Gabriel Lepousez

Zsolt Csaba

Véronique Bernard

Catherine Loudes

Catherine Videau

Joelle Lacombe

Jacques Epelbaum

Cécile Viollet
email: cecile.viollet@inserm.fr

Paul E Sawchenko

Somatostatin interneurons delineate the inner part of the external plexiform layer in the mouse main olfactory bulb

Keywords: SRIF, neuropeptide, GABA, Van Gehuchten, EPL, reciprocal synapse SRIF, neuropeptide, GABA, Van Gehuchten, EPL, reciprocal synapse

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Introduction

The olfactory bulb (OB), the first central relay in olfactory processing, is a good model for deciphering the function of interneurons. Within its well-defined cytoarchitectural organisation, numerous interneurons contribute to the processing of sensory information, through direct or indirect innervation of olfactory inputs and mitral/tufted cells [START_REF] Shepherd | The Synaptic Organization of the Brain[END_REF][START_REF] Shipley | Functional organization of olfactory system[END_REF]. Periglomerular and granule cells, interneurons which populate the glomerular (GL) and granule cell (GCL) layers respectively, have been extensively studied [START_REF] Shepherd | The Synaptic Organization of the Brain[END_REF].

Directly innervating the principal cells, these cells are classically considered first order neurons.

Remaining interneurons are classified as second order interneurons, since they only interact with first order neurons [START_REF] Schneider | Laminar distributions of interneurons in the main olfactory bulb of the adult hamster[END_REF].

Morphological analysis of interneurons, combined with descriptions of their neurochemical content for calcium-binding proteins or neuropeptides, has allowed for a better understanding of their diversity, notably in the telencephalon (for a review, see [START_REF] Ascoli | Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex[END_REF][START_REF] Klausberger | Brain-state-and cell-type-specific firing of hippocampal interneurons in vivo[END_REF][START_REF] Markram | Interneurons of the neocortical inhibitory system[END_REF]. Among neuropeptides, somatostatin (SRIF) is extensively expressed in a large subset of GABAergic interneurons throughout the telencephalon which specifically innervate principal cells in most brain regions (reviewed in [START_REF] Viollet | Somatostatinergic systems in brain: Networks and functions[END_REF]. Through the activation of five specific G protein-coupled receptors, sst1 to 5, somatostatin is involved in various central functions from classical neuroendocrine control to cognitive functions. In the olfactory bulb, SRIF-ir interneurons have been described in several mammalian species [START_REF] Davis | Localization of methionine-enkephalin, substance P, and immunoreactivities in the main olfactory bulb of the hamster[END_REF][START_REF] Gutièrrez-Mecinas | Characterization of somatostatin-and cholecystokinin-immunoreactive periglomerular cells in the rat olfactory bulb[END_REF][START_REF] Matsutani | Distribution of neuropeptidelike immunoreactivities in the guinea pig olfactory bulb[END_REF][START_REF] Ohm | Somatostatin-14-like immunoreactive neurons and fibres in the human olfactory bulb[END_REF][START_REF] Scott | Short axon cells of the rat olfactory bulb display 34 NADPH-diaphorase activity, neuropeptide Y-like immunoreactivity, and somatostatinlike immunoreactivity[END_REF][START_REF] Seroogy | Somatostatin-like immunoreactivity in rat main olfactory bulb: extent of coexistence with neuropeptide Y-, tyrosine hydroxylase-and vitamin D-dependent calcium binding protein-like immunoreactivities[END_REF]. In these species, SRIF is systematically found in deep short-axon cells of the GCL. It has also been reported in few rare EPL interneurons in guinea pigs, periglomerular cells in rats and, to a lesser extent, periglomerular cells in humans.

Surprisingly the presence of SRIF-ir interneurons in the mouse olfactory bulb had not yet been described. Since mice are becoming increasingly important as models of nervous system function and since neuroanatomical divergence between mouse and rat olfactory bulb exists (Kosaka andKosaka, 2007a, Kosaka andKosaka 2007b), we undertook to map the somatostatinergic system in the mouse main olfactory bulb (MOB) using light microscopic immunohistochemical and ultrastructural studies. The results indicate that SRIF interneurons in the iEPL are in a key position to modulate the bulbar synaptic network.

Animals

Two-three month-old male wild-type (n=23) and homozygous SRIF knockout (KO,n=3) littermates were obtained from a somatostatin knockout strain [START_REF] Zeyda | Impairment in motor of somatostatin null mutant mice[END_REF] backcrossed onto a C57Bl/6J genetic background for thirteen generations. Swiss (n=3) and FVB/V (n=3) mice were obtained from Charles Rivers (L'Arbresles, France). Animals were housed and bred in our Research Center animal facilities under a 12h/12h light/dark cycle. All animal procedures complied with French laws regarding animal experimentation (Decree n°87-848, 19 October 1987 and the Ministerial Decree of 19 April 1988) and recommendations of a local ethical committee.

Antibodies characterization

The main characteristics of the primary and secondary antibodies used in the present study are summarized in Table 1.

The calbindin D-28k (CB) antibody #300 specifically stains the 45 Ca-binding site of calbindin D-28K(MW 28 kDa, IEP 4.8) in a two-dimension immunoblot of rabbit cerebellum extract. Using radioimmunoassay, it detects calbindin D-28k in rat brain tissue with a sensitivity of 10 ng/assay and an affinity of 1.6 x 10 -12 M/L [START_REF] Celio | Monoclonal antibodies directed against the calcium binding protein Calbindin D-28k[END_REF]. In mouse olfactory bulb, the predominant labeling of the glomerular layer described herein was also reported using other mouse (Kosaka et al., 2005) and rabbit [START_REF] Parrish-Aungst | Quantitative analysis of neuronal diversity in the mouse olfactory bulb[END_REF] calbindin antibodies.

The calretinin (CR) antibody #AB1550 recognizes a 31kD band in western blot and a spot corresponding to the expected size and isoelectric point for calretinin in a two-dimension immunoblot of rat cerebellum [START_REF] Winsky | Calretinin mRNA and immunoreactivity in the medullary reticular formation of the rat: colocalization with glutamate receptors[END_REF]. The immunohistochemical pattern obtained in rat brain sections is consistent with the one obtained with a well characterized rabbit antiserum and it disappears after preabsorption with purified rat recombinant calretinin [START_REF] Winsky | Calretinin mRNA and immunoreactivity in the medullary reticular formation of the rat: colocalization with glutamate receptors[END_REF]. As previously reported in another study in mouse [START_REF] Parrish-Aungst | Quantitative analysis of neuronal diversity in the mouse olfactory bulb[END_REF], the labeling pattern observed in our study is similar to those observed with other goat (Kosaka and Kosaka, 2007a), mouse [START_REF] Batista-Brito | The distinct temporal origins of olfactory bulb interneuron subtypes[END_REF]Kosaka and Kosaka, 2007a), and rabbit 5 (Kosaka and Kosaka, 2007a;Kosaka et al., 2005) calretinin specific antibodies in the murine MOB.

The parvalbumin (PV) antibody #AB9314 detects a single 12 kDa band in immuno blots of soluble proteins from rat and mouse whole brain and precipitates a single PV band from whole brain homogenates. Specificity tests revealed no cross-reactivity with calmodulin, calbindin-D28k or calretinin (Pr. Ken Baimbridge, Univ. of British Columbia, Vancouver, Canada, personal communication). In the present study, it labels the EPL and IPL/superficial GCL layers, as also observed using other mouse [START_REF] Parrish-Aungst | Quantitative analysis of neuronal diversity in the mouse olfactory bulb[END_REF] and rabbit (Kosaka and Kosaka, 2008) parvalbumin antibodies.

The Tyrosine Hydroxylase (TH) antiserum #AB152 recognizes a single band around 62 kDa in immunoblotted brain tissues, consistent with the size of the Tyrosine Hydroxylase protein (Manufacturer's technical information). Herein, it strongly labels the glomerular layer in the mouse MOB, as previously observed, using other rabbit [START_REF] Batista-Brito | The distinct temporal origins of olfactory bulb interneuron subtypes[END_REF]Kosaka et al., 2005;Kosaka and Kosaka, 2008;[START_REF] Parrish-Aungst | Quantitative analysis of neuronal diversity in the mouse olfactory bulb[END_REF], mouse [START_REF] Panzanelli | GABAergic phenotype of periglomerular cells in the rodent olfactory bulb[END_REF] or sheep (Kosaka and Kosaka, 2007b) Tyrosine Hydroxylase antibodies. This staining pattern is also consistent with the transgene expression patterns obtained either in Tyrosine Hydroxylase-lacZ or Tyrosine Hydroxylase-GFP mice [START_REF] Saino-Saito | Differentiation of the dopaminergic phenotype in the olfactory system of neonatal and adult mice[END_REF]. Furthermore, in an experimental model for olfactory deprivation (Viollet et al., unpublished data), Tyrosine Hydroxylase staining strongly decreased as previously reported in olfactory deprivation studies [START_REF] Baker | Adult naris closure profoundly reduces tyrosine hydroxylase expression in mouse olfactory bulb[END_REF].

The goat somatostatin antibody #D-20 labels a 17 kD human recombinant somatostatin transfected protein by western blotting (Manufacturer's technical information). The sequence of the immunizing peptide used to raise this antibody is unavailable in the manufacturer's information. The sequence given in Table 1 was obtained by mass spectrometry analysis of the blocking peptide (sc-7819 P; Santa Cruz Biotechnology, Santa Cruz, CA) [START_REF] Cox | β-adrenergic receptors are differentially expressed in distinct interneuron subtypes in the rat hippocampus[END_REF]. The staining pattern obtained with this antibody in rat MOB is identical to (data not shown) the one previously reported with another rabbit SRIF antiserum [START_REF] Hwang | Age-related change of somatostatin-immunoreactive neurones in the main olfactory bulb of the rat[END_REF] and SRIF immunoreactivity was totally absent in brain sections processed from SRIF KO mice (Supplemental figure S1).

The specificity of the rabbit somatostatin polyclonal antiserum IS-7 [START_REF] Ciofi | Sexual dimorphism in the organization of the rat hypothalamic infundibular area[END_REF] was tested by preabsorption with increasing concentrations of SRIF28 [1][2][3][4][5][6][7][8][9][10][11][12], SRIF28[1-14] or SRIF28, and all three peptides abolished staining (P. Ciofi, personal communication). The The glutamic acid decarboxylase (GAD) 65/67 antibody #G1563 specifically recognizes a doublet of bands at 65-67 kDa when immunoblotting rat brain extracts. The staining disappears in the presence of GAD65/67 peptide (human, amino acids 579-594 with N-terminally added lysine) (Manufacturer's technical information). An identical staining pattern was reported in the mouse MOB using this antibody (Kosaka and Kosaka, 2007a;Kosaka et al., 2005;Kosaka and Kosaka, 2007b).

The glial fibrillary acidic protein antibody #Z0334 recognizes the native (49 kDa) and proteolytic (40-47 kDa) bands on a western blot of adult mouse brain [START_REF] García-Sevilla | Neurofilament proteins and cAMP pathway in brains of mu-, delta-or kappa-opioid receptor gene knockout mice: effects of chronic morphine administration[END_REF]. The reported distribution of GFAP in mouse olfactory bulb [START_REF] Monnier | Structural and immunohistological modifications in olfactory bulb of the staggerer mutant mouse[END_REF] is identical to the one described herein.

The specificity controls performed by ICC and RIA of the cholecystokinin (CCK) antiserum IS-15/8 show expected cross-reaction with gastrin [START_REF] Ciofi | Distribution of cholecystokinin-like-immunoreactive neurons in the guinea pig forebrain[END_REF]. CCK labeling is mainly found in large somata at the EPL/GL border (presumably in medium-sized tufted cells), as also reported using a mouse monoclonal antibody (Kosaka et al., 2005;Kosaka and Kosaka, 2007b).

