity was stopped by incubation of sections in 0.1 M glycine followed by a 3% H2O2 treatment. DIG epitopes were detected with HRP anti-DIG Fab fragments at 1:1000 and revealed with TSA kit (Perkin Elmer) using Cy3 tyramide at 1:200. Nuclear staining was performed with 4' 6-diamidino-2-phenylindole (DAPI). The distribution of all probes is in agreement with those previously described in the mouse and human brain, confirming the reliability of our FISH procedures.

FISH image acquisition

All slides were scanned on a NanoZoomer 2.0-HT (Hamamatsu Photonics, Hamamatsu City, Japan) at 20x resolution. Laser intensity and time of acquisition was set separately for each riboprobe. Images were analyzed using the NDP.view2 software (Hamamatsu Photonics). Regions of interest were identified according to the Paxinos mouse brain atlas [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]. Positive cells refer to a staining in a cell body clearly above background and surrounding a DAPI-stained nucleus. Co-localization was determined by the presence of the signals for both probes in the soma of the same cell. The entire region of interest was evaluated. For illustration purposes, the NanoZoomer images were exported in TIFF format using NDP viewer. Images were corrected for contrast, cropped on Photoshop CS6, and assembled on Illustrator CS6.

RESULTS

Topographic expression of hevin mRNA in the adult mouse brain

First, we investigated the distribution of hevin transcript in adult mouse brain coronal sections using a mixture of three radiolabeled oligonucleotides (Table 2). As shown on film autoradiography, hevin mRNA was found throughout the brain with varying levels of intensity (Fig. 1a). Estimates of hevin mRNA expression levels in major brain structures are shown in Table 3. A strong expression was observed in cortical regions with the highest density in layers II/III and V of cortex; hippocampal subdivisions including presubiculum, and dentate gyrus; lateral and medial globus pallidus; and specific thalamic nuclei including anterodorsal thalamus, lateral habenula, laterodorsal thalamus, reticular thalamus, subthalamic nucleus and zona incerta. Many nuclei in the midbrain and in the hindbrain contained a high density of hevin signal, including the dorsolateral geniculate, motor nuclei III and IV, red nucleus, superior and inferior colliculus; Bergman glial cells of cerebellum; and pontine reticular nucleus, reticulotegmental nucleus of the pons, superior olive nucleus, tegmental nucleus, and motor nuclei V, VII, X and XII in the hindbrain. Lower intensity of hevin mRNA expression was observed in the rest of brain, in particular in the anterior olfactory nucleus, striatum, CA1 and CA3 of the hippocampus, and the molecular layer of the cerebellum. Hevin mRNA was poorly expressed in the white matter. Fluorescent ISH with a DIG-labeled hevin cRNA probe in the mouse brain confirmed this regional pattern of expression (Figs. 2-10). The regional pattern of expression of hevin was similar in male and female mice at both 9 and 12 weeks old (data shown only for a 12-week-old male).

Hevin and its close homologue SPARC share 53% identity overall and 62% identity over their follistatin-like and calcium-binding domains, suggesting functional conservation of this region [START_REF] Girard | Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC[END_REF]. As shown on film autoradiography, SPARC mRNA showed a radically different distribution from that of hevin mRNA in mouse brains (compare Fig. 1b and1a). SPARC mRNA was expressed at low levels, with little regional differences except in claustrum, vertical diagonal band, lateral globus pallidus, hypothalamus and substantia nigra pars reticulata, which showed high intensity levels.

Phenotypes of hevin mRNA-expressing cells in the adult mouse brain

Enrichment of hevin signal in specific brain structures suggests that different cellular populations may express hevin. To precisely identify the chemical phenotype of hevinexpressing cells, we used a DIG-labeled probe directed against hevin mRNA and fluoresceinlabeled probes to detect mRNA encoding markers of astrocytes and various types of neurons (Table 2).

Astrocytes

We used the glutamate/aspartate transporter (GLAST), also known as excitatory amino acid transporter 1, a specific marker of astrocytes [START_REF] Cahoy | A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function[END_REF]. We observed an almost complete co-localization of hevin with GLAST mRNA signals (arrowheads in Fig. 2). Accordingly, astrocytes in almost every brain region expressed hevin, as illustrated for cortex (Fig. 2a), striatum (Fig. 2b), lateral septum (Fig. 2c), hippocampus (Fig. 2d), hypothalamus (Fig. 2e) and cerebellum (Fig. 2f). Surprisingly, in two brain areas, the septohippocampal nucleus and the dorsal part of the lateral septum, both signals were detected but did not colocalize (Fig. 2c). Hevin astrocytic expression across the entire brain is listed in Table 4.

In almost all brain regions, hevin was also expressed in a large portion of GLASTnegative cells (arrows in Fig. 2a-d), which is consistent with its neuronal expression [START_REF] Kucukdereli | Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC[END_REF]Lively and Brown 2008c;[START_REF] Lively | SC1/hevin and reactive gliosis after transient ischemic stroke in young and aged rats[END_REF][START_REF] Lloyd-Burton | SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS[END_REF][START_REF] Mckinnon | Extracellular matrix-associated protein Sc1 is not essential for mouse development[END_REF][START_REF] Mendis | SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain[END_REF][START_REF] Risher | Astrocytes refine cortical connectivity at dendritic spines[END_REF]. Qualitatively, the staining in these GLAST-negative cells was different, i.e. a larger somatic staining surrounding the nucleus.

Neurons display a wide diversity of neurotransmitter content, projections, biophysical properties and functional roles and can be divided into excitatory glutamatergic neurons, inhibitory GABAergic neurons, and modulatory monoaminergic and cholinergic neurons. The regional distribution of these hevin-positive and GLAST-negative cells in several cortical layers, specific thalamic nuclei and other subcortical areas suggested that hevin could be expressed in both glutamatergic and GABAergic neurons. To test this hypothesis, we used markers of glutamatergic and GABAergic neuronal populations.

Glutamatergic neurons

The vast majority of neurons in the CNS are excitatory glutamatergic neurons that can be identified by the expression of the vesicular glutamate transporters VGLUT1 and VGLUT2. VGLUT1 and VGLUT2 transcripts allow the identification of cortical and subcortical glutamatergic neurons, respectively [START_REF] Herzog | The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons[END_REF]. Overall, we identified three cellular populations: neurons that expressed both hevin and VGLUT mRNA (arrowhead in Fig. 3 for VGLUT1 and Fig. 4 for VGLUT2), non-glutamatergic cells that expressed hevin mRNA alone (arrow in Fig. 3 and4), and VGLUT-positive neurons that did not express hevin mRNA (star in Fig. 3 and4).

