J Mol Neurosci (2015) 55:480–491 DOI 10.1007/s12031-014-0369-5

Colocalization of Cannabinoid Receptor 1 with Somatostatin and Neuronal Nitric Oxide Synthase in Rat Brain Hypothalamus Shenglong Zou & Rishi K Somvanshi & Seungil Paik & Ujendra Kumar

Received: 19 June 2014 / Accepted: 26 June 2014 / Published online: 8 July 2014 # Springer Science+Business Media New York 2014

Abstract Despite several overlapping functions of cannabinoid receptor 1 (CB1 receptor), somatostatin (SST), and neuronal nitric oxide synthase (nNOS) in the hypothalamus, nothing is currently known whether CB1 receptor-positive neurons coexpress SST and nNOS. In the present study, we describe the colocalization of CB1 receptor with SST and nNOS in the rat brain hypothalamus. In the hypothalamus, the distributional patterns and colocalization of CB1 receptor with SST and nNOS were selective and region specific. CB1 receptor and SST exhibited comparable colocalization (80 %, followed by 60 and 30 % in PVN and VMH, respectively. In contrast, SST- and nNOS-positive neurons displayed comparable colocalization (>55 %) in PeVN and VMH, followed by PVN (~20 %). In the median eminence, CB1 receptor-, SST-, and nNOS-like immunoreactivity was mostly confined to the nerve fibers. The morphological colocalization of CB1 receptor with SST and nNOS shed new light on the understanding of their roles in regulation of physiological and pharmacological response to certain stimuli in hypothalamic nuclei specifically in food intake and energy balance. Keywords Cannabinoid receptor 1 . Somatostatin . Neuronal nitric oxide synthase . Hypothalamus . Immunohistochemistry

S. Zou : R. K. Somvanshi : S. Paik : U. Kumar (*) Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada e-mail: [email protected]

Abbreviations AN Arcuate nucleus CB1 receptor Cannabinoid receptor 1 CNS Central nervous system CRF Corticotrophin-releasing factor GABA γ-Aminobutyric acid GHRH Growth hormone-releasing hormone GPCR G protein-coupled receptor ME Median eminence NGS Normal goat serum nNOS Neuronal nitric oxide synthase NO Nitric oxide NPY Neuropeptide Y PeVN Periventricular nucleus PMOC Pro-opiomelanocortin PNS Peripheral nervous system PVN Paraventricular nucleus SST Somatostatin SSTR Somatostatin receptor VMH Ventromedial hypothalamic nucleus

Introduction The hypothalamus exhibits complex array of function in response to various stimuli and is known to be a prominent physiological regulator of the central nervous system (CNS). Several neurotransmitters, neuropeptides and receptor proteins are well expressed in the hypothalamus. Among them, endocannabinoids play crucial role in various biological activities, including appetite stimulation, energy balance, pain relief, cognition, memory, anxiety, motor behavior, and autonomic and neuroendocrine responses (Marsicano et al. 2003; Mechoulam and Lichtman 2003; van der Stelt et al. 2002; Calignano et al. 1998; Walker and Hohmann 2005). The