Labeling with the neuropeptide Y (NPY) antiserum IS-12 is abolished after preabsorption with increasing concentrations of NPY peptide [START_REF] Ciofi | Expression of neuropeptide Y precursorimmunoreactivity in the hypothalamic dopaminergic tubero-infundibular system during lactation in rodents[END_REF]. Furthermore, the staining is the same with an antibody against the C-terminal part of the NPY precursor (C-PON, [START_REF] Ciofi | Expression of neuropeptide Y precursorimmunoreactivity in the hypothalamic dopaminergic tubero-infundibular system during lactation in rodents[END_REF].

The staining specificity of the vasoactive intestinal peptide (VIP) antiserum IS-21 was previously described [START_REF] Maubert | Co-localization of vasoactive intestinal polypeptide and neuropeptide Y immunoreactivities in the nerve fibers of the rat adrenal gland[END_REF]. VIP antiserum was preadsorbed with homologous (VIP) or heterologous antigens (NPY and C-PON) at various concentrations. Abolition of the VIP staining was observed only with the homologous antigen. The absence of cross-reactivity of the antiserum for several non-related peptides (substance P, SRIF 1-28 , arginine-vasopressin, oxytocin and angiotensin II) was also demonstrated by radioimmunoassay.

The Post-Synaptic Density 95K protein (PSD-95) antibody #MA1-046 detects a 95 kDa protein in rat brain extracts (Kornau et al., 1995 and Manufacturer's instructions). Using this antibody, staining in the OB is the same as the one observed with the #05-494 mouse antibody (Upstate Biotech., Lake Placid, NY, USA) whose specificity was confirmed by the extinction of the 95 kDa signal detected by immunoblotting in brain extracts when using PSD-95 KO mice [START_REF] Rasband | Clustering of neuronal potassium channels is independent of their interaction with PSD-95[END_REF].

In rat CNS, the staining pattern obtained with the guinea-pig vesicular GABA transporter (VGAT) antibody #AB5855 is in agreement with the pattern described using other VGAT antisera [START_REF] Belenky | Heterogeneous expression of gammaaminobutyric acid and gamma-aminobutyric acid-associated receptors and transporters in the rat suprachiasmatic nucleus[END_REF]. This staining is abolished by preadsorption with the immunogen peptide (Manufacturer's instructions). In a recent study in mouse MOB, another rabbit VGAT antiserum (#131002, Synaptic Systems, Göttingen, Germany) and the antibody #AB5855 displayed an identical staining pattern in doubleimmunofluorescence experiments [START_REF] Panzanelli | GABAergic phenotype of periglomerular cells in the rodent olfactory bulb[END_REF]. Antibody #131002 recognizes a doublet of bands migrating at the expected size for VGAT in brain tissue and heterologous expression systems [START_REF] Takamori | Immunoisolation of GABA-specific synaptic vesicles defines a functionally distinct subset of synaptic vesicles[END_REF].

Tissue preparation for light and fluorescent microscopy

Mice were deeply anesthetized following intraperitoneal injection of sodium pentobarbital (180 mg/kg, Ceva Sante Animal, Libourne, France) and transcardially perfused with 0.9% NaCl then 4% paraformaldehyde (PFA) in 0.1M phosphate buffer pH=7.4 (PB). Brains were quickly removed, post-fixed overnight in 4% PFA, incubated in 30% sucrose in phosphate buffered saline (PBS) for 24 hours, then quickly frozen with ice-cold isopentane and stored at -80°C. MOB coronal sections were prepared using a freezing microtome (CM 1325, Leica, Heidelberg, Germany).

MOB somatostatin interneurons distribution and morphological analysis.

Chromogenic somatostatin immunohistochemistry was performed using the standard ABC-DAB method on 40 µm-thick floating serial sections (inter-slice interval: 120 µm). After incubation with 1% H 2 O 2 in PBS, sections were incubated for one hour with 5% normal donkey serum (NDS, Biowest, Nuaillé, France) and 5% bovine serum albumin (BSA, Sigma, St Louis, MO, USA) in PBS containing 0.3% Triton X-100 (PBST). They were then incubated with goat anti-somatostatin antibody in PBST/1% NDS/1% BSA for six days at 4°C, rinsed with PBS, then incubated with biotinylated anti-goat secondary antibody in PBST/1% NDS/1% BSA for 90 1). After rinses, ABC Elite reagent (Vector laboratories, Burlingame, CA, USA) was applied to the sections and revelation was performed with 3-3'-diaminobenzidine (DAB, Sigma; 0.5 mg/ml in PBS/0.02% H 2 O 2 ) for one minute.

Sections were then counterstained with cresyl violet, and mounted for light microscopic observation. Immunohistochemical specificity controls for somatostatin labeling consisted of incubation in absence of the primary anti-somatostatin antibody.

The analysis of somatostatin-ir cell relative distribution in the olfactory bulb was performed on three mice (12 sections each, mean number of analyzed SRIF-ir cells per animal: 1195.9±20.4). Given the relatively low density of SRIF-ir cells in the olfactory bulb, all SRIF-ir somata were counted in each layer of the DAB-processed MOB sections (section thickness, 40 µm) under a 40x objective using a Lumenera Infinity 2 CCD color camera (Lumenera, Ottawa, Canada) linked to the image analysis mapping software (Mercator, Explora Nova, La Rochelle, France).

For camera lucida analysis SRIF-ir cells were randomly sampled from the DAB-processed sections (n=53 cells for iEPL ; n=30 cells for GCL, from three animals) and observed under an Olympus BH2 light microscope (Olympus, Tokyo, Japan). The projected contours of SRIF-ir neurons showing well stained processes were traced using a camera lucida with 40x or 20x immersion planapochromatic objectives (Olympus, Tokyo, Japan) for iEPL and GCL cells respectively (magnification 500x and 250x). Drawings were scanned with a flat bed scanner (resolution: 300 dpi) and transfered to a personal computer. Measurement of the area, area equivalent diameter, perimeter, minor and major diameters of the soma, number of primary dendrites (exit points from the soma) and dendritic branching points were performed from each individual immunoreactive neuron using NIH ImageJ 1.33 (NIH, Bethesda, MD, USA). Sholl crossing analysis was performed using a transparent mask displaying concentric circles (intercircle interval: 10 µm) centered on each individual soma. The furthest dendrite intercept (ie the radius of the largest virtual sphere centered on the soma, still intercepted by the dendrite) was expressed as a mean ± standard error of the mean (SEM).

Illustrations correspond to the projected contours of DAB stained somata of representative SRIF-ir neurons, camera lucida drawn using a 100x oil immersion objective lens (magnification 1250) and scanned with a flat bed scanner (resolution: 300 dpi). Images were adjusted for levels using Photoshop (CS2, Adobe Systems, San Jose, CA, USA). No correction for tissue shrinkage Immunofluorescence double-labeling and cell counts.

For simultaneous detection of somatostatin with other markers (see Table 1) fluorescent immunohistochemistry was performed using standard methods on 30 µm-thick serial sections.

After blocking with 5% NDS/5% BSA in PBST, sections were incubated for 48 hours at 4°C in PBST/1% NDS/1% BSA with mixtures of primary antibodies raised in distinct species for somatostatin and each marker (see details and final concentrations in Table 1). After rinsing, a mixture of appropriate fluorescence-conjugated secondary antibodies in the same buffer (Table 1) was applied for 90 minutes at room temperature. Sections were mounted with Fluoromount mounting medium (Southern Biotech, Birmingham, AL, USA). In the case of GAD 65/67 immunohistochemistry, cryostat-cut 16 µm-thick MOB sections (2800 Frigocut E, Reichert-Jung, Leica, Hedelberg, Germany) were processed as described above, except that incubation with both primary antibodies lasted one week and that detergent was omitted in all steps. Fluorescence signals were detected using an epifluorescence microscope (Axioplan 2, Carl Zeiss, Jena, Germany) fitted with standard filters for the visualization of A488 and CY3 fluorochroms. The relative distribution of SRIF-ir cells in the MOB did not statistically differ between fluorescenceprocessed and DAB-processed SRIF immunohistochemistry experiments (data not shown).

Immunohistochemical specificity controls for somatostatin labeling were previously mentioned. In the case of multiple labeling, specificity controls included omission of the primary antibodies to evaluate the non-specific binding of the secondary antibodies as well as incubation with one primary but both secondary antibodies to demonstrate the absence of cross labeling.

To assess the neurochemical nature of the iEPL and GCL somatostatinergic cells, SRIF-ir cells were analyzed for coexpression with different interneuronal markers (see Table 3) . The total number of cells immunoreactive for each marker (Marker-ir population) or somatostatin (SRIF-ir population) was counted in both layers and all SRIFir cells were individually checked for presence (SRIF/marker) or absence (SRIF alone) of coexpression. All data were averaged from counts derived from three mice, with six sections being analyzed in each animal (section thickness 30 µm, inter-slice interval: 180 µm). Numbers of analyzed cells per animal and colocalization percentages (averaged from the colocalization 3 as mean percentage ± SEM. The proportion of cells expressing the marker also positive for SRIF is expressed as « percent colocalization in Marker population » whereas the proportion of SRIF-ir cells coexpressing a marker is expressed as « percent colocalization in SRIF population».

For each marker, colocalization with SRIF immunoreactivity was confirmed by confocal microscopy on a representative sample of cells (n=25). Single laser beams (wavelengths 488 and 543 nm) were alternately used using a TCS SP2 confocal imaging system equipped with Ar 488nm and HeNe 543-nm lasers (Leica Microsystem, Heidelberg, Germany). For each optical section, double-fluorescence images were acquired under a 40x oil-immersion objective (numeric aperture 1.25) in sequential mode to avoid potential contamination by linkage specific fluorescence emission cross-talk to collect images for both fluorescent signals. These analyses allowed 1) to confirm the correct penetration of both antibodies through the whole section and 2) to validate somatostatin/marker colocalization in a three-dimensional space. Confocal analysis was also used to assess the colocalization of NPY with somatostatin in GCL fibers. Panels displayed in Fig. 3 correspond to five (A,E) or ten (B,C,D,F) serial optical slices spaced by 200-400 nm stacked along the z-axis with maximum intensity projection mode. Once acquired, confocal pictures were exported into Photoshop software (CS2, Adobe Systems, San Jose, CA, USA) and adjusted for levels, luminosity and contrast. Final composites were assembled using Illustrator software (CS2, Adobe Systems).

Electron microscopy.

Pre-embedding immunocytochemical procedures were performed for SRIF detection at the ultrastructural level. Mice were fixed using gravity-driven transcardially perfusion with 10 ml of 0.9% NaCl, followed by 50 ml of PB/ 4% PFA/ 0.05% glutaraldehyde (Sigma) except for panel E which corresponds to PB/ 2% PFA/ 0.5% glutaraldehyde perfusion conditions. Brains were postfixed overnight in 4% PFA at 4°C. Seventy µm coronal sections were cut using a vibratome (VT1000S, Leica, Heidelberg, Germany) and collected in 0.01 M PBS, pH 7.4.

Sections were equilibrated in 30% sucrose and 10% glycerol in PB, subjected to three freeze/thaw cycles using cold isopentane and rinsed in PBS. Sections were incubated sequentially in 1% sodium borohydride (NaBH4) in PBS for 5 minutes, rinsed with PBS, and processed for 11 somatostatin chromogenic immunohistochemistry (see above) except that primary antibody incubation was overnight and DAB revelation lasted 3-10 min. Sections were post-fixed first in 1% glutaraldehyde, then in 1% osmium tetroxide in PB for 10 minutes at room temperature.