The proportion of each population varied between brain structures. In the cortex, hevin mRNA, which was distributed on cortical layers I to VI, co-localized with VGLUT1-mRNA across layers II to VI (arrowhead in Fig. 3a). In all layers, a substantial amount of hevin mRNA-expressing cells did not express VGLUT1 mRNA (arrow in Fig. 3a). In the hippocampus, hevin mRNA was found in glutamatergic granule cells of dentate gyrus but was rarely visualized in pyramidal glutamatergic neurons of CA1-3 (Fig. 3b), as also evidenced by the weak signal in CA1-3 observed with ISH experiments (Fig. 1a, Table 3). In the olfactory bulb, hevin mRNA was observed in VGLUT1-positive glutamatergic neurons of mitral cell layer, but not in those of the anterior olfactory nucleus (Fig. 3c). In the cerebellum, hevin mRNA was absent from VGLUT1-positive glutamatergic neurons of the granular cell layer (not shown).

Subcortical areas expressed varying levels of hevin mRNA (Fig. 1a, Table 3). We found that hevin mRNA co-localized with VGLUT2 mRNA in several glutamatergic thalamic nuclei such as anterodorsal thalamus (Fig. 4a1), dorsal lateral geniculate (Fig. 4b1), ventral posteromedial thalamus (Fig. 4b2), subthalamus, and zona incerta (not shown). Hevin is also found in midbrain nuclei such as the dorsal part of periaqueductal gray (Fig. 4c1), red nucleus (Fig. 4c3), interpeduncular nucleus, and prerubral field (not shown), and nuclei in the pons such as the vestibular nucleus (Fig. 4d). Despite the clear co-localization of hevin and VGLUT2 in these nuclei, hevin was absent from VGLUT2-positive neurons in many other brain regions, e.g., paraventricular thalamus (Fig. 4a2), ventral anterior thalamus (Fig. 4a3), and ventral part of periaqueductal gray (Fig. 4c2). In the hypothalamus, hevin mRNA was only expressed in astrocytes, except in the lateral mammillary nucleus, where hevin colocalized with VGLUT2-positive neurons (not shown). In regions where hevin was expressed only in some glutamatergic neurons, dual labeled cells did not delineate any neuroanatomical boundary, as they were randomly intermingled with hevin-negative glutamatergic neurons. See Table 4 for a summary of the co-localization between hevin and VGLUT1 and VGLUT2 mRNA across the mouse brain.

GABAergic neurons

Throughout the brain, many cells expressing hevin mRNA did not co-localize with GLAST (arrows in Fig. 2a-d), VGLUT1 (arrows in Fig. 3a-c) or VGLUT2 mRNAs (arrows in Fig. 4ad), suggesting that GABAergic inhibitory neurons express hevin mRNA. Double FISH experiments performed to detect hevin and GAD67 (also called GAD1, glutamic acid decarboxylase) mRNAs, a marker of GABAergic neurons, showed an almost complete colocalization in the cortex (Fig. 5a1), reticular thalamus (Fig. 5a2), and lateral globus pallidus (Fig. 5a3). GABAergic neurons, which are the major source of inhibitory input in CNS, display a wide diversity of morphologies, biophysical properties, molecular markers and functional roles [START_REF] Mendez | Assortment of GABAergic plasticity in the cortical interneuron melting pot[END_REF][START_REF] Tepper | Heterogeneity and diversity of striatal GABAergic interneurons[END_REF]. To further characterize the phenotype of hevin-positive GABAergic neurons, we used well-validated molecular markers of these neurons (Table 2).

Based on hevin mRNA strong expression in the mouse globus pallidus and reticular thalamic nucleus (Fig. 1a and5), two parvalbumin-enriched nuclei, we hypothesized that hevin would be present specifically in parvalbumin-expressing projection neurons. In the globus pallidus and reticular thalamic nucleus, parvalbumin neurons project to substantia nigra pars reticulata and various thalamic nuclei, respectively [START_REF] Clemente-Perez | Distinct Thalamic Reticular Cell Types Differentially Modulate Normal and Pathological Cortical Rhythms[END_REF]). In the rest of the brain, the majority of parvalbumin-expressing cells are local interneurons. We found that distribution of parvalbumin mRNA paralleled hevin mRNA in the entire brain (Fig. 6). Hevin and parvalbumin transcripts co-localized in almost all brain regions studied, including cortex (Fig. 6a), striatum (Fig. 6b), reticular thalamus (Fig. 6c1), lateral globus pallidus (Fig. 6c2), hippocampus (Fig. 6d), red nucleus (Fig. 6e1), and substantia nigra pars reticulata (Fig. 6e2). Surprisingly, in the cerebellum, hevin was absent from parvalbuminpositive Purkinje cells and interneurons of the molecular layer (star in Fig. 7). Using cell typespecific mRNA ribosomal purification, we confirmed the lack of hevin mRNA expression in parvalbumin-positive neurons of the cerebellum (Supplementary Fig. 1). In contrast, qRT-PCR revealed enrichment in hevin mRNA in parvalbumin-positive neurons of dorsal striatum and hippocampus (Supplementary Fig. 2). Likewise hevin mRNA was enriched after ribosomal purification in astrocytes of hippocampus (Fig. S2). These results suggest that hevin mRNA is being translated into protein in both parvalbumin neurons and astrocytes.

In addition to parvalbumin, we used four common molecular markers of GABAergic neurons: somatostatin, neuropeptide Y, cholecystokinin, and calbindin, which segregate from parvalbumin-positive neurons, in particular in the cortex and hippocampus [START_REF] Zeisel | Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq[END_REF]. The vast majority of somatostatin-positive neurons were sparsely distributed in cortex, hippocampus, striatum, lateral septum, basal nucleus, dorsal portion of reticular nucleus, periventricular hypothalamic areas, lateral geniculate nucleus, and few nuclei in brainstem, consistent with the reported distribution [START_REF] Johansson | Molecular cloning of SC1: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/BM40/SPARC[END_REF]. Almost all somatostatinexpressing neurons were negative for hevin mRNA, illustrated in the cortex (Fig. 8a1), striatum (Fig. 8b1), and ventral reticular thalamus (Fig. 8e1). However, hevin mRNA was found in some somatostatin-positive neurons in the lateral septum (Fig. 8c1), reticular thalamus (Fig. 8d1), parabrachial nucleus, reticulotegmental nucleus of the pons, and the oral part of the pontine reticular nucleus (not shown).