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cannabinoids are also linked to multiple sclerosis, schizophrenia, and depression (Robson 2001). Such different arrays of biological effects in CNS and peripheral nervous system (PNS) are mediated by two cannabinoid receptor subtypes, namely, cannabinoid receptors 1 and 2 (CB1 and CB2 receptor), which belong to the superfamily of G protein-coupled receptors (GPCRs). CB1 receptor is highly expressed in CNS in a region-specific manner in different neuronal populations, while CB2 receptor is predominantly present in peripheral tissues. Despite low expression in the hypothalamus, CB1 receptor is crucially involved in regulation of food intake and energy balance through hypothalamic circuits (Bellocchio et al. 2008; Di Marzo and Matias 2005; Silvestri and Di Marzo 2013). The levels of endocannabinoids increase in the rat hypothalamus during fasting and return to normal after food intake (Hanus et al. 2003). The stimulation of appetite is observed after direct injection of endocannabinoid into rat ventromedial hypothalamic nucleus (VMH) and abolished by the administration of CB1 receptor antagonist (Jamshidi and Taylor 2001). Also, in target tissues, the biological effects of endocannabinoids have also been shown independent of CBs (De Petrocellis and Di Marzo 2010). These observations indicate the possibility of direct and indirect roles of other hormones in association with CB1 receptor in regulation of appetite. Somatostatin (SST), also known as somatotropin releaseinhibiting factor, was first isolated from the hypothalamus as growth hormone inhibitory peptide (Brazeau et al. 1973). SST-positive neurons are present throughout the brain with enriched SST-like immunoreactivity in the hypothalamus, specifically in the periventricular nucleus (PeVN) constituting 80 % of SST immunoreactivity in CNS (Finley et al. 1981). Relatively low expression of SST occurs in the paraventricular nucleus (PVN), suprachiasmatic nucleus, arcuate nucleus (AN), and VMH (Johansson et al. 1984). SSTs via five different receptor subtypes play various crucial roles in mammalian brain including neurodegenerative and neuropsychological disorders, pain relief, inflammation, and nociception (Kumar and Grant 2009). In the hypothalamus, SST not only serves hypophysiotropic function, but also influences several other neuronal functions through local circuits. For instance, SST regulates the release of growth hormone-releasing hormone (GHRH), corticotropin-releasing factor (CRF), norepinephrine, and SST itself (Patel 1999). Recent studies have described potential role of SST in inhibition of ghrelin, one of the major hormones affecting food intake and energy balance, indicating the role of SST in regulation of appetite (Seoane et al. 2007; Silva et al. 2005). Also, the presence of SST receptors (SSTRs) in 35 % pro-opiomelanocortin (POMC) messenger RNA (mRNA)-containing neurons in AN might support this notion (Fodor et al. 1998). Nitric oxide (NO) is the most prominent second messenger in CNS and PNS, where it is produced mainly by neuronal NO

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synthase (nNOS). Previous studies have shown that nNOSpositive neurons are well expressed in the hypothalamus, the brain region displaying highest concentration other than cerebellum (Rodrigo et al. 1994). In the hypothalamus, nNOS is associated with several physiological functions, including energy balance and regulation of appetite (Calabrese et al. 2007). Inhibition of nNOS leads to decreased food intake and weight loss while food deprivation results in enhanced nNOS activity (O’Shea and Gundlach 1996; Ueta et al. 1995). In addition, nNOS has also been implicated in modulation of several appetite-related hormones, including ghrelin, neuropeptide Y (NPY), and leptin. Ghrelin and NPY have been shown to increase nNOS in the hypothalamus while leptin does the opposite (Gaskin et al. 2003; Morley et al. 1999). Furthermore, ghrelin-induced feeding is blocked by nNOS inhibitor, and leptin-regulated body energy control requires hypothalamic nNOS-positive neurons (Gaskin et al. 2003; Leshan et al. 2012). The comparable distributional pattern and overlapping functional roles suggest that CB1 receptor in the hypothalamus might colocalize with SST and nNOS. However, to date, no study has shown whether SST and nNOS are coexpressed in CB1 receptor-positive neurons in the hypothalamus. On the other hand, SST and nNOS have been shown to express in the same neuron in cortical and striatal regions. However, whether they colocalize in the hypothalamus is not known. Accordingly, in the present study, we investigated CB1 receptor colocalization with SST and nNOS in the hypothalamus. Taken together, our results showed region-specific colocalization of CB1 receptor with SST and nNOS, as well as colocalization of SST and nNOS in the rat brain hypothalamus.

Materials and Methods Animals Male Sprague-Dawley rats (body weight 200–250 g) were obtained from The University of British Columbia animal care unit. The protocols regarding animal care were followed in compliance with the principles of the Canadian Council on Animal Care and were approved by the University of British Columbia Animal Care Committee. Materials SST mouse monoclonal antibody (Cat. No. sc-74556) and CB1 receptor goat polyclonal antibody (Cat. No. sc-10066) were purchased from Santa Cruz (Dallas, TX). nNOS rabbit polyclonal antibody (Cat. No. AB5380) was purchased from Millipore (Billerica, MA). Normal goat serum (NGS) was purchased from Vector Laboratories (Burlingame, CA).