After PB rinses, sections were dehydrated in ascending series of ethanol concentrations, which included 1% uranyl acetate in 70% ethanol. They were then treated twice with propylene oxide for 10 minutes, equilibrated overnight in Durcupan ACM (Fluka, Sigma), mounted on glass slides and cured at 60°C for 48 h. Areas of interest were cut out from the slide and glued to blank cylinders of resin. Immunoreactive samples were carefully identified on thick sections and cut in semithin sections (500 nm) and then in ultrathin sections (45 nm) with an ultramicrotome (Ultracut S, Reichert, Leica, Heidelberg, Germany). Consecutive ultrathin sections were collected on 200-mesh copper grids (AGAR Scientific, U.K.) and stained with lead citrate before examination with a Philips CM100 electron microscope (Philips, Suresnes, France). Once acquired, all images presented herein were exported and adjusted for levels, luminosity and contrast with Photoshop software (CS2, Adobe Systems, San Jose, CA, USA). Final composites were assembled using Illustrator software (CS2, Adobe Systems).

Triple-labeling of somatostatin dendrites with synaptic markers.

To combine multiple labeling of somatostatin with glutamatergic and GABAergic synaptic markers, immunofluorescence was performed according to [START_REF] Schneider-Gasser | Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons[END_REF]. Briefly, mice were anesthetized with pentobarbital (180 mg/kg) and transcardially perfused with a 4°C normal artificial cerebrospinal fluid solution containing (in mM): 124 NaCl, 3 KCl, 2 CaCl2, 1.3 MgSO 4 , 25 NaHCO 3 , 1.2 NaH 2 PO 4 , and 10 D-glucose, pH 7.3. The brain was rapidly removed and 200 µm-thick horizontal slices were immediately cut with a vibrating microslider (World Precision Instruments, Sarasota, FL, USA). Slices were immediately transferred to cold 4% PFA for 30 min, rinsed twice in PBS and incubated in PBS/30% sucrose overnight. Tissue-Tek-(Sakura Finetek France, Villeneuve d'Ascq, France) embedded slices were then cut using a cryostat into 14 µm-thick sections and mounted onto glass Superfrost Plus slides. Multiple immunohistochemical labeling was performed as mentioned above, using a combination of goat SRIF, mouse PSD-95 and guinea-pig vesicular GABA transporter antibodies, followed by coincubation with the appropriate fluorescent secondary antibodies (see 12 (SP2, Leica, Heidelberg, Germany) under a 63x objective (numeric aperture: 1.4) to select representative well delineated somatostatin dendrites. Series of 10-15 confocal sections spaced by 300-500 nm were acquired using sequential acquisition of separate wavelength channels (laser wavelengths : 488, 543 and 633 nm). The signal was treated with line averaging to integrate the signal collected over four frames to reduce noise. When necessary, background noise was reduced using "smooth edge" image filter (Leica Confocal Software LCS, Leica, Heidelberg, Germany).

The colocalization pattern of total and somatostatin-ir dendrite-associated VGAT and PSD-95 immunoreactive clusters was determined from 50 confocal sections (resolution: 60 nm/pixel) using Imaris software (Bitplane, Zurich, Switzerland). For each section, each singlelabeled VGAT or PSD-95 immunoreactive cluster was identified by a segmentation algorithm (intensity threshold: 15-25 % of the highest intensity value, surface threshold: 0.05 µm 2 ) and their number and size were calculated. A colocalization algorithm was then used to identify the proportions of VGAT/PSD-95 colocalized clusters as well as the proportion of surface shared by the two markers. In the case of dendrite-associated somatostatin labeling, which did not correspond to the entire surface of dendrites, we considered the clusters as being included in SRIF-ir dendrite using a conservative estimate corresponding to a surface threshold of 0.1 µm 2 , ie 25% of mean PSD-95 surface.

Statistical Analysis

Neuroanatomical and neurochemical data were analyzed using Statview 5.0 software (SAS, Cary, NC, USA). Somatic parameters of iEPL and GCL types of neurons were compared using ANOVA and SRIF-ir cell densities were compared between the different layers using repeated-measures ANOVA. Results are expressed as mean ± SEM. Distribution normality of each subgroup of SRIF-ir neurons was tested for the soma equivalent diameter using the Shapiro-Wilk test with StatEL software (Ad Science, Paris, France). Neurochemical data expressed as proportions were tested using a t-test. Chi-square analysis was used to compare the distribution of VGAT and PSD95 clusters in the total population vs in SRIF-ir dendrites.

Somatostatin-ir in the mouse MOB

As shown in Fig. 1A, the somatostatin-ir pattern is layer-specific. Somata and processes are mainly found in the inner sublayer of the EPL (88.5±0.6% of total SRIF-ir cells, Fig. 1, panels A and C1-C3). Both adjacent layers of the iEPL, the mitral cell layer (MCL) and the outer sublayer of the EPL (oEPL), display low immunoreactivity (2.8±0.3% and 3.4±0.5% respectively) with some rare somata sending their processes into the iEPL (see arrowheads, Fig. 1A). Somatostatin-ir labeling of the MCL and EPL layers delimits a unique ribbon-like staining pattern in the MOB accounting for 94.7% of total SRIF cells. Somatostatin staining is also found in the granule cell layer (GCL), in a few strongly labeled large cells (3.1±0.3%; Fig. 1,panels A and B1-B3) as well as in a high density of fibers that spread throughout the GCL and along the inner plexiform layer (IPL). In the glomerular layer (GL) a few processes and faintly stained somata (2.1±0.1%, open arrow, Fig. 1A) are observed. Finally, no significant cellular staining is visible in the IPL (<0.1%). Most immunoreactive cellular profiles display morphological characteristics of interneurons (Fig. 1, panels B1-B3, C1-C3) and not large-bodied mitral/tufted cells or asymmetric star-like glial figures. Olfactory axons are not stained. Such an immunoreactivity pattern, observed in the C57Bl/6J olfactory bulb, was also found in two other murine strains commonly used, the Swiss and FVB/V strains. This pattern is totally absent in SRIF KO mice (Suppl. Fig. S1).

Morphological analysis of SRIF-ir cells in the mouse iEPL and GCL

Morphometric analyses of camera lucida drawings reveal that iEPL SRIF-ir cells (Fig. 2, top part) constitute a homogeneous population whose main cellular attributes are summarized in Table 2. These cells show small round to piriform somata, localized in a regular manner along the inner part of the EPL. Their soma area equivalent diameter fits to a normal distribution (n=53, Shapiro-Wilk test, W=0.99, p=0.8). A few thick primary processes emerge from the soma. They have numerous large varicosities and rarely bear spines. Their dendritic field is relatively small with few branching processes that elaborate in close proximity to the cell body (mean furthest The few inframitral SRIF-ir neurons mostly found scattered in the deep part of the murine GCL are large and multipolar (Fig. 1 A, B1-3). Their somata are ovoid to fusiform, displaying homogeneous characteristics as detailed in Table 2. The area equivalent diameter of the soma also fits to a normal distribution (n=30, Shapiro-Wilk test, W=0.98, p=0.8). Two to seven primary processes develop into poorly ramified lengthy neurites (Fig. 2, bottom part) oriented in parallel to GCL striations and projecting into the IPL, running up to 400 µm from the soma (mean furthest dendrite Sholl intercept, 209.3±16.1 µm, n=30, Fig. 2, bottom part). These processes are not spiny but show a few small varicosities. GCL SRIF-ir neurons thus share some morphological properties with deep short-axon cells (see below in Discussion).

In addition to identified cellular processes, SRIF-ir fibers that are not connected to visible perikarya are observed in the GCL. They also progress in parallel to the striations of the GCL and IPL, bear few small varicosities and branch very rarely.

Neurochemical characterization of SRIF-ir interneurons in the mouse MOB

The neurochemical properties of iEPL and GCL SRIF-ir cell populations were characterized using fluorescent double immunostaining procedures. We analyzed the extent of colabeling between somatostatin and markers of bulbar interneurons or SRIF interneurons in other telencephalic regions (see Table 1 for experimental details, Table 3 for results).

In the iEPL, immunostaining for GAD65/67 reveals a dense fiber mesh corresponding mainly to GCL granule cell dendritic projections to the EPL, with small to medium-sized GABAergic somata. All SRIF-ir somata are also GAD65/67-ir (Fig. 3A, Table 3), accounting for 13.9% of the iEPL GABAergic population. Concerning calcium-binding proteins commonly used as markers for GABAergic neurons, calretinin is found in small to medium-sized neurons all over the iEPL. All SRIF-ir interneurons are calretinin-ir (Fig. 3B, Table 3), constituting a third of the total calretinin population of the iEPL.

Parvalbumin is also found in the iEPL in small to medium-sized neurons scattered in the layer. Only half of iEPL SRIF-ir neurons (51.1%) coexpress parvalbumin. These double- 3).

Finally immunoreactivity for calbindin in the iEPL is found in rare medium-sized neurons which are immunonegative for somatostatin (Table 3). SRIF-ir neurons were further characterized by analyzing their colocalization with typical interneuronal markers. In the iEPL, vasoactive intestinal peptide is predominantly found in small to medium-sized neurons. Nearly all SRIF-ir interneurons are also VIP-ir and they account for 37.5% of the VIP-ir population of the iEPL (Fig. 3D, Table 3). On the other hand, SRIF-ir interneurons do not express neuropeptide Y which is barely present in the iEPL. Cholecystokinin is mostly expressed in large somata in the iEPL and never colocalized with SRIF immunoreactivity. Similarly the dopaminergic marker tyrosine hydroxylase which labels small to medium-sized iEPL neurons (Table 3) is not expressed in SRIF-ir neurons. Finally, SRIF staining was never found associated with glial fibrillary acidic protein immunoreactivity which occurs in star-like immunoprofiles in the iEPL (data not shown).

In the GCL, GAD65/67 cellular staining corresponds to packed intrinsic populations of GABAergic interneurons, including granule cells (Fig. 3E). The few SRIF-ir deep shortaxon cells are all labeled by the GAD65/67 antibody (Table 3). Concerning calcium-binding proteins, a high level of CR immunoreactivity is found in the GCL, especially in a population of superficial granule cells as well as in sparse superficial large multipolar cells. This pattern does not overlap with the SRIF-ir pattern (Table 3). Parvalbumin immunoreactivity is mostly found in large multipolar neurons restricted to the superficial part of the GCL and only 1.7% of SRIF-ir cells demonstrate PV coexpression (Table 3).

Calbindin immunoreactivity is found in medium-sized multipolar neurons with highly ramified dendrites lying in the superficial part of the GCL as well as in deeper large fusiform multipolar cells (data not shown). One third of the SRIF-ir population shows CB Connectivity of the SRIF-ir iEPL interneurons SRIF-ir elements were further analyzed at the ultrastructural level in the iEPL (Fig. 4). SRIF-ir profiles exhibit dendritic ultrastructural features including the presence of polymorph vesicles and mitochondria, especially near the synaptic sites. Gap junctions were never observed in SRIF-ir profiles. Most SRIF-ir elements make synaptic contacts with transversally-or longitudinally-cut large dendritic trunks whose radial orientation, size, regular outline, electron lucent appearance and presence of vesicles (alveolate and round synaptic) and multivesicular bodies meet the criteria for mitral/tufted (M/T) cell dendrites [START_REF] Price | The Mitral and Short Axon Cells of the Olfactory Bulb[END_REF]. The frequent presence of presynaptic clusters of round vesicles in M/T dendrites apposing prominent post-synaptic densities in SRIF-ir elements (see black arrows, Fig. 4, panels A, B, C, E, F) demonstrated that SRIF interneurons receive asymmetrical contacts from M/T cell dendrites. Some asymmetrical contacts from M/T dendrites onto SRIF-immunonegative elements were also observed (black arrow, Fig. 4D). In most cases, the M/T dendrites, presynaptic to SRIF-ir elements, were found in close proximity to large clusters of flattened vesicles in the SRIF-ir terminals, characteristic of GABAergic spines or buttons (Fig. 4 [START_REF] Panzanelli | Pre-and postsynaptic GABA receptors at reciprocal dendrodendritic synapses in the olfactory bulb[END_REF][START_REF] Schneider-Gasser | Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons[END_REF]. As shown in Fig. 5, fluorescent clusters showing PSD-95 and VGAT immunolabeling are very abundant in the EPL (Fig. 5, panels A and C). PSD-95 clusters are approximately twice as abundant as VGAT clusters (see Table 4), but they are significantly smaller (VGAT: 1.11± 0.03 µm 2 , PSD-95: 0.39±0.01 µm 2 , mean surface±SEM).