Neuropeptide Y-positive neurons were scattered in cortex, hippocampus, and striatum, and were almost nonexistent in thalamus, hypothalamus, midbrain, cerebellum, and brainstem, in agreement with their known distribution [START_REF] Chronwall | The anatomy of neuropeptide-Y-containing neurons in rat brain[END_REF]. Hevin mRNA was found in few neuropeptide Y-positive neurons: in layer II to VI of the cortex (Fig. 8a2), in the dentate gyrus (Fig. 8f2), CA3 region of hippocampus, basolateral amygdala, and medial amygdaloid nucleus (not shown).

Cholecystokinin-positive neurons were mostly found in cortex, amygdala, olfactory bulb, hippocampus, several thalamic nuclei, geniculate nucleus and substantia nigra compacta, and were almost absent in striatum, cerebellum and brainstem, as previously described [START_REF] Savasta | Cellular and regional distribution of the glutamate transporter GLAST in the CNS of rats: nonradioactive in situ hybridization and comparative immunocytochemistry[END_REF]. Hevin mRNA was expressed only in cholecystokinin-positive neurons in the mitral cell layer of the olfactory bulb, anterior olfactory nucleus, and laterodorsal thalamic nucleus (not shown). No co-localization was observed in the hippocampus (Fig. 8f3), Edinger-Westphal nucleus (Fig. 8i3), cortex, nuclei of the basal ganglia complex, and amygdala (not shown).

Calbindin-positive neurons were distributed throughout the brain and found in olfactory bulb, cortex, hilus of dentate gyrus, lateral and medial septum, basal nucleus, thalamic and hypothalamic nuclei, midbrain and hindbrain nuclei. This expression pattern is consistent with published reports [START_REF] Celio | Calbindin D-28k and parvalbumin in the rat nervous system[END_REF]). In all these regions, hevin mRNA was only present in calbindin-expressing neurons in the posterior thalamic nuclear group (Fig. 8h4), vestibular nucleus (Fig. 8j4), mesencephalic trigeminal nucleus (Fig. 8k4), reticulotegmental nucleus of the pons (Fig. 8l4) and principal sensory trigeminal nucleus (not shown). Hevin mRNA was also found in some calbindin interneurons of CA3 and CA1 layers of hippocampus (Fig. 8f4), inferior colliculus, and anterior pretectal nucleus (Fig. 8g4), and mostly absent in calbindin interneurons of the cortex (Fig. 8a4), olfactory bulb, striatum and all other thalamic, hypothalamic, midbrain and hindbrain nuclei (not shown). See Table 4 for a summary of the co-localizations between hevin mRNA and each marker of GABAergic neuron subtype.

Cholinergic neurons

In situ hybridization revealed that hevin was strongly expressed in the brainstem, in particular in motor nuclei (Fig. 1a). These nuclei are composed of large cholinergic neurons identifiable with the marker choline acetyltransferase (ChAT). We found that ChAT-positive neurons were distributed mostly in subcortical structures in the basal forebrain, midbrain and brainstem, in agreement with previous publications [START_REF] Armstrong | Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase[END_REF]. Hevin mRNA co-localized with ChAT in all cholinergic neurons of motor nuclei III (Fig. 9C), IV, V (Fig. 9d), VI, VII (Fig. 9f) and XII (Fig. 9g), but not in cholinergic neurons of motor nuclei X and nucleus ambiguus (Fig. 9g). In contrast, in the midbrain and forebrain, hevin was absent from all cholinergic neurons, including the lateral tegmental nucleus (Fig. 9e), pedunculopontine tegmental nucleus and basal forebrain (Fig. 9a1,b), which comprises the nucleus of the diagonal band, the magnocellular preoptic nucleus, the substantia innominata and the globus pallidus. Cholinergic interneurons in the striatum were also negative for hevin (Fig. 9a2).

Monoaminergic neurons

The regional distribution of hevin mRNA showed that it was not enriched in monoaminergic nuclei, i.e., substantia nigra, ventral tegmental area, raphe nucleus and locus coeruleus (Fig. 1). We thus hypothesized that hevin was absent from monoaminergic neurons. We used tyrosine hydroxylase as a specific marker of catecholaminergic neurons and tryptophane hydroxylase type 2 as a specific marker of serotoninergic neurons. No co-localization was observed between hevin and tyrosine hydroxylase in the substantia nigra pars compacta, ventral tegmental area (star in Fig. 10a), and locus coeruleus (star in Fig. 10b), demonstrating that hevin mRNA is absent from dopaminergic and noradrenergic neurons. Likewise, in raphe nuclei, hevin mRNA did not co-localize with tryptophane hydroxylase 2 (star in Fig. 10c), showing that hevin is also absent from serotoninergic neurons.

Phenotypes of hevin mRNA-expressing cells in adult human prefrontal cortex and caudate nucleus

In order to determine if the specific cellular profile of hevin mRNA expression was conserved in the human brain, we performed double FISH on post-mortem prefrontal cortical and caudate samples using GLAST, GAD67, PV, and VGLUT1, as markers for astrocytes, GABAergic, parvalbumin, and glutamatergic neurons, respectively. A similar pattern was observed between the different subjects. In the prefrontal cortex, hevin mRNA was present in most astrocytes (arrowhead in Fig. 11a), GABAergic neurons (arrowhead in Fig. 11b), and more specifically in parvalbumin neurons (arrowhead in Fig 11c). In the human cortex, hevin mRNA was also expressed in glutamatergic neurons (arrowhead in Fig 11d). A number of VGLUT1-positive neurons did not express hevin (star, Fig. 11d), showing that hevin expression is not specific to glutamatergic neurons in the human prefrontal cortex. Interestingly, hevin was differentially distributed in cortex (Fig. 11e), as observed in the mouse. In the caudate nucleus, hevin mRNA was expressed in astrocytes (arrowhead in Fig 12a ) and parvalbumin interneurons (arrowhead in Fig 12b).