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Fluorescein isothiocyanate (FITC) and Cy3-conjugated goat anti-mouse, goat anti-rabbit, and donkey anti-goat secondary antibodies were obtained from Jackson Laboratories (West Grove, PA). All other reagents and chemicals were of analytical grade and obtained from various commercial sources. Immunocytochemistry Rats were perfused with 4 % paraformaldehyde freshly prepared in 0.1 M phosphate-buffered saline. The brains were taken out and postfixed in same perfusion solution for 2 h. Forty-micrometer-thick free-floating brain sections passing through the hypothalamus were processed for indirect double-label immunofluorescence colocalization as described previously (Kumar 2007). Briefly, the rat brain sections were

Fig. 1 Photomicrographs illustrating the specificity of CB1 receptor antibody and immunofluorescence in HEK-293 cells, hypothalamus section, and tissue lysate. Wild-type and CB1 receptor-transfected HEK-293 cells were incubated with CB1 receptor antibody at a dilution of 1:500 overnight at 4 °C, followed by Cy3-conjugated secondary antibody (dilution 1:800) for 2 h at room temperature. In wild-type cells, no receptor-like immunoreactivity was detected (a), whereas transfected cells exhibited strong cell surface immunoreactivity for CB1 receptor (arrows, b). CB1 receptor-transfected cells probed only with secondary antibody in absence of primary antibody were devoid of specific fluorescence (c). In the lower panel, hypothalamic section stained for SST

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incubated in 5 % NGS and 0.2 % Triton X100 in Tris-buffered saline (TBS) for 1 h at room temperature. The sections were then incubated overnight at 4 °C in a humid atmosphere with anti-goat CB1 receptor (1:200) and anti-mouse SST (1:200) or anti-rabbit nNOS (1:200) antibodies in TBS containing 1 % NGS. The brain sections were then washed in TBS for three times and incubated for 2 h in presence of FITC and Cy3conjugated secondary antibodies for the final color development. Following three subsequent washes in TBS, the brain sections were mounted onto the slide and photographed using Carl Zeiss confocal microscope (LSM 700, Axio Observer). All the photographs composites were made using Adobe Photoshop (San Jose, CA), and merged images showing colocalization were generated in ZEN Lite blue edition software (Jena, Germany).

showed only green fluorescence (d) but not red (e). No bleach through was detected. In g, representative Western blot analysis showed CB1 receptor expression in rat hypothalamus tissue lysate. Rat hypothalamus tissue lysate was processed for Western blot analysis as described in the “Materials and Methods” section. In the presence of CB1 primary antibody, two strong bands were detected at the expected molecular sizes of 64 and 53 kDa, respectively, representing the expression of CB1 receptor in the hypothalamus. No band was observed in the absence of receptor-specific antibody (right panel). Scale bar=20 μm (a–c) and 40 μm (d–f)

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proteins were analyzed for colocalization in each combination. The mean percent of neurons showing colocalization was calculated from total neurons, and results are presented as mean±SE (n=5).

2 Representative confocal photomicrographs illustrating the colocalization of SST and CB1 receptors in the hypothalamus. Rat brain sections were incubated overnight with SST monoclonal (1:200) and antigoat CB1 receptor antibodies (1:200). Following three washes in TBS, sections were incubated with goat anti-mouse FITC and donkey anti-goat Cy3-conjugated secondary antibodies as described in the “Materials and Methods” section. Note three distinct neuronal populations displaying either colocalization or individual staining for SST and CB1 receptors in different hypothalamic regions. Neurons positive to SST (arrowheads), CB1 receptor (tilted arrows), and colocalization (arrows) in merged images are indicated in respective panel. Neuronal processes and nerve fibers showing colocalization are indicated by asterisks in individual merged panel. In PVN, strong colocalization was observed (arrows, c). In PeVN, neurons expressing either SST (arrowheads) or CB1 receptors (tilted arrows) and colocalization (arrows) were seen (d–f). The majority of neurons in VMH were either positive to only CB1 receptors (tilted arrows) or displaying colocalization (arrows, g–i). Neuronal cell bodies in AN (j–l) and innervated nerve fibers in ME (m–o) exhibited strong colocalization between CB1 receptor and SST. In addition, neuronal processes and nerve fibers were seen positive to CB1 receptor throughout the hypothalamus (asterisks, c, f). Scale bar=40 μm

Western Blot Analysis CB1 receptor immunoreactivity in hypothalamic tissue lysate was determined using Western blotting analysis as previously described (Somvanshi and Kumar 2014). Briefly, 15 μg of tissue protein was solubilized in Laemmli sample buffer and fractionated on 10 % SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membrane. Membrane was probed with goat anti-CB1 receptor antibody (1:500) overnight. Following three subsequent washes, the membrane was incubated in donkey anti-goat secondary antibody, and signals were developed with chemiluminescence and photographed using FluorChem software (Alpha Innotech).