Numerous VGAT and PSD-95 clusters clearly overlap (arrowheads in Fig. 5, enlarged in Fig. 5 panel C). While most VGAT-ir clusters are colocalized with PSD-95-ir clusters (94.5±1.2 %, Table 4, Fig. 5, panels A and C), less than half of PSD-95 clusters colocalize with VGAT clusters (43.5±0.9%). This colocalization involves 63% of the PSD-95-ir cluster surface compared to 26% of the VGAT cluster surface (Table 4). The present results indicate that somatostatin expression delineates a sublayer in the EPL of the murine main olfactory bulb. Unlike in other species, nearly 95% of bulbar SRIFimmunoreactive neurons correspond to small interneurons restricted to the inner half of the EPL that arborize locally. Ultrastructural and confocal immunohistochemical data show that SRIF-ir interneurons directly interact with mitral cell dendrites, involving dendrodendritic reciprocal synapses. This provides an anatomical basis for a neuromodulatory role of the peptide on the granule-mitral cell dendritic interactions which are fundamental to olfactory processing.

Superimposition of SRIF

Somatostatin in deep short-axon cells: comparative analysis and functional implications

Somatostatin had been described in the olfactory bulb of the hamster and the rat [START_REF] Davis | Localization of methionine-enkephalin, substance P, and immunoreactivities in the main olfactory bulb of the hamster[END_REF][START_REF] Gutièrrez-Mecinas | Characterization of somatostatin-and cholecystokinin-immunoreactive periglomerular cells in the rat olfactory bulb[END_REF][START_REF] Scott | Short axon cells of the rat olfactory bulb display 34 NADPH-diaphorase activity, neuropeptide Y-like immunoreactivity, and somatostatinlike immunoreactivity[END_REF][START_REF] Seroogy | Somatostatin-like immunoreactivity in rat main olfactory bulb: extent of coexistence with neuropeptide Y-, tyrosine hydroxylase-and vitamin D-dependent calcium binding protein-like immunoreactivities[END_REF]) mostly as SRIFpositive deep short-axon cells (dSACs) and fibers which are consistently found in the GCL [START_REF] Scott | Short axon cells of the rat olfactory bulb display 34 NADPH-diaphorase activity, neuropeptide Y-like immunoreactivity, and somatostatinlike immunoreactivity[END_REF][START_REF] Seroogy | Somatostatin-like immunoreactivity in rat main olfactory bulb: extent of coexistence with neuropeptide Y-, tyrosine hydroxylase-and vitamin D-dependent calcium binding protein-like immunoreactivities[END_REF]. In rats, and to a lesser extent in men and frogs, SRIF-ir was also present in some periglomerular cells and superficial short-axon cells [START_REF] Davis | Localization of methionine-enkephalin, substance P, and immunoreactivities in the main olfactory bulb of the hamster[END_REF][START_REF] Gutièrrez-Mecinas | Characterization of somatostatin-and cholecystokinin-immunoreactive periglomerular cells in the rat olfactory bulb[END_REF][START_REF] Inagaki | Distribution of somatostatin in the frog brain, Rana catesbiana, in relation to location of catecholamine-containing neuron system[END_REF][START_REF] Ohm | Somatostatin-14-like immunoreactive neurons and fibres in the human olfactory bulb[END_REF][START_REF] Scott | Short axon cells of the rat olfactory bulb display 34 NADPH-diaphorase activity, neuropeptide Y-like immunoreactivity, and somatostatinlike immunoreactivity[END_REF] while those cells were not found in guinea pig and hamster [START_REF] Davis | Localization of methionine-enkephalin, substance P, and immunoreactivities in the main olfactory bulb of the hamster[END_REF][START_REF] Matsutani | Distribution of neuropeptidelike immunoreactivities in the guinea pig olfactory bulb[END_REF].

We postulate here that the SRIF-ir cells of the mouse GCL also belong to the dSAC type on the basis of the morphological features previously defined using Golgi impregnation [START_REF] Price | The Mitral and Short Axon Cells of the Olfactory Bulb[END_REF][START_REF] Ramon Y Cajal | Histologie du système nerveux de l'homme et des vertébrés[END_REF][START_REF] Schneider | Laminar distributions of interneurons in the main olfactory bulb of the adult hamster[END_REF]: it should be noted though that early neuroanatomical descriptions lacked quantitative and morphometric data [START_REF] Price | The Mitral and Short Axon Cells of the Olfactory Bulb[END_REF][START_REF] Ramon Y Cajal | Histologie du système nerveux de l'homme et des vertébrés[END_REF][START_REF] Schneider | Laminar distributions of interneurons in the main olfactory bulb of the adult hamster[END_REF] or primarily involved species other than the mouse [START_REF] Larriva-Sahd | The accessory olfactory bulb in the adult rat: a cytological study of its cell types, neuropil, neuronal modules, and interactions with the main olfactory system[END_REF][START_REF] López-Mascaraque | Structure of the olfactory bulb of the hedgehog (Erinaceus europaeus): a Golgi study of the intrinsic organization of the superficial layers[END_REF][START_REF] Shipley | Functional organization of olfactory system[END_REF]. Inframitral SRIF-ir cells are located in the deep GCL, close to the subependymal layer, usually oriented parallel to olfactory bulb layers. They display a large-sized and ovoid soma, from which emerge several dendrites, running at least 150 µm from the soma, and extend very long processes which can be followed up to 400 µm. Nevertheless, we could not classify these SRIF-ir dSAC as either horizontal, vertical, Blanes or Golgi cell types -initially identified in the rodent MOB on the basis of the shape, location and size of the soma, orientation of the [START_REF] Price | The Mitral and Short Axon Cells of the Olfactory Bulb[END_REF][START_REF] Schneider | Laminar distributions of interneurons in the main olfactory bulb of the adult hamster[END_REF][START_REF] Shipley | Functional organization of olfactory system[END_REF] -since their morphological features did not exclusively or completely match any of these subgroups.

Horizontal and vertical cells are projecting neurons, mainly localized in the superficial GCL and IPL. Their soma are fusiform and bipolar, with horizontal cells arborizing in parallel to GCL lamination while vertical cell dendrites arborize perpendicularly to GCL lamination [START_REF] López-Mascaraque | Structure of the olfactory bulb of the 32 hedgehog (Erinaceus europaeus): description of cell types in the granular layer[END_REF][START_REF] Price | The Mitral and Short Axon Cells of the Olfactory Bulb[END_REF][START_REF] Schneider | Laminar distributions of interneurons in the main olfactory bulb of the adult hamster[END_REF]. Their localization and shape clearly differ from GCL SRIF-ir cells. Golgi and Blanes cells, considered as locally projecting neurons, are more frequently found in the deep GCL, like SRIF-ir cells.

Nevertheless, they usually display a random multipolar dendritic arborization, while SRIF-ir neurites arborize along the main orientation of the bulbar layers. Furthermore, whereas Blanes cells show numerous dendrites emerging from all sides of the soma, the latter arborize relatively close to the soma (-100 µm) and frequently bear spines [START_REF] Price | The Mitral and Short Axon Cells of the Olfactory Bulb[END_REF][START_REF] Schneider | Laminar distributions of interneurons in the main olfactory bulb of the adult hamster[END_REF]. This is in contrast to the long and aspiny SRIF-ir dendrites we observed in the mouse GCL. Smaller dSAC subpopulations had also been described in other species, such as deep stellate cells in the monkey [START_REF] Alonso | Chemical anatomy of the macaque monkey olfactory bulb: NADPH-diaphorase/nitric oxide synthase activity[END_REF], piriform and fusiform cells in the hedgehog [START_REF] Briñón | Calretinin-, neurocalcin-, and parvalbumin-immunoreactive elements in the olfactory bulb of the hedgehog (Erinaceus europaeus)[END_REF] and giant cells in the rat [START_REF] Briñón | Calbindin D-28kpositive neurons in the rat olfactory bulb. An immunohistochemical study[END_REF]; none of which share the morphological features of SRIF-ir GCL cells. Thus, SRIF-ir cells likely correspond to a distinct subtype of deep short-axon cell. Such a dSAC profile was reproducibly reported after SRIF immunodetection in rats [START_REF] Gutièrrez-Mecinas | Characterization of somatostatin-and cholecystokinin-immunoreactive periglomerular cells in the rat olfactory bulb[END_REF][START_REF] Scott | Short axon cells of the rat olfactory bulb display 34 NADPH-diaphorase activity, neuropeptide Y-like immunoreactivity, and somatostatinlike immunoreactivity[END_REF][START_REF] Seroogy | Somatostatin-like immunoreactivity in rat main olfactory bulb: extent of coexistence with neuropeptide Y-, tyrosine hydroxylase-and vitamin D-dependent calcium binding protein-like immunoreactivities[END_REF], guinea pigs [START_REF] Matsutani | Distribution of neuropeptidelike immunoreactivities in the guinea pig olfactory bulb[END_REF], hamsters [START_REF] Davis | Localization of methionine-enkephalin, substance P, and immunoreactivities in the main olfactory bulb of the hamster[END_REF] and humans [START_REF] Ohm | Somatostatin-14-like immunoreactive neurons and fibres in the human olfactory bulb[END_REF][START_REF] Smith | Immunohistochemical analyses of the human olfactory bulb[END_REF]. Moreover, mouse SRIF-ir deep short-axon cells coexpress NPY as previously reported in rats [START_REF] Gall | Distribution of VIP-and NPY-like immunoreactivities in rat main olfactory bulb[END_REF][START_REF] Scott | Short axon cells of the rat olfactory bulb display 34 NADPH-diaphorase activity, neuropeptide Y-like immunoreactivity, and somatostatinlike immunoreactivity[END_REF][START_REF] Seroogy | Somatostatin-like immunoreactivity in rat main olfactory bulb: extent of coexistence with neuropeptide Y-, tyrosine hydroxylase-and vitamin D-dependent calcium binding protein-like immunoreactivities[END_REF]. Taken together, these results suggest that SRIF expression in dSAC of the GCL is conserved across mammalian species. Since NPYergic and calbindin-positive phenotypes identify most somatostatinergic interneurons in the forebrain (for a review, see [START_REF] Viollet | Somatostatinergic systems in brain: Networks and functions[END_REF], bulbar GCL SRIF cells also share common features of all SRIF interneurons. The fact that SRIF-ir fibers observed in the GCL colocalize with NPY staining also strongly suggests that they may originate from GCL SRIF-ir dSAC neurons. However, many SRIF-ir fibers could not be traced to identified cells, and their cellular origin remains uncertain. Thus, GCL SRIF-ir fibers may also have an extrinsic origin since a subpopulation of raphe serotoninergic neurons projecting to the 20 olfactory bulb and notably to the GCL also contains somatostatin, at least in the rat [START_REF] Araneda | Raphe serotonergic neurons projecting to the olfactory bulb contain galanin or somatostatin but not neurotensin[END_REF].