DISCUSSION

In contrast to other matricellular proteins, hevin is strongly expressed in the adult brain, both in rodents [START_REF] Hambrock | SC1/hevin. An extracellular calcium-modulated protein that binds collagen I[END_REF][START_REF] Johansson | Molecular cloning of SC1: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/BM40/SPARC[END_REF][START_REF] Mckinnon | Extracellular matrix-associated protein Sc1 is not essential for mouse development[END_REF][START_REF] Soderling | Cloning and expression of murine SC1, a gene product homologous to SPARC[END_REF]) and humans [START_REF] Girard | Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC[END_REF]. The present study provides the first detailed description of hevin distribution in the brain and its expression in phenotypically identified neuron subtypes (summarized in Fig. 13). We did not identify any brain region that did not express hevin. This extensive expression of hevin throughout all brain regions is explained by its ubiquitous expression in astrocytes. Overall, our results reveal that hevin cellular phenotype in the brain is highly heterogeneous. Depending on the brain structure, hevin will be primarily present in astrocytes, parvalbumin neurons, other GABAergic inhibitory neuron subtypes, glutamatergic neurons, cholinergic motor neurons, or a combination of these cell types. Importantly, we show that hevin cellular expression profile is similar in the prefrontal cortex and caudate nucleus between mouse and human, suggesting a conserved cellular specificity between the two species. Whether this observation can be extended to other brain areas remains to be established in future studies. Furthermore, despite similarities in mRNA expression, hevin translation might also be differentially regulated between human and mouse, leading to differences in protein level. Our regional distribution study in mice reveals a strong expression for hevin in the cortex, hippocampus, basal ganglia complex, diverse thalamic nuclei and in cholinergic motor neurons in the brainstem. The discrete and specific distribution of hevin mRNA suggests that hevin might be involved in the synaptic plasticity underlying hippocampal-dependent processes such as learning and memory, and cortico-basal ganglia-thalamo-cortical loop processes such as goal-directed behaviors, decision-making and fine motor control.

Hevin neuronal expression has been overlooked in previous studies, overshadowed by its expression in almost all astrocytes, which represent nearly half of brain cells (Herculano-Houzel 2014; [START_REF] Markiewicz | The role of astrocytes in the physiology and pathology of the central nervous system[END_REF][START_REF] Nedergaard | New roles for astrocytes: redefining the functional architecture of the brain[END_REF]. Transcriptome studies using fluorescence-activated cell sorting or bacterial artificial chromosome-translating ribosome affinity purification analysis missed the discrete but strong neuronal expression profile of hevin [START_REF] Cahoy | A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function[END_REF][START_REF] Morel | Molecular and Functional Properties of Regional Astrocytes in the Adult Brain[END_REF]). In addition to its widespread expression in astrocytes, we show here that hevin mRNA is remarkably and consistently observed in one GABAergic neuronal subtype, parvalbumin-expressing projection neurons and parvalbumin-expressing local interneurons. As a result, in the mouse brain, hevin mRNA is robustly found in the reticular thalamus and lateral globus pallidus, and in the substantia nigra pars reticulata, regions of high density of long-range projection neurons, and local inhibitory interneurons, respectively. Hevin was also found to be expressed in a restricted number of other subpopulations of GABAergic neurons expressing calbindin, somatostatin, neuropeptide Y and cholecystokinin mRNA, compared to parvalbumin-expressing neurons.

Additionally, hevin mRNA was abundantly found in subgroups of glutamatergic neurons in cortical and subcortical structures, as well as cholinergic neurons of brainstem motor nuclei. A recent study showed that, in humans, hevin protein is expressed in large neurons of the IIIrd, Vth, VIth, VIIth, IXth, Xth, XIth, XIIth motor nuclei in the brainstem and spinal cord [START_REF] Hashimoto | SPARCL1-containing neurons in the human brainstem and sensory ganglion[END_REF]. This corroborates our observation of strong hevin expression in cholinergic neurons of motor nuclei in mouse, except in motor nucleus X and nucleus ambiguus, where hevin was only expressed in astrocytes.

In order to evaluate the singularity of hevin mRNA distribution compared to other matricellular proteins, we determined the distribution of SPARC mRNA. Our study shows radically different distributions for SPARC and hevin mRNAs. SPARC specific expression in glial cell types [START_REF] Mendis | SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain[END_REF][START_REF] Vincent | SPARC is expressed by macroglia and microglia in the developing and mature nervous system[END_REF]) matches the regional distribution of the astrocyte marker GLAST in the rodent brain, in accordance with higher density of astrocytes in the lateral septum, medial preoptic area, and hypothalamus [START_REF] Cui | Blunted mGluR Activation Disinhibits Striatopallidal Transmission in Parkinsonian Mice[END_REF][START_REF] Emsley | Astroglial heterogeneity closely reflects the neuronal-defined anatomy of the adult murine CNS[END_REF][START_REF] Savasta | Cellular and regional distribution of the glutamate transporter GLAST in the CNS of rats: nonradioactive in situ hybridization and comparative immunocytochemistry[END_REF]. Expression of both hevin and SPARC in astrocytes and the absence of obvious phenotypes in SPARC or hevin knock-out mice have suggested that the two proteins might compensate for each other [START_REF] Brekken | SPARC, a matricellular protein: at the crossroads of cell-matrix[END_REF]. However, their opposite effects on synaptogenesis [START_REF] Jones | Astrocytes control glutamate receptor levels at developing synapses through SPARC-beta-integrin interactions[END_REF][START_REF] Kucukdereli | Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC[END_REF], and on inflammatory processes [START_REF] Blakely | Astrocyte matricellular proteins that control excitatory synaptogenesis are regulated by inflammatory cytokines and correlate with paralysis severity during experimental autoimmune encephalomyelitis[END_REF], and as shown here, their strikingly different regional and cellular brain distribution, suggest a peculiar role for hevin in adult CNS.