Little is known about the role of GCL deep short-axon cells. Early electron microscopy studies showed that they innervate local interneurons, either granule cells or other dSACs [START_REF] Fj | VIP-containing deep short-axon cells of the olfactory bulb innervate interneurons different from granule cells[END_REF][START_REF] Price | The Mitral and Short Axon Cells of the Olfactory Bulb[END_REF], suggesting that SRIF interneurons may also participate in selective GABAergic local circuits involved in the modulation of principal cells.

The demonstration of the monosynaptic inhibition of granule cells by Blanes cells indeed stressed the point of the functional importance of SACs in the MOB circuitry for the global dishinhibition of the output neurons via feed-forward inhibition of granule cells [START_REF] Pressler | Blanes cells mediate persistent feedforward inhibition onto granule cells in the olfactory bulb[END_REF]. Two recent studies improved our knowledge concerning dSAC in the MOB [START_REF] Eyre | Distinct Deep Short-Axon Cell Subtypes of the Main Olfactory Bulb Provide Novel Intrabulbar and Extrabulbar GABAergic Connections[END_REF][START_REF] Eyre | Molecular diversity of deep short-axon cells of the rat main olfactory bulb[END_REF]. They showed that dSACs unexpectedly represent a large population of cells (a quarter of the mitral cell population), which innervate all layers of the MOB. They can be subdivided into several main categories according to the location of their soma in the GCL, axodendritic distribution, neurochemical repertoire and synaptic connectivity. As a common feature, all dSACs participate to the inhibition of GABAergic interneurons. But each dSAC subpopulation very selectively innervates a given type of intrinsic interneurons (i.e. periglomerular interneurons, granule cells) or even projects to higher olfactory area, involving novel intra-and extra-bulbar GABAergic projections [START_REF] Eyre | Distinct Deep Short-Axon Cell Subtypes of the Main Olfactory Bulb Provide Novel Intrabulbar and Extrabulbar GABAergic Connections[END_REF][START_REF] Fj | VIP-containing deep short-axon cells of the olfactory bulb innervate interneurons different from granule cells[END_REF][START_REF] Pressler | Blanes cells mediate persistent feedforward inhibition onto granule cells in the olfactory bulb[END_REF]. As previously suggested by the heterogeneity of VIP-ir [START_REF] Fj | VIP-containing deep short-axon cells of the olfactory bulb innervate interneurons different from granule cells[END_REF], NOS-ir (Kosaka and Kosaka, 2007b) and PV-ir (Kosaka and Kosaka, 2008) GCL interneurons in rat and mice, the neurochemical characterization of dSAC did not recapitulate a former morphological classification [START_REF] Eyre | Molecular diversity of deep short-axon cells of the rat main olfactory bulb[END_REF]. Neuropeptideexpressing subpopulations were found to be scarce: GCL SRIF-ir population was indeed estimated below 1% of the total dSAC [START_REF] Eyre | Molecular diversity of deep short-axon cells of the rat main olfactory bulb[END_REF] suggesting that these cells may not have been recorded in the initial study reporting the functional properties of the main dSACs subpopulations [START_REF] Eyre | Distinct Deep Short-Axon Cell Subtypes of the Main Olfactory Bulb Provide Novel Intrabulbar and Extrabulbar GABAergic Connections[END_REF]. SRIF-ir dSAC subpopulation may then constitute a distinct dSAC subtypes with selective synaptic connectivity. In contrast to the GCL population, the concentration of SRIF interneurons in the iEPL had never been described to date in mammalian species and seems unique to the murine MOB. A few sparse SRIF-ir cells were reported in the EPL of guinea pigs [START_REF] Matsutani | Distribution of neuropeptidelike immunoreactivities in the guinea pig olfactory bulb[END_REF]. In rats, and to a lesser extent in humans, some SRIF-ir cells were found scattered in the GL [START_REF] Gutièrrez-Mecinas | Characterization of somatostatin-and cholecystokinin-immunoreactive periglomerular cells in the rat olfactory bulb[END_REF][START_REF] Hwang | Age-related change of somatostatin-immunoreactive neurones in the main olfactory bulb of the rat[END_REF]Kosaka and Kosaka, 2005). Such variability between the EPL and GL SRIF content across mammalian species clearly contrasts with the constancy of somatostatin detection in dSAC in the GCL, as discussed above. Cytological and morphological parameters of the mouse iEPL SRIF-ir cells show that they constitute an homogeneous interneuronal population, whose characteristics are reminiscent of Van Gehuchten interneurons (Ramon y Cajal, 1911;[START_REF] Schneider | Laminar distributions of interneurons in the main olfactory bulb of the adult hamster[END_REF], a subpopulation of axonless intrinsic neurons of the EPL initially described using Golgi impregnation in the cat [START_REF] Van Gehuchten | Mémoire sur le système nerveux[END_REF]. Van Gehuchten interneurons are characterized by a medium-sized fusiform soma, from which emerge one or two thick and highly varicosed dendrites which arborize at the vicinity of the cell body (less than 100 µm) in a non-oriented way, forming a dense ovoid dendritic field [START_REF] Briñón | Calbindin D-28kpositive neurons in the rat olfactory bulb. An immunohistochemical study[END_REF][START_REF] Hamilton | Properties of external plexiform layer interneurons in mouse olfactory bulb slices[END_REF][START_REF] Schneider | Laminar distributions of interneurons in the main olfactory bulb of the adult hamster[END_REF]. They differ from other EPL interneurons, namely horizontal cells, satellite cells, superficial short-axon cells and multipolar cells. Horizontal and satellite cells have small round somata (8-10 µm, mean diameter in mouse EPL) and display axon-like terminals [START_REF] Kosaka | Calcium-binding protein parvalbuminimmunoreactive neurons in the rat olfactory bulb. 1. Distribution and structural features in adult rat[END_REF][START_REF] López-Mascaraque | Structure of the olfactory bulb of the hedgehog (Erinaceus europaeus): a Golgi study of the intrinsic organization of the superficial layers[END_REF]. Horizontal cells are bipolar interneurons with long smooth processes running tangentially to the MCL, whereas satellite cells display highly varicosed processes that precisely envelop the soma and segments of mitral cell dendrites. Superficial short-axon cells have a large soma (around 14 µm, mean diameter in mouse EPL, Kosaka et al., 2008;Kosaka and Kosaka, 2008), positioned mainly in the outer part of the EPL. They display a large dendritic field together with an axon-like process that spreads in the EPL [START_REF] Kosaka | Calcium-binding protein parvalbuminimmunoreactive neurons in the rat olfactory bulb. 1. Distribution and structural features in adult rat[END_REF]Kosaka and Kosaka, 2008).

Somatostatin-ir

Finally, multipolar cells also have medium-size somata but these cells display a higher number of primary dendrites and a larger dendritic field (>100 µm) than Van Gehuchten cells. In various species Van Gehuchten interneurons are scattered in the whole EPL [START_REF] Briñón | Neurocalcin immunoreactivity in the rat main olfactory bulb[END_REF].

Immunostaining for VIP neuropeptide [START_REF] Crespo | Vasoactive intestinal polypeptide-containing elements in the olfactory bulb of the hedgehog (Erinaceus europaeus)[END_REF][START_REF] Gall | Distribution of VIP-and NPY-like immunoreactivities in rat main olfactory bulb[END_REF] or parvalbumin allowed descriptions of Van Gehuchten interneurons in rats [START_REF] Briñón | Neurocalcin immunoreactivity in the rat main olfactory bulb[END_REF][START_REF] Crespo | Vasoactive intestinal polypeptide-containing elements in the olfactory bulb of the hedgehog (Erinaceus europaeus)[END_REF][START_REF] Kosaka | Calcium-binding protein parvalbuminimmunoreactive neurons in the rat olfactory bulb. 1. Distribution and structural features in adult rat[END_REF][START_REF] Toida | Electron microscopic serialsectioning/reconstruction study of parvalbumin-containing neurons in the external plexiform layer of the rat olfactory bulb[END_REF], hedgehogs [START_REF] Crespo | Parvalbumin-29 containing interneurons do not innervate granule cells in the olfactory bulb[END_REF] and mice (Kosaka and Kosaka, 2008;[START_REF] Parrish-Aungst | Quantitative analysis of neuronal diversity in the mouse olfactory bulb[END_REF]. The present neurochemical analysis of Van Gehuchten cells in the iEPL confirms their GABAergic, VIPergic and PV-ir phenotype but also reveals an atypical colocalization of somatostatin with these classical markers. Indeed, in other telencephalic regions, somatostatin is usually coexpressed with NPY and calbindin, rarely with calretinin and parvalbumin, and never with VIP (for a review, see [START_REF] Viollet | Somatostatinergic systems in brain: Networks and functions[END_REF]. Such a striking difference may be due to a different developmental origin: hippocampal and cortical SRIF interneurons both originate from the median ganglionic eminence [START_REF] Danglot | The development of hippocampal interneurons in rodents[END_REF][START_REF] Métin | Cell and molecular mechanisms involved in the migration of cortical interneurons[END_REF][START_REF] Wonders | The origin and specification of cortical interneurons[END_REF] whereas most bulbar interneurons originate from the lateral ganglionic eminence and the dorsal telencephalon [START_REF] Bovetti | Spatio-temporal specification of olfactory bulb interneurons[END_REF][START_REF] Kohwi | A subpopulation of olfactory bulb GABAergic interneurons is derived from Emx1-and Dlx5/6-expressing progenitors[END_REF][START_REF] Ventura | Dorsal radial glia generate olfactory bulb interneurons in the postnatal murine brain[END_REF][START_REF] Young | Subventricular zone stem cells are heterogeneous with respect to their embryonic origins and neurogenic fates in the adult olfactory bulb[END_REF].

Regarding the putative functional role of Van Gehuchten cells in MOB circuitry, serial electron microscopy studies showed that VIP and/or parvalbumin processes directly innervate mitral/tufted cells [START_REF] Crespo | Parvalbumin-29 containing interneurons do not innervate granule cells in the olfactory bulb[END_REF][START_REF] Crespo | Vasoactive intestinal polypeptide-containing elements in the olfactory bulb of the hedgehog (Erinaceus europaeus)[END_REF][START_REF] Toida | Electron microscopic serialsectioning/reconstruction study of parvalbumin-containing neurons in the external plexiform layer of the rat olfactory bulb[END_REF]. This challenges the classical scheme according to which EPL interneurons only contact granule cells [START_REF] Schneider | Laminar distributions of interneurons in the main olfactory bulb of the adult hamster[END_REF]. In a combined physiological and biocytin-staining study, [START_REF] Hamilton | Properties of external plexiform layer interneurons in mouse olfactory bulb slices[END_REF] confirmed that murine Van Gehuchten neurons were axonless GABAergic cells. These cells exhibit high frequency spontaneous excitatory post-synaptic currents, suggesting inputs from mitral/tufted cells [START_REF] Hamilton | Properties of external plexiform layer interneurons in mouse olfactory bulb slices[END_REF]. Moreover, depolarizing currents applied to these cells evoke trains of action potentials [START_REF] Hamilton | Properties of external plexiform layer interneurons in mouse olfactory bulb slices[END_REF]. Recently, markers of the axon initial segment have been localized on the dendritic processes of parvalbumin Van Gehuchten cells, providing anatomical support for the occurrence of dendritic spike generation in these cells, despite their lack of an axon (Kosaka et al., 2008;Kosaka and Kosaka, 2008). These data, together with the connectivity of iEPL somatostatinergic terminals as reported here, strongly suggest that iEPL axonless somatostatin interneurons directly interact through dendrodendritic contacts with their synaptic partners: the mitral/tufted cells. We cannot categorically rule out that some ascending SRIF-ir axons might also contact mitral/tufted cells. However, the present results, in accordance with [START_REF] Eyre | Distinct Deep Short-Axon Cell Subtypes of the Main Olfactory Bulb Provide Novel Intrabulbar and Extrabulbar GABAergic Connections[END_REF], indicate that ascending SRIF-ir axons are far less abundant than local SRIF-ir dendrites in the iEPL. Therefore anaxonic iEPL somatostatinergic cells likely represent the predominant source of SRIF-ir terminals on mitral/tufted cell dendrites. The present study then confirms that iEPL Van Gehuchten interneurons must be considered as first order neurons since they exert their inhibitory action directly on principal cells. Interestingly, in the other forebrain regions studied so far (hippocampus, neocortex, amygdala, neostriatum, basal forebrain, [START_REF] Viollet | Somatostatinergic systems in brain: Networks and functions[END_REF]. This canonical synaptic organisation of somatostatin interneurons is thus conserved in the mouse main olfactory bulb, since somatostatinergic interneurons directly innervate the basal compartment of mitral/tufted cells.

Moreover, the dendritic processes of somatostatin Van Gehuchten interneurons spread into a spatially limited domain in the EPL which fits with the diameter of the functional column previously described in viral tracing studies [START_REF] Willhite | Viral tracing identifies distributed columnar organization in the olfactory bulb[END_REF]. Somatostatin interneurons could then interact with mitral/tufted cells which are associated with a given glomerulus and thus contribute to the tuning of their activity.

The strict delineation of the inner lamina of the EPL by SRIF interneurons suggests a primary interaction with mitral cells. Indeed, principal cells and granule cells segregate within the EPL [START_REF] Shepherd | The Synaptic Organization of the Brain[END_REF][START_REF] Shipley | Functional organization of olfactory system[END_REF]: mitral cell lateral dendrites preferentially develop in the deep lamina of the EPL where they contact dendrites from deep granule cells, whereas tufted cell lateral dendrites arborize in the outer part of the EPL [START_REF] Mori | Membrane and synaptic properties of identified neurons in the olfactory bulb[END_REF][START_REF] Mori | Distribution of dendrites of mitral, displaced mitral, tufted, and granule cells in the rabbit olfactory bulb[END_REF][START_REF] Orona | Different granule cell populations innervate superficial and deep regions of the external plexiform layer in rat olfactory bulb[END_REF] and contact superficial GC dendrites [START_REF] Greer | Plasticity of dendrodendritic microcircuits following mitral cell loss in the olfactory bulb of the murine mutant Purkinje cell degeneration[END_REF][START_REF] Mori | Membrane and synaptic properties of identified neurons in the olfactory bulb[END_REF][START_REF] Mori | Distribution of dendrites of mitral, displaced mitral, tufted, and granule cells in the rabbit olfactory bulb[END_REF][START_REF] Orona | Different granule cell populations innervate superficial and deep regions of the external plexiform layer in rat olfactory bulb[END_REF]. The occurrence of distinct repertoires of GABA-A [START_REF] Panzanelli | Pre-and postsynaptic GABA receptors at reciprocal dendrodendritic synapses in the olfactory bulb[END_REF] and AMPA [START_REF] Montague | Differential distribution of ionotropic glutamate receptor in the rat olfactory bulb[END_REF] receptor subunits in the inner and the outer laminae of the EPL further suggests that mitral and tufted cells participate in distinct synaptic microcircuits through different signaling pathways. Functionally, tufted cells would be associated with low-threshold perception of odorants [START_REF] Christie | Tufted cell dendrodendritic inhibition in the olfactory bulb is dependent on NMDA receptor activity[END_REF][START_REF] Ezeh | Organization of inhibition in the rat olfactory bulb external plexiform layer[END_REF][START_REF] Nagayama | Mitral and tufted cells differ in the decoding manner of odor maps in the rat olfactory bulb[END_REF] whereas mitral cells, via lateral inhibition, would participate in odor discrimination [START_REF] Lledo | Information processing in the mammalian olfactory system[END_REF][START_REF] Yokoi | Refinement of odor molecule tuning by dendrodendritic inhibition in the olfactory bulb[END_REF]. According to their peculiar distribution in the bulbar network, somatostatin interneurons may preferentially interact with mitral-deep granule cell microcircuits and thus modulate olfactory discrimination processes.

Somatostatin release and olfactory processing

In contrast to other neurochemical markers in the MOB, somatostatin is expressed in a morphologically and spatially homogeneous subpopulation of interneurons. Our immunohistochemical and immunocytological evidences support the direct interaction of iEPL synapses. This suggests an important role for this peptide in modulating neural networks involved in sensory and learning processes. In most telencephalic regions neuropeptide-expressing interneuronal systems are crucial for maintaining brain functioning under normal and pathophysiological conditions [START_REF] Baraban | Interneuron Diversity series: Interneuronal neuropeptides-endogenous regulators of neuronal excitability[END_REF]. In particular, somatostatin systems are involved in learning and memory processes [START_REF] Gastambide | Hippocampal SSTR4 somatostatin receptors control the selection of memory strategies[END_REF][START_REF] Viollet | Somatostatinergic systems in brain: Networks and functions[END_REF] and may represent putative endogenous agents [START_REF] Baraban | Interneuron Diversity series: Interneuronal neuropeptides-endogenous regulators of neuronal excitability[END_REF][START_REF] Moneta | Somatostatin receptor subtypes 2 and 4 affect seizure susceptibility and hippocampal excitatory neurotransmission in mice[END_REF]. Many studies have revealed the neuronal tetrodotoxin-sensitive and Ca2+-dependent of SRIF [START_REF] Arancibia | In vivo release of somatostatin from rat median eminence after local K+ infusion or delivery of nociceptive stress[END_REF][START_REF] Iversen | Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro[END_REF][START_REF] Vezzani | Brain somatostatin: a candidate inhibitory role in seizures and epileptogenesis[END_REF] and have demonstrated the preferential release of the peptide under high neuronal activity, in particular high frequency bursting or even epileptic seizures [START_REF] Bartfai | Regulation of the release of coexisting neurotransmitters[END_REF][START_REF] Christenson | Co-localized GABA and somatostatin use ionic mechanisms to hyperpolarize target neurons in the lamprey spinal cord[END_REF][START_REF] Csaba | Plasticity of somatostatin somatostatin sst2A receptors in the rat dentate gyrus during kindling epileptogenesis[END_REF][START_REF] Vezzani | Somatostatin release is enhanced in the hippocampus of partially and fully kindled rats[END_REF].

In vitro, Van Gehuchten cells exhibit high frequency spontaneous and evoked excitatory currents (40-100 Hz) related to mitral/tufted cell inputs [START_REF] Hamilton | Properties of external plexiform layer interneurons in mouse olfactory bulb slices[END_REF]. High mitral/tufted cell activity observed during odorant stimulation may then trigger somatostatin release. In most telencephalic regions somatostatin immunoreactivity is found in dendrites [START_REF] Epelbaum | Somatostatin in the central nervous system: physiology and pathological modifications[END_REF].

Several studies have highlighted the fact that dendrites are a major source of peptide release in the brain (for a recent review, [START_REF] Ludwig | Dendritic peptide release and peptide-dependent behaviours[END_REF]. Here, we demonstrate that iEPL somatostatin dendrites mainly interact with M/T cell dendrites. The occurrence of calciuminduced GABA release from somatostatinergic Van Gehuchten dendrites may create an even more favorable context for peptide release. Like other neuropeptides, somatostatin is likely to act via volume transmission (diffusion in a three-dimensional fashion within the extracellular space) and may exert global inhibitory effects on the synaptic network, even on distant synapses.

Through the activation of hyperpolarizing currents or inhibition of calcium channels, somatostatin mainly acts on glutamatergic sites, either presynaptic or postsynaptic [START_REF] Csaba | Cellular biology of somatostatin receptors[END_REF][START_REF] Csaba | Plasticity of somatostatin somatostatin sst2A receptors in the rat dentate gyrus during kindling epileptogenesis[END_REF][START_REF] Viollet | Somatostatinergic systems in brain: Networks and functions[END_REF]. Such effects are related to the subtype and cellular localization of the somatostatin receptors involved. Using autoradiographical techniques sst2, sst3 and sst4 binding sites were detected in the EPL as well as sst2 and sst4 binding sites in the GCL [START_REF] Videau | Characterisation of [125I]-Tyr0DTrp8-somatostatin binding in sst1-to sst4-and 35 SRIF-gene-invalidated mouse brain[END_REF]. Interestingly the bulk of somatostatin immunoreactivity and binding sites is found in the EPL. This layer consists of a dense neuropilic network of granule and mitral cell dendrites interacting through reciprocal dendrodendritic Earlier ultrastructural studies reported that nearly all synapses upon M/T cell dendrites were found to be reciprocal [START_REF] Price | The Mitral and Short Axon Cells of the Olfactory Bulb[END_REF], as visualized here by the abundance of colocalized VGAT/PSD-95 immunopositive clusters. Since somatostatin can inhibit both presynaptic and postsynaptic sites, it may then directly modulate mitral cell-granule cell synaptic interactions, and consequently alter the resulting oscillatory dynamics [START_REF] Lagier | Interplay between local GABAergic interneurons and relay neurons generates gamma oscillations in the rat olfactory bulb[END_REF]. The EPL is also innervated by feedback centrifugal fibers, including cholinergic, serotoninergic or noradrenergic inputs [START_REF] Shepherd | The Synaptic Organization of the Brain[END_REF][START_REF] Shipley | Functional organization of olfactory system[END_REF] and somatostatin is known to influence monoaminergic release in many brain regions [START_REF] Beal | The effect of somatostatin on striatal catecholamines[END_REF][START_REF] Garcia-Sevilla | Effect of intracerebroventricularly administered somatostatin on brain monoamine turnover[END_REF][START_REF] Göthert | Somatostatin selectively inhibits noradrenaline release from hypothalamic 30 neurones[END_REF][START_REF] Hathway | Somatostatin potently stimulates in vivo striatal dopamine and gamma-aminobutyric acid release by a glutamate-dependent action[END_REF][START_REF] Tanaka | Somatostatin facilitates the serotonin release from rat cerebral cortex, hippocampus and hypothalamus slices[END_REF][START_REF] Vécsei | Somatostatin28(15-28), but not somatostatin28(1-12), affects central monoaminergic neurotransmission in rats[END_REF].

Since peptides can act on synapses distant from their release sites, a putative action of somatostatin on such neuromodulatory elements cannot be ruled out.

Conclusion

In summary, the present study identifies a novel and species-selective SRIF interneuronal population in the iEPL of the mouse olfactory bulb, ideally inserted in the bulbar synaptic network to exert a functional modulatory role on olfactory signal processing. These iEPL somatostatin interneurons display selective neurochemical and anatomical properties which clearly differ from those previously reported for the main telencephalic somatostatin interneurons. These results substantiate the variability of the inhibitory interneuron repertoires between mouse and rat species, as recently reported for other markers (Kosaka and Kosaka, 2007a;Kosaka and Kosaka, 2007b). Finally iEPL somatostatinergic Van Gehuchten cells interact through dendrodendritic synaptic interactions with the principal cells of the MOB, suggesting a local peptide release driven by mitral-tufted cell activity. Further molecular and physiological studies are in progress to elucidate how somatostatin modulates olfactory synaptic networks and physiology. A, B, C, D : Representative pictures of ABC-DAB-processed sagittal sections of mouse main olfactory bulb showing the absence of labeling in SRIF-KO mice (B, D) as compared to wild-type mice (A, C) using the goat D-20 somatostatin antibody (see Table 1). Similar results were obtained using the IS-7/51 antiserum (data not shown). A, B : cresyl violet counterstaining ; C,D, without counterstaining. Scale bars : 100 µm. 176x145mm (300 x 300 DPI)

  totally absent in brain sections from somatostatin KO mice (data not shown).

  the analysis of ABC-DAB sections.