Implications for the role of hevin in the adult brain

The mRNA expression profile determined here is coherent with the distribution of hevin protein previously defined in both the mouse [START_REF] Kucukdereli | Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC[END_REF]Lively and Brown 2008c;[START_REF] Lively | SC1/hevin and reactive gliosis after transient ischemic stroke in young and aged rats[END_REF][START_REF] Lloyd-Burton | SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS[END_REF][START_REF] Mckinnon | Extracellular matrix-associated protein Sc1 is not essential for mouse development[END_REF][START_REF] Mendis | SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain[END_REF][START_REF] Risher | Astrocytes refine cortical connectivity at dendritic spines[END_REF]) and the human [START_REF] Hashimoto | SPARCL1-containing neurons in the human brainstem and sensory ganglion[END_REF] brain. Additionally, our study reveals the phenotypic heterogeneity of the cells expressing hevin mRNA in both species. Hevin was previously detected in GFAP-positive astrocytes and in small subsets of NeuN-positive neurons in mouse hippocampus and cortex [START_REF] Lloyd-Burton | SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS[END_REF][START_REF] Risher | Astrocytes refine cortical connectivity at dendritic spines[END_REF], suggesting that its mRNA is translated in at least some cell-types and brain regions. To directly test this hypothesis, we used cell-type-specific isolation of mRNAs bound to tagged-ribosomes in mice [START_REF] Ingolia | Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling[END_REF]) to challenge whether hevin was actively translated in parvalbumin neurons and astrocytes in mice, two cell types with consistent hevin mRNA expression. These experiments confirmed that hevin mRNA is actively translated in astrocytes and demonstrated its translation in parvalbumin interneurons of hippocampus and striatum, reinforcing the significance of our study and suggesting that the protein hevin may play a role in these cells in the adult brain. Hevin in astrocytes was shown to be required for synaptic stabilization of excitatory synapses, as well as for synaptic refinement during development [START_REF] Risher | Astrocytes refine cortical connectivity at dendritic spines[END_REF][START_REF] Singh | Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1alpha and NL1 via Hevin[END_REF], two processes implicated in synaptic plasticity. Accordingly, lack of hevin prevents ocular dominance plasticity, which relies on synaptic rearrangement in the visual cortex [START_REF] Singh | Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1alpha and NL1 via Hevin[END_REF]. Although the role of astrocytic hevin in the adult brain is unclear, neuroanatomical studies show that hevin is ideally localized in these cells to modulate adult synaptic plasticity, as in the developing brain [START_REF] Singh | Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1alpha and NL1 via Hevin[END_REF]. Indeed, electron microscopy and confocal imaging have localized hevin in perisynaptic astrocytic processes of adult rodent brain [START_REF] Jones | Astrocytes control glutamate receptor levels at developing synapses through SPARC-beta-integrin interactions[END_REF][START_REF] Kucukdereli | Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC[END_REF]Lively and Brown 2008a;[START_REF] Risher | Astrocytes refine cortical connectivity at dendritic spines[END_REF][START_REF] Singh | Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1alpha and NL1 via Hevin[END_REF][START_REF] Weaver | Processing of the matricellular protein hevin in mouse brain is dependent on ADAMTS4[END_REF]. Perisynaptic astrocytic processes are tightly connected with the majority of synapses [START_REF] Ventura | Three-dimensional relationships between hippocampal synapses and astrocytes[END_REF], where they regulate glutamatergic neurotransmission [START_REF] Anderson | Astrocyte glutamate transport: review of properties, regulation, and physiological functions[END_REF], spine maturation [START_REF] Nishida | Direct astrocytic contacts regulate local maturation of dendritic spines[END_REF], and ultimately control synaptic plasticity [START_REF] Panatier | Glia-derived Dserine controls NMDA receptor activity and synaptic memory[END_REF]). More importantly, calcium signals in astrocytic processes closely follow nearby neuronal activity evoked by sensory stimulation in awake mice [START_REF] Shigetomi | Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses[END_REF][START_REF] Stobart | Cortical Circuit Activity Evokes Rapid Astrocyte Calcium Signals on a Similar Timescale to Neurons[END_REF]. Because hevin possess several calcium-binding sites [START_REF] Girard | Modulation of endothelial cell adhesion by hevin, an acidic protein associated with high endothelial venules[END_REF][START_REF] Hambrock | SC1/hevin. An extracellular calcium-modulated protein that binds collagen I[END_REF], it could be engaged in the response to calcium signals in astrocytic processes, hence affecting synaptic plasticity in response to modifications of neuronal activity in the adult brain. In support of a role of hevin in adult synaptic plasticity, hevin's binding partner neuroligin has been implicated in activity-dependent plasticity [START_REF] Chubykin | Activitydependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2[END_REF]). Furthermore, the activity of hevin is controlled by extracellular proteases [START_REF] Weaver | Proteolysis of the matricellular protein hevin by matrix metalloproteinase-3 produces a SPARC-like fragment (SLF) associated with neovasculature in a murine glioma model[END_REF][START_REF] Weaver | Processing of the matricellular protein hevin in mouse brain is dependent on ADAMTS4[END_REF], which are released upon neuronal activity and have been implicated in hippocampal long-term potentiation [START_REF] Huntley | Synaptic circuit remodelling by matrix metalloproteinases in health and disease[END_REF].

At first sight, it seems difficult to find a common denominator among the variety of neuron types expressing hevin. Interestingly, hevin is consistently absent from all monoaminergic neurons and forebrain cholinergic neurons. These neurons have a functionally different organization in the brain compared to glutamatergic and GABAergic neurons. Aminergic and cholinergic cell bodies are restricted to a small number of nuclei in the hindbrain, midbrain, hypothalamus and basal forebrain but their axons project widely throughout the forebrain. This organization allows these neurotransmitters to modulate the activity of diverse circuits. It thus appears that the expression of hevin in neurons is restricted to populations mediating fast excitatory and inhibitory synaptic transmission ensured by glutamatergic and GABAergic neurons, respectively. As proposed for astrocytes, intracellular calcium signals induced by neuronal activity could regulate hevin and in turn strengthen synaptic plasticity in glutamatergic and GABAergic neurons. In addition, hevin expression in almost all parvalbumin neurons, independently of their morphological differences, extent of axonal projection, differences in connectivity, presence of perineuronal nets and electrophysiological properties [START_REF] Alberi | The calcium-binding protein parvalbumin modulates the firing 1 properties of the reticular thalamic nucleus bursting neurons[END_REF][START_REF] Mendez | Assortment of GABAergic plasticity in the cortical interneuron melting pot[END_REF][START_REF] Rossier | Cortical fast-spiking parvalbumin interneurons enwrapped in the perineuronal net express the metallopeptidases Adamts8, Adamts15 and Neprilysin[END_REF], is in favor for a role in the specific properties of parvalbumin-expressing cells. Excitatory synapses onto parvalbumin neurons are modulated by the post-synaptic cell adhesion molecule neuroligin 3 [START_REF] Polepalli | Modulation of excitation on parvalbumin interneurons by neuroligin-3 regulates the hippocampal network[END_REF], which is a binding partner of hevin [START_REF] Singh | Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1alpha and NL1 via Hevin[END_REF]). Deletion of neuroligin 3 in parvalbumin interneurons of the hippocampus alters network activity by reducing gamma oscillations and sharp wave ripples, which specifically impairs contextual fear extinction [START_REF] Polepalli | Modulation of excitation on parvalbumin interneurons by neuroligin-3 regulates the hippocampal network[END_REF]. Thus, hevin could regulate network activity by modulating excitatory inputs into parvalbumin interneurons. Further studies will be necessary to determine the functional significance of hevin in parvalbumin neurons and other specific neuronal populations, using conditional cell-specific targeting of hevin expression.

Dysregulation of hevin protein levels has been associated with neurodegenerative and psychiatric diseases: hevin is among the most upregulated proteins detected in the cerebrospinal fluid of patients with Alzheimer's, Parkinson's disease, and multiple sclerosis [START_REF] Hammack | Proteomic analysis of multiple sclerosis cerebrospinal fluid[END_REF][START_REF] Yin | Neuronal pentraxin receptor in cerebrospinal fluid as a potential biomarker for neurodegenerative diseases[END_REF]. Conversely, hevin protein levels are downregulated in postmortem prefrontal cortex samples of depressed patients [START_REF] Zhurov | Some co-localization is observed between hevin and neuropeptide Y mRNAs in the cortex and hippocampus (arrowhead, a2, f2). Most neuropeptide Y-positive cells in the cortex, striatum and dentate gyrus do not express hevin (stars, a2, b2, f2). In the dentate gyrus (f3) and the Edinger-Westphal nucleus (i3), no co-localization is observed between hevin-positive cells (arrows) and cholecystokinin-positive neurons (stars). Partial co-localization is observed between calbindin and hevin mRNAs in the hindbrain and brainstem (arrowhead, h4, j4, and l4)[END_REF]. Hevin could contribute to the pathophysiology of these disorders by altering synaptic strength and morphology [START_REF] Bernardinelli | Structural plasticity: mechanisms and contribution to developmental psychiatric disorders[END_REF][START_REF] Braak | Cortico-basal ganglia-cortical circuitry in Parkinson's disease reconsidered[END_REF]. Stress-induced depressive-like behaviors rely on changes in glutamate signaling, spine morphology and synaptic plasticity [START_REF] Christoffel | IkappaB kinase regulates social defeat stress-induced synaptic and behavioral plasticity[END_REF][START_REF] Christoffel | Excitatory transmission at thalamo-striatal synapses mediates susceptibility to social stress[END_REF][START_REF] Vialou | DeltaFosB in brain reward circuits mediates resilience to stress and antidepressant responses[END_REF]. In agreement with a role of hevin in these processes, we previously found that hevin is induced in resilience to stress and is sufficient to induce an antidepressant response in adult mice [START_REF] Vialou | DeltaFosB in brain reward circuits mediates resilience to stress and antidepressant responses[END_REF]. This supports our hypothesis of a major role for hevin in higher cognitive functions in the adult.

Conclusions

Our neuroanatomical study in mouse and human brain distinguishes hevin from other matricellular proteins, and reveals a specific pattern of expression of hevin in subsets of neuronal populations throughout the adult brain. The cellular distribution of hevin is similar between human and mouse brain, suggesting an evolutionary conserved role for hevin in mammal adult CNS function. The function of hevin might depend on the specific cell type in which it is present. Hevin could contribute to adult synaptic plasticity mediated by astrocytes whereas its expression in parvalbumin neurons points to a fundamentally novel role in the regulation of network activity. This characterization of hevin expression profile will help dissect novel functions for this unique matricellular protein in the adult brain.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. In addition, all applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Informed consent

No consent is required for using leftover body material for scientific purposes from medicolegal autopsies. 3 for hevin regional distribution). b. Serial brain sections labeled with oligonucleotide antisense radiolabeled probes specific for SPARC in mouse brain. SPARC mRNA is expressed at low levels, but enriched in subcortical structures, in particular in the hypothalamus. See list of abbreviations Fig. 2 Hevin mRNA is found in astrocytes expressing GLAST mRNA in mouse brain. At low magnification, hybridization signal for hevin (red) and GLAST (green) mRNA is observed throughout all regions of the brain, including cortex (a), striatum (b), lateral septum (c), hippocampus (d), hypothalamus (e), and cerebellum (f). At high magnification, photomicrographs show co-localization between hevin and GLAST (arrowhead) in each area.

A substantial number of GLAST-negative cells express hevin mRNA (arrow) in cortex (a), striatum (b), lateral septum (c) and hippocampus (d), while in hypothalamus and cerebellum, hevin is found in astrocytes only (e-f). Hevin mRNA is absent from GLAST-positive astrocytes in the dorsal part of the lateral septum (star, c). Note that astrocytic hevin mRNA staining is somatic and is also present along glial processes in cerebellum (arrowhead, f) and that hevin is absent from progenitor cells in the subgranular layer of dentate gyrus, which express GLAST (star, d). DAPI signal is also presented (blue staining in the nucleus). See list of abbreviations In the hippocampus, hevin is expressed in glutamatergic granular neurons of the dentate gyrus (arrowhead, b4) and in a few glutamatergic pyramidal neurons of CA2 (b2). Hevin mRNA is absent from VGLUT1-positive neurons of CA1-CA3 and hilus of dentate gyrus (star, b1-b3). c. In the olfactory bulb, hevin signal co-localizes with the VGLUT1-positive mitral cells (arrowhead) but not with VGLUT1-positive glutamatergic neurons of the anterior olfactory nucleus and glomerular layer (arrow). In addition to hevin staining in astrocytes identified by fine processes and small soma size, many cells expressing hevin mRNA do not co-localize with VGLUT1 mRNA in the three regions depicted here (arrow). DAPI signal is also presented (blue staining in the nucleus). See list of abbreviations Neurons expressing hevin mRNA in anterodorsal thalamus (a1), lateral posterior thalamus and dorsolateral geniculate (b1), ventral posteromedial thalamus (b2), periaqueductal gray (c1), red nucleus (c3) and vestibular nucleus (d1) are all VGLUT2positive glutamatergic neurons (arrowhead). In other thalamic regions, hevin mRNA labeling (arrow) does not co-localize with VGLUT2 neurons (star), e.g., in paraventricular thalamus (a2), reuniens thalamus (a3), and nucleus of Darkschewitsch (c2). Cells expressing hevin mRNA are observed in reticular thalamic nucleus, which is devoid of VGLUT2-positive neurons (a). DAPI signal is also presented (blue staining in the nucleus). See list of abbreviations Fig. 5 Characterization of hevin mRNA expression in GABAergic neurons expressing GAD67 mRNA in mouse brain. Hybridization signal for hevin (red) and GAD67 (green) mRNA is observed in the cortex (a1), reticular thalamus (a2), and lateral part of the globus pallidus (a3). High magnification images show an almost complete co-localization between hevin and GAD67 mRNAs (arrowhead, a1-a3). A small number of GAD67 neurons do not express hevin (star, a1, a2). In the cortex, a majority of hevin-positive cells do not co-localize with GAD67 mRNA signal (arrow, a1-a3). DAPI signal is also presented (blue staining in the nucleus). See list of abbreviations