  , n=3) are given in Table

  intercept : 77.0±2.6 µm, n=53) and remain restricted to the iEPL (Fig.1A, 2 top part). Somatostatin-ir axon-like structures were not detected. Altogether, these morphological characteristics correspond to those reported for Van Gehuchten cells (see below in Discussion).

  to 59.1% of the iEPL parvalbumin population (Fig.3C, Table

  staining, mainly corresponding to the deep CB-ir population. NPY is found in large fusiform multipolar neurons lying deep in the GCL, close to the subependymal layer. As shown in Fig 3F, SRIF-ir and NPY-ir populations strongly overlap : 89.5% of SRIF-ir deep short-axon cells express NPY while 81.9% of NPY-ir cells also express somatostatin (Table3). Interestingly most SRIF-ir fibers in the GCL are also NPY-ir [90% colocalization in SRIF fibers, 84% in NPY fibers, (n=195), Fig.3F]. Tyrosine Hydroxylase immunoreactivity rare large cells and does not label SRIF-ir neurons. Finally, cells immunoreactive for VIP or CCK were not detected in mouse GCL, ruling out any colocalization of somatostatin with these two markers.

  , panels B, F). Symmetric synapses formed by SRIF-ir elements onto M/T dendrites were also occasionally identified on the basis of both pre-and post-synaptic membrane specializations in the presence of round or flattened vesicles on the presynaptic side (see open black arrows, Fig. 4, panels B, D). These symmetrical synapses onto M/T cell dendrites were in general less readily recognized because DAB staining can interfere with the identification of synaptic vesicles. The occurrence of asymmetrical excitatory synapses onto a GABAergic spine that in turn synapse on mitral cells (as shown in Fig. 4 B) strongly suggests that SRIF-ir cells establish dendrodendritic reciprocal synapses onto M/T cell dendrites.In order to estimate the degree of occurrence of reciprocal synapses on SRIF-ir dendrites, the relative distribution of VGAT-positive clusters (a marker for presynaptic GABAergic terminals) and PSD-95-positive clusters (a signature for mature glutamatergic postsynaptic quantified. Physical proximity between a GABAergic presynaptic site and a glutamatergic postsynaptic site suggests the existence of a reciprocal synapse

  -ir profiles with VGAT and PSD-95 double staining indicates that colocalized VGAT/PSD-95 clusters are observed within SRIF-ir dendritic processes (arrowheads, Fig. 5, panels B and D). Proportions of VGAT and PSD-95 colocalization remains the same in SRIF-ir dendrites as found in the whole optical section (Table 4, Chi-square test for VGAT and PSD-95 respectively, ns). Occasionally, SRIF-ir processes are observed at the border of a large immunonegative profile typical of a M/T cell dendrite (Fig. 5, panels A and B) and include some VGAT/PSD-95 colocalized clusters (open arrowheads, Fig. 5B).

  presence or absence of dendritic spines[START_REF] López-Mascaraque | Structure of the olfactory bulb of the 32 hedgehog (Erinaceus europaeus): description of cell types in the granular layer[END_REF][START_REF] Price | The Mitral and Short Axon Cells of the Olfactory Bulb[END_REF]

  Van Gehuchten interneurons : comparative analysis and integration in the MOB circuitry

  , somatostatinergic interneurons systematically innervate their respective principal cells, targeting either their apical or basal dendritic compartments as shown in neocortex and hippocampus (reviewed in

  the principal cells of the MOB through dendrodendritic reciprocal

Figure 1 :

 1 Figure legends:

Figure 2 :

 2 Figure 2 : Morphological analysis of MOB SRIF neurons.

Figure 3 :

 3 Figure 3 : Neurochemical signature of iEPL and GCL SRIF-ir interneurons.

Figure 4 :

 4 Figure 4: Connectivity of the SRIF-ir dendrites in the iEPL.

Figure 5 :

 5 Figure 5: Colocalization of GABAergic presynaptic and glutamatergic postsynaptic proteins in SRIF-ir dendrites in the iEPL.

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 12345 Figure 1 : Distribution of SRIF immunoreactivity in the murine main olfactory bulb. A: Representative ABC-DAB-processed coronal section of mouse main olfactory bulb showing SRIF immunoreactivity in the different cell layers. Note the ribbon of stained cells whose somata and neurites are confined to the inner half of the EPL (iEPL). Counterstaining: cresyl violet. Arrowheads : SRIF-ir labeled cells in MCL and oEPL, open arrow: SRIF-ir labeled soma in GL. Scale bar: 100 µm. B1, B2, B3 : Representative pictures of SRIF-ir cells in the mouse GCL. Scale bars: 25 µm. C1, C2, C3 : Representative pictures of SRIF-ir cells in the mouse iEPL. MCL is visble at the lower part of each panel. Scale bars: 25 µm Abbreviations : EPL, External Plexiform Layer; GCL, Granule Cell Layer; GL, Glomerular Layer; iEPL, inner part of the EPL; IPL, Internal Plexiform Layer; MCL, Mitral Cell Layer; oEPL, outer part of the EPL; ONL, Olfactory Nerve Layer. 172x101mm (600 x 600 DPI)

Table 1

 1 for antibodies details). After mounting, sections were submitted to confocal observation

	F o
	r
	P
	e e r
	R e v i e w

Table 2 . Morphological features of the SRIF-ir main populations in the MOB

 2

	Morphological parameters	iEPL SRIF + cells (n=53)	GCL SRIF + cells (n=30)
	Soma area (µm 2 )	66.0 ± 2.2	139.9±7.3
	Soma perimeter (µm)	30.4 ± 0.5	45.4 ± 1.3
	Soma area equivalent diameter (µm)	9.1±0.2	13.2±0.4
	Soma major diameter (µm)	10.7 ± 0.2	16.1± 0.5
	Soma minor diameter (µm)	9.1 ± 1.3	10.9 ± 0.3
	Primary dendrites number	1.9 ± 0.1	3.4 ± 0.2
	Branching point number	4.6 ± 0.3	4.1 ± 0.4
	Total dendritic length (µm)	254.8 ± 11.0	610.5 ± 47.8
	Furthest Sholl intercept (µm)	77.0 ± 2.9	209.3 ± 16.1

Table 3 . Chemical properties of iEPL and GCL somatostatin-ir cells

 3 Data were averaged from three mice, with six consecutive 30 µm-thick coronal sections being analyzed per animal (inter-slice interval : 180 µm), except for GAD65/67 for which 16 µm-thick sections were used (*see Materials and Methods). Values correspond to mean ± SEM. 0: absence of colocalization; ε: negligible; n.d.: not determined.

			Analyzed cells per animal		Percent colocalization
	Interneuronal Marker	Marker-ir population	SRIF-ir population	SRIF/marker	SRIF alone	in Marker population	in SRIF population
	Inner External Plexiform Layer (iEPL)
	GAD 65/67*	1007.5±50.6	142.0±5.7	141.3±6.1	0.8±0.4	13.9±0.8	99.4±0.3
	Calretinin	1155.5±102.5	413.8±44.8	413.4±44.6	0.4±0.2	35.7±0.7	99.9±0.1
	Parvalbumin	373.0±40.4	428.2±39.8	218.9±21.4	209.3±18.8	59.1±3.2	51.1±0.7
	Calbindin	52.6±8.6	446.8±42.5	0	446.8±42.5	0	0
	Vasoactive Intestinal Peptide	866.3±70.7	336.0±28.1	324.8±26.6	11.1±2.6	37.5±0.1	96.7±0.6
	Neuropeptide Y	0.4±0.2	443.1±30.8	0	443.1±30.8	0	0
	Cholecystokinin	436.8±30.0	400.2±31.8	0	400.2±31.8	0	0
	Tyrosine Hydroxylase	30.4±5.8	446.0±69.0	0	446.0±69.0	0	0
	Granule Cell Layer (GCL)
	GAD 65/67*	n.d.	6.5±0.6	6.5±0.6	0	ε ε ε ε	100
	Calretinin	n.d.	8.4±3.0	0	8.4±3.0	0	0
	Parvalbumin	41.4±8.4	10.4±2.2	0.2±0.2	10.2±2.3	0.3±0.3	1.7±1.7
	Calbindin	15.4±2.6	14.8±0.3	4.8±0.6	9.9±0.6	32.6±4.2	32.7±4.1
	Vasoactive Intestinal Peptide	0	14.8±1.6	0	14.8±1.6	0	0
	Neuropeptide Y	11.3±2.3	10.5±2.5	9.4±2.2	1.1±0.3	81.9±3.8	89.5±0.7
	Cholecystokinin	0	14.2±1	0	14.2±1	0	0
	Tyrosine Hydroxylase	7.2±3.9	10.3±0.7	0	10.3±0.7	0	0

Journal of Comparative Neurology

John Wiley & SonsJournal of Comparative Neurology

John Wiley & Sons

Acknowledgments

The authors wish to acknowledge A. Cougnon for taking care of the mice, Drs G. Tramu and P. Ciofi (Inserm UMR 862, Bordeaux) for providing SRIF, VIP and CCK antisera, Dr M. Darmon (Inserm UMR 894, Paris) for providing the anti-PSD-95 antiserum, Prof. K.G. Baimbridge (Univ. of British Columbia, Vancouver, Canada) for providing information concerning the specificity of the anti-parvalbumin antiserum, Dr. S. Yang (Inserm UMR 894, Paris) and M. Palloto (Department of Anatomy, Pharmacology, and Forensic Medicine, Univ. Italy) for technical help with the immunodetection of synaptic markers, the Inserm Fer à Moulin Institute imaging facility (Inserm UMR 839, Paris) for expert help with electron microscopy, Dr S. Peineau (Inserm UMR 676, Paris) and Dr N. Ramoz (Inserm UMR 894, Paris) for helpful discussions and Dr M. Grubb for careful reading of the manuscript.

1 This study was supported by Inserm and MENRT (fellowship to G.L.).

F o r P e e r R e v i e w

F o r P e e r R e v i e w


Bernard Boitrel

Stéphane Le Gac

Match-Mismatch Effects in Twofold Transfer of Chirality within a Möbius Metallo-Receptor

come

Match-mismatch effects in two-fold transfer of chirality within a Mobius metallo-receptor