FIGURESFig. 1

 1 FIGURES

Fig. 3

 3 Fig. 3 Characterization of hevin mRNA expression in VGLUT1-positive glutamatergic neurons in mouse brain. Hybridization signal for hevin (red) and VGLUT1 (green) mRNA is observed in cortex (a), hippocampus (b) and olfactory bulb (c). a. High magnification images show only partial co-localization (arrowhead) in VGLUT1-positive glutamatergic neurons scattered throughout cortical layers II to VI. Neurons expressing only VGLUT1 mRNA (star) are also present. b.In the hippocampus, hevin is expressed in glutamatergic granular neurons of the dentate gyrus (arrowhead, b4) and in a few glutamatergic pyramidal neurons of CA2 (b2). Hevin mRNA is absent from VGLUT1-positive neurons of CA1-CA3 and hilus of dentate gyrus (star, b1-b3). c. In the olfactory bulb, hevin signal co-localizes with the VGLUT1-positive mitral cells (arrowhead) but not with VGLUT1-positive glutamatergic neurons of the anterior olfactory nucleus and glomerular layer (arrow). In addition to hevin staining in astrocytes identified by fine processes and small soma size, many cells expressing hevin mRNA do not co-localize with VGLUT1 mRNA in the three regions depicted here (arrow). DAPI signal is also presented (blue staining in the nucleus). See list of abbreviations

Fig. 4

 4 Fig.4Characterization of hevin mRNA expression in VGLUT2-positive glutamatergic neurons in mouse brain. Hybridization signal for hevin (red) and VGLUT2 (green) mRNA is observed in the thalamus (a-b) as well as in various nuclei of the hindbrain (c) and brainstem (d). Neurons expressing hevin mRNA in anterodorsal thalamus (a1), lateral posterior thalamus and dorsolateral geniculate (b1), ventral posteromedial thalamus (b2), periaqueductal gray (c1), red nucleus (c3) and vestibular nucleus (d1) are all VGLUT2positive glutamatergic neurons (arrowhead). In other thalamic regions, hevin mRNA labeling (arrow) does not co-localize with VGLUT2 neurons (star), e.g., in paraventricular thalamus (a2), reuniens thalamus (a3), and nucleus of Darkschewitsch (c2). Cells expressing hevin mRNA are observed in reticular thalamic nucleus, which is devoid of VGLUT2-positive neurons (a). DAPI signal is also presented (blue staining in the nucleus). See list of abbreviations

Fig. 6 Fig. 7 Fig. 8

 678 Fig.6Hevin mRNA is expressed in parvalbumin neurons and neurons in mouse brain. Hybridization signal for hevin (red) and parvalbumin (green) mRNA is observed throughout the brain, including cortex (a), striatum (b), reticular thalamus (c1), lateral globus pallidus (c2), hippocampus (d), and several midbrain nuclei (e). High magnification images show a complete co-localization between hevin and parvalbumin mRNAs in these regions (arrowhead, a-e). All parvalbumin-positive interneurons express hevin mRNA in layers II to VI of cortex (a), striatum (b), all hippocampal subfields, substantia nigra (e2), and red nucleus (e1). All parvalbumin-positive projection neurons in reticular thalamic nucleus (c1) and

Table 1

 1 Demographic characteristics, post-mortem delay (PMD), cause of death and brain pH of subjects included in the study

	Subject Gender Age PMD Cause of death	Brain pH
	1	Male	82	3	Natural/cardiorespiratory failure ND
	2	Female	57	11	Natural/hemorrhage	ND
	3	Male	44	5	Natural/cardiorespiratory failure 6.2
	4	Male	38	3	Natural/cardiorespiratory failure 6.2
	5	Male	21	3	Accident/fall from height	6.34
	6	Male	19	7	Accident/fall from height	6.7
	7	Male	31	11	Homicide/knife wounds	6.53
	8	Male	49	13	Accident/traffic	6.4
	9	Male	71	8	Accident/fall from height	5.92
	10	Male	21	35	Natural/cardiorespiratory failure ND
	11	Male	53	40	Homicide/gun wounds	ND
	12	Male	35	41	Accident/drowning	ND

Table 2

 2 Oligonucleotides and cRNA probes used in present study. Gene symbol, common gene name, NCBI accession number, size and position are indicated GAD67 glutamic acid decarboxylase 67, GLAST glutamate/aspartate transporter, SPARC secreted protein acidic and rich in cysteine, VGLUT vesicular glutamate transporter type