Chirality inherent to Möbius topology has recently regained interest, boosted by the booming of Möbius aromaticity. 1 As predicted by Heilbronner almost 60 years ago, 2 cyclic πconjugated systems featuring half-a twist follow aromaticity rules opposite to planar systems, and therefore 4n Möbius (hetero)annulenes exhibit variable degrees of aromaticity. 3 Major advances in the design of Möbius π-systems were accomplished over the last decade with the broad family of expanded porphyrins, [START_REF] Tanaka | For recent reviews[END_REF][START_REF] Stępień | Selected leading references for Möbius hexaphyrins[END_REF] that present several advantages: straightforward preparation, relatively good stability, wide range of metal coordination, strong light absorption (eventually in the NIR region), two-photon absorption, and last but not least, conformational flexibility that can be controlled via different stimuli. The latter property makes these macrocycles attractive to build up Möbius adaptative systems, such as molecular receptors that can feature induced-fit or allosteric processes. 6 Indeed, the strong interplay between conformation, (anti)aromaticity, and chirality may afford large and tunable chiroptical activity variation, of particular interest for sensing systems. Mastering Möbius chirality is thus crucial to control and enrich the properties of ensuing molecular systems. To do so, transfer of chirality from a chiral source to a Möbius ring, aiming at asymmetric winding of the π-system, deserves intense investigations. To the best of our knowledge, only few systems have been reported to date, all involving a [28]hexaphyrin scaffold (Figure 1). Osuka et al. reported the very first asymmetric preparation of a Möbius hexaphyrin Pd(II) complex using a chiral palladium salt, with a moderate 23 % e.e. (Figure 1a). 5c In a different approach relying on dynamic transfer of chirality, 7 our group has shown more efficient stereoselective controls of the Möbius twist (up to 76 % d.e.), either from metalbound molecules (Figure 1b) 6a or from covalently attached sources (Figure 1c). 6c Pursuing our efforts, we describe herein relatively simple Zn(II) hexaphyrin complexes undergoing twofold transfer of chirality upon simultaneous binding of two chiral molecules (Figure 1d), demonstrating an efficient allosteric tuning of the resulting chiroptical activity. [28]Hexaphyrin 2 (Scheme 1), functionalized with 2acetamidophenyl and pentafluorophenyl groups in alternate meso positions, was designed aiming at simpler receptors than those bearing a tripodal cap (Figure 1b). 6a This particular mesosubstitution pattern affords electron withdrawing groups enhancing the hexaphyrin scaffold stability, and H bond donor moieties promoting Zn(II) complexation through a second sphere of coordination with amino/carboxylato ligands. 6a Compound 2 was prepared in one step from the known [26]hexaphyrin 1 (Scheme 1). Reduction of both the nitro groups by Pd(0)/C catalyzed hydrogenation, and the hexaphyrin a. Univ Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes)-UMR 6226, Rennes F-35000, France b. E-mail: stephane.legac@univ-rennes1.fr, bernard.boitrel@univ-rennes1.fr. Electronic Supplementary Information (ESI) available: experimental part and NMR spectra. See DOI: 10.1039/x0xx00000x core ([26] -> [28] π-system), 6a followed by an acylation reaction of the amino moieties with acetyl chloride, led to the desired [28]hexaphyrin 2 in moderate yield upon silica gel chromatography purification (23 %). HRMS analysis indeed confirmed the reduced state of the hexaphyrin scaffold featuring a [28] π-system. 2 exhibits an intense blue colour in solution, and its UV-vis absorption spectrum is characteristic of an aromatic species, with a sharp intense Soret-like band at 606 nm and four Q-like bands spanning from 730 to 1060 nm (SI). 8 Its 1 H NMR spectrum in CDCl3 exhibits marked broadening at 298 K, indicating a conformationally flexible compound (SI). [START_REF]Möbius aromatic conformations frozen on the NMR time scale[END_REF] These experimental observations are consistent with a predominant aromatic Möbius conformation undergoing fluxional equilibrium (fast exchange process on the NMR time scale), as previously observed with related compounds. 5b,6a,10 Atropisomerism involving the meso 2-acetamidophenyl groups likely contributes as well to this ill-resolved 1 H NMR spectrum. Zn(II) insertion in 2 was investigated by 1 H NMR experiments in 9:1 CDCl3/CD3OD. In the presence of DIPEA, addition of Zn(OTf)2 led only to partial consumption of 2 affording minor unidentified species with non-trivial resonances in the highfield region (Figure 2b). In contrast, subsequent addition of TBAOAc led instantaneously to complete disappearance of 2 and to the formation of several species featuring dissymmetric patterns (Figure 2c), the two major ones (ca. 1:1 ratio) being partially characterized by 2D NMR spectra. They display three sets of βpyrrolic protons, around -2 ppm (blue triangles, inner β-pyr), 4.5 ppm (green triangles, twisted β-pyr) and 7-8 ppm (red triangles, outer β-pyr). These patterns correspond to aromatic Möbius conformations, rigidified on the NMR time scale by Zn(II) complexation, with a typical diatropic ring current shielding the inside and deshielding the outside of the hexaphyrin core. 5b,6a 2D ROESY NMR experiment indicates that shielded signals at -1 ppm correspond to acetate molecules oriented inward (Figure 2c, blue dot, ∆δ = -2.9 ppm), in agreement with the binding of a 'ZnOAc' moiety at a dipyrrin site (Scheme 1, 2.ZnOAc), as in related tren-capped hexaphyrin complexes. 6a,11 The 2acetamidophenyl group adjacent to the twisted pyrrole (bound to zinc) is projected towards the hexaphyrin core and thus experiences partial shielding (Figure 2c, orange dots). This feature likely reinforces the dissymmetry at the level of the metal centre. More detailed NMR characterization is hampered by the complexity of the spectrum. core (Figure 2d, labelled "A", "B" and "C"). 13 In addition to a Zn(II) acetato ligand oriented inward (∆δ ~ -3 ppm, Figure 2 inset), 2D ROESY NMR experiment (SI) evidences concomitant binding of a butylamino ligand, oriented outward. Indeed, only the protons in α position of the coordinated nitrogen atom undergo slight shielding (∆δ ~ -0.7 ppm, Figure 2 inset), consistently with an aliphatic chain pointing away from the inner core of the Möbius hexaphyrin (Scheme 1, 2.ZnOAc NH2Bu ). These observations are in agreement with related tren-capped hexaphyrin complexes, in which a 4-coordinate Zn(II) (tetrahedral geometry) orients carboxylato and amino ligands respectively inward and outward. 6a During the course of our investigations towards twofold transfer of chirality to the Möbius twisted π-system, we collected interesting results with two other ligands, N-Boc protected (S)-Proline (BocProOH) as inward guest and (R/S)methylbenzylamine (MBA) as outward guest. With their resulting complexes, 1 H NMR spectroscopy does not provide direct access to chirality induction (d.e. values) due to too complex sets of NMR patterns arising from atropisomerism. In contrast, electronic circular dichroism (ECD) spectroscopy is a straightforward and efficient method to evaluate and compare P/M twist stereoselectivity, since atropisomers of the same P (/M) configuration will simply sum up their CD spectra. To assess twofold transfer of chirality, five situations were thus compared by both 1 H NMR and ECD spectroscopies (Figure 3ae), all generated from the same mixture of 2, DIPEA and Zn(OTf)2 (CDCl3/CD3OD 9:1, 298 K): (i) Whereas addition of (S)-BocProOH led to an ill-defined NMR spectrum and an ECD one of weak intensity (Figure 3a), subsequent introduction of BuNH2 led to the expected mixture of Zn(II) Möbius complexes with (S)-BocProO -inward and BuNH2 outward (Figure 3b; ∆δ values in the SI). 14,15 An important 5-fold increase of the ECD intensity (λ = 624 nm), displaying negative Please do not adjust margins cotton effect, indicates marked chirality transfer favouring a M twist. 16 Such a behaviour corresponds to a cooperative process, requiring concomitant binding of the two guests;

(ii) Addition of EtCO2H and (R)-MBA led to a similar result, i.e. Zn(II)-bound carboxylato and amino ligands are respectively oriented inward and outward and support transfer of chirality favouring a M twist (Figure 3e, same CD spectrum as Figure 3b). Thus, chirality transfer can proceed from inward [(S)-BocProO -] or outward [(R)-MBA] guests with comparable efficiency, provided that their complementary (achiral) guest is present (respectively BuNH2 and EtCO2 -);

(iii) Addition of (S)-BocProOH and (S)-MBA, that respectively favour M and P twists, led to an ill-defined 1 H NMR spectrum 17 and to an ECD spectrum of weak intensity (Figure 3c; 10-fold decrease vs Figure 3b). In contrast, addition of (S)-BocProOH and (R)-MBA, that both favour a M twist, led to a very well defined 1 H NMR spectrum (two predominant Möbius Zn(II) complexes in ca. 2:1 ratio, Figure 3d), and to an ECD spectrum of higher intensity than those of the two separated chiral ligands (Figure 3d, 50 % increase vs Figure 3b and3e). Hence, an inward guest favouring a M twist exhibits a mismatch effect with an outward guest favouring a P twist, leading to weak recognition and poor diastereoselectivity, but exhibits a match effect with an outward guest also favouring a M twist, leading to a remarkable increase of the diastereoselectivity. The same trend is observed when comparing (R)-MBA and (S)-BocProOH vs EtCO2H (Figure 3d vs 3e, match effect). These match-mismatch effects evidence an overall communication between the two sources of chirality, meaning that chirality transfers do not take precedence over one another. This feature nicely allows up or down regulation of the chiroptical activity of the Möbius π-system owing to allosterism, relying on the configuration of the two guests. This phenomenon, to date restricted to Möbius porphyrinoids, is possible because of the conformational equilibrium between the two Möbius enantiomers, allowing (twofold) transfer of chirality under thermodynamic control. Such dynamic metalloreceptors featuring highly responsive chiroptical activity are attractive to build up chirality sensing systems.

Figure 1 .

 1 Figure 1. Transfer of chirality (red/blue arrows) in Möbius aromatic [28]hexaphyrin metal complexes: a) at the transition state of a metal insertion process [M = Pd(II)]; 5c b) from a metal-bound molecule in a capped-hexaphyrin metallo-receptor [M = Zn(II)]; 6a c) from two sources in covalently bridged hexaphyrin-cyclodextrin hybrids [M = Zn(II)]; 6c d) from two metal-bound molecules in a hexaphyrin metallo-receptor [M = Zn(II)].

Scheme 1 .

 1 Scheme 1. Synthesis of[28]hexaphyrin 2 (Ar = C6F5) and subsequent Zn(II) insertion in the presence of two different guest molecules (for the sake of clarity, only one possible isomer [Möbius conformation and 2-acetamidophenyl orientation] is displayed; for consistency with related previous work, 6a a monodentate coordination of OAc is arbitrarily depicted): i) H2 (80 bars), Pd(0)/C, AcOEt, RT, 15 h; then AcCl, TEA, THF, 0 °C, 30 min; ii) Zn(OTf)2, DIPEA, TBAOAc, CDCl3/CD3OD 9:1 (298 K); iii) BuNH2 (298 K).

  12 Subsequent addition of BuNH2 led to a new set of NMR patterns with at least the three major species retaining the characteristic resonances of Zn(II) Möbius complexes with a 2acetamidophenyl group projected towards the hexaphyrin

Figure 2 . 1 H

 21 Figure2.1 H NMR study of the metallation of 2 with Zn(II) (CDCl3/CD3OD 9:1, 298 K, 500 MHz): a) 2 at 4.4 mM; b) upon addition of 1.5 equiv. of Zn(OTf)2 and 5 equiv. of DIPEA; c) upon addition of 3 equiv. of TBAOAc; d) upon addition of 5 equiv. of BuNH2 (at 278 K; signals labelled "A", "B" and "C" belong to the three major species). S = solvent. Inset : ∆δ = δbound-δfree.

Figure 3 .

 3 Figure 3. Assessment of the P/M stereoselective formation of Möbius isomers, from 1 H NMR (top) and ECD (down) studies. Metallation of 2 with Zn(OTf)2/DIPEA (CDCl3/CD3OD 9:1, 298 K, 500 MHz) performed in the presence of different guest molecules: a) (S)-BocProO -; b) BuNH2 and (S)-BocProO -; c) (S)-MBA and (S)-BocProO -; d) (R)-MBA and (S)-BocProO -; e) (R)-MBA and EtCO2 -. S = solvent. Proton labelling: same as Figure 2. NMR initial concentration of 2: 4.4 mM; ECD total concentration of 2: 44 µM (∆ε values and proportions of each species are not accessible).

The UV-vis-NIR absorption spectrum of 2.ZnOAc is characteristic of a Möbius aromatic [28]hexaphyrin, the Soretlike band being red shifted compared to 2, see the SI.

13 Roughly, the same ratio of A, B and C (4:2.5:1) is measured from different signals (inner and twisted β-pyrrolic protons, CH3 protons of the acetato ligands, aromatic protons of the meso 2-acetamidophenyl substituent oriented inward).

Conflicts of interest

There are no conflicts to declare.

Notes and references