	Probe	Gene symbol	Common gene name	NCBI accession #	Size (bp)	5' Position
	Oligonucleotide	Sparcl1	Hevin (mouse)	NM_010097	35	756, 1081, 1734
	(ISH mouse)	Sparc	Sparc (mouse)	NM_009242	35	175, 753, 1300
	cRNA	Sparcl1	Hevin (mouse)	NM_010097	1299	126
	(FISH mouse)	Slc1a3	GLAST (mouse)	NM_148938	658	1350
		Slc17a7	VGLUT1 (rat)	NM_053859	508	115
		Slc17a6	VGLUT2 (mouse)	NM_080853	930	2315
		Gad1	GAD67 (rat)	NM_017007	539	195
		Pvalb	Parvalbumin (mouse)	NM_013645	517	74
		Calb1	Calbindin1 (mouse)	NM_009788	1122	268
		Cck	Cholecystokinin (mouse)	NM_031161	483	251
		Npy	Neuropeptide Y (mouse)	NM_023456	453	13
		Sst	Somatostatin (mouse)	NM_009215	401	143
		Chat	Choline acetyltransferase (rat)	NM_001170593 540	524
		Th	Tyrosine hydroxylase (rat)	NM_012740	998	456
		Tph	Tryptophane hydroxylase (mouse)	NM_009414	986	268
	cRNA	Sparcl1	Hevin (human)	NM_001128310 908	1611
	(FISH human)	Slc1a3	GLAST (human)	NM_004172.4	958	3113
		Slc17a7	VGLUT1 (human)	NM_020309	972	2082
		Gad1	GAD67 (human)	NM_000817	896	365
		Pvalb	Parvalbumin (human)	NM_002854.2	415	1

Table 3

 3 Relative densities of hevin mRNA expression in the mouse brain by ISH. Qualitative estimates of hevin mRNA expression were made based on the signal strength of the hybridization signal in a given structure. Rating scale for hevin mRNA labeling intensity: very high (++++), high (+++), moderate (++), low (+) Calb calbindin, Cck cholecystokinin, GLAST glutamate/aspartate transporter, NPy neuropeptide Y, PV parvalbumin, Sst somatostatin, VGLUT vesicular glutamate transporter

	Brain region	Intensity	Brain region	Intensity
	Olfactory bulb		Thalamus-Hypothalamus (continued)
	Anterior olfactory nucleus	+	Interanteromedial thalamic nucleus	++
	Glomerular layer	+++	Lateral habenula	++++
	Mitral cell layer	++	Laterodorsal thalamic nucleus	++++
	Cortex		Mediodorsal thalamic nucleus	++
	Layer I	++	Paraventricular thalamic nucleus	+++
	Layer II-III	+++	Subthalamic nucleus	++++
	Layer IV	++	Reticular thalamic nucleus	++++
	Layer V	++	Ventral posteromedial thalamic nucleus	++
	Layer VI	++	Zona incerta	++++
	Hippocampus		Midbrain
	Granular layer of dentate gyrus	++++	Anterior pretectal nucleus	+++
	Molecular layer of dentate gyrus	+	Dorsal lateral geniculate nucleus	++++
	Polymorphic layer/Hilus	++	Interpeduncular nucleus	++
	CA3 Pyramidal cell layer	+	Nucleus of Darkschewitsch	+++
	CA3 Stratum oriens	+	Oculomotor nucleus (III)	++++
	CA3 Stratum radiatum	+	Periaqueductal gray	++
	CA1 Pyramidal cell layer	+	Prerubral field	++
	CA1 Stratum oriens	+	Red nucleus	++++
	CA1 Stratum radiatum	+	Substantia nigra pars reticulata	+++
	CA2 Pyramidal cell layer	+++	Superior colliculus	++++
	Presubiculum	++++	Cerebellum
	Subiculum	++	Bergmann glial cells	++++
	Septohippocampal nucleus	+	Molecular layer	+
	Septum		Granule cell layer	+
	Lateral septum	++	Deep cerebellar nuclei	++
	Medial septum	+++	Pons
	Basal ganglia		Abducens nucleus (VI)	++++
	Caudate putamen	+	Facial nucleus (VII)	++++
	Lateral globus pallidus	++++	Gigantocellular reticular nucleus	+++
	Medial globus pallidus	++++	Lateral parabrachial nucleus	+++
	Ventral pallidum	+++	Mesencephalic trigeminal nucleus	++
	Amygdala		Motor trigeminal nucleus (V)	++++
	Basolateral amygdala	++	Pontine reticular nucleus, oral part	++++
	Central amygdala	++	Principal sensory trigeminal nucleus	++++
	Medial amygdaloid nucleus	++	Reticulotegmental nucleus of the pons	++++
	Thalamus-Hypothalamus		Spinal trigeminal nucleus	++++
	Anterodorsal thalamic nucleus	++++	Superior olive nucleus	++++
	Anteroventral thalamic nucleus	++	Tegmental nucleus	++++
	Hypothalamus	+++	Vestibular nucleus	++++

Acknowledgements

This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), and Sorbonne Université, and by grants from the Brain & Behavior Research Foundation (NARSAD Young Investigator Award to VV, #17566), FP7 Marie Curie Actions Career Integration Grant (FP7-PEOPLE-2013-CIG #618807 to VV), Promouvoir l'Excellence de la Recherche à Sorbonne Université (PER-SU 2014 to VV), Agence Nationale de la Recherche (ANR JCJC 2015 Hevinsynapse to VV), the Basque Government (IT616/13 to JJM), Fondation pour la Recherche Médicale (DEQ20160334919 to EV), Fundación Vital (2018 to AME) and the European Foundation for Alcohol Research (EA 18 19 to LFC). The authors thank Etienne Audinat for the PV-Cre mice and Glenn Dallerac for the GFAP-CreER T2 . EP was a recipient of Marie Curie Intra-European Fellowship (IEF327648). LC has benefited from support by the Labex EpiGenMed (Investissements d'avenir #ANR-10-LABX-12-01). We thank Catalina Betancur for helpful discussions and comments on the manuscript; the staff members of the Basque Institute of Legal Medicine for processing the post-mortem human brain samples; Stéphane Fouquet, David Godefroy, and Marie-Laure Niepon of the Imaging Facility at Institut de la Vision; Annick Prigeant of the Histology Facility at Institut du Cerveau et de la Moelle; and Chooyoung Baek and Audrey Pondaven for their help with the FISH experiments.

Table 4 Identity of hevin-expressing cells determined by double FISH in the mouse brain. For each marker, (+) indicates that all cells express hevin mRNA, (+/-) indicates that only some cells express hevin mRNA, while some cells do not, and (-) indicates a lack of co-localization with hevin mRNA. Cells are left blank in the absence of the cellular marker. Monoaminergic nuclei are not indicated