Results Specificity of Immunoreactivity and Colocalization In the present study, to determine the specificity of CB1 receptor antibody, wild-type and CB1 receptor-transfected HEK-293 cells were used. As shown in Fig. 1a, CB1 receptor-like immunoreactivity was not seen in wild-type HEK-293 cells whereas CB1 receptor-transfected cells showed intensive receptor-like immunoreactivity at cell surface (Fig. 1b). On the other hand, CB1 receptor-transfected cells in absence of primary antibody were devoid of receptorlike immunoreactivity (Fig. 1c). To further characterize the possibility of any bleach through, rat brain sections were processed for SST primary antibody and then exposed to both FITC and Cy3-conjugated secondary antibodies. Specific staining was detected in green channel but not in red channel (Fig. 1d–f). We also characterized the specificity of CB1 receptor antibody in rat hypothalamic tissue lysate using Western blot. As shown in Fig. 1g left panel, two strong bands were observed at 64 and 53 kDa, respectively, representing the glycosylated and non-glycosylated form of CB1 receptor, whereas no such band was detected in the absence of primary antibody. Furthermore, the specificity of CB1 receptor antibody used in this study has been demonstrated by Sanford et al. by immunohistochemistry using antibody preadsorbed with blocking peptide (Sanford et al. 2008). Several studies using this antibody revealed a similar distributional pattern of

Quantitative Analysis The relative percentage distribution of neurons displaying colocalization between SST, CB1 receptor, and nNOS was determined as previously described (Kumar 2007). Fortymicrometer coronal rat brain sections passing through the hypothalamus were double-labeled for SST, CB1 receptor, and nNOS and processed for quantitative analysis using Carl Zeiss confocal microscope (LSM 700, Axio Observer) at×20 magnification. Neurons were considered immunoreactive if the staining of soma was distinctively stronger than background. Five to ten sections obtained from five different rat brains were subjected for quantification. Eight to ten areas in interested regions were randomly selected for neuron counting in each section. Only neuronal cell bodies showing intact morphology and immunoreactivity were counted. No dot or nerve fiber-like structures were included in this quantitative analysis. A total of 250~ 300 neurons positive to given

Fig. 3 Quantitative analysis of neurons coexpressing SST and CB1 receptors in three hypothalamic nuclei. Neurons positive to SST and/or CB1 receptor were counted from PeVN, PVN, and VMH, and data were presented as the percentage of neurons coexpressing SST and CB1 receptors in neurons positive to either SST or CB1 receptor. Data were collected from five rat brains, and a total number of 250~300 neurons were analyzed. Bars present the mean±SE (n=5)

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VMH, 66 and 25 % neurons displayed colocalization between SST and CB1, respectively.

4 Confocal photomicrographs depicting colocalization of CB1 receptor with nNOS-positive neurons in the rat brain hypothalamus. nNOS-like immunoreactivity was identified using FITC-conjugated secondary antibodies (green) whereas CB1 receptor-positive neurons were recognized in presence of Cy3-conjugated (red) secondary antibodies. In merged images, neurons expressing either nNOS or CB1 receptor or colocalization are indicated by arrowheads, tilted arrows, and arrows, respectively, while neuronal processes and nerve fibers showing colocalization are indicated by asterisks. As shown in PVN (a–c), PeVN (d–f), and VMH (g–i), not all neurons positive to nNOS or CB1 receptor displayed colocalization. Three different neuronal populations positive to either nNOS or CB1 receptor or both were frequently seen in most hypothalamic regions except AN (j–l). Nerve fibers in ME displaying nNOS or CB1 receptor-like immunoreactivity were devoid of colocalization (m–o). Scale bar=40 μm

CB1 receptor immunoreactivity which mainly confined in soma and cytoplasm (Kalifa et al. 2011). Taken these results together, the immunoreactivity seen in presence of selective antibodies was considered specific. Colocalization of CB1 Receptor and SST in the Hypothalamus Indirect immunofluorescence analysis of CB1 receptor-like immunoreactivity revealed that CB1 receptor was well expressed in all major nuclei of the hypothalamus. The intensity of receptor-like immunoreactivity varied from mild to strong. In PVN, CB1 receptor-positive neurons showed strong colocalization with SST, whereas some neurons expressing SST were devoid of CB1 receptor immunoreactivity (Fig. 2a–c). SST-positive neurons were expressed in high density in PeVN when compared to other regions. As shown in Fig. 2d–f, in PeVN, large percentage of SST-positive neurons exhibited colocalization with CB1 receptor. In neuronal cells, SST-like immunoreactivity was primarily confined in apical ending of neuronal cell bodies with strong colocalization with CB1 receptor (Fig. 2f). Furthermore, neuronal processes displayed variable degree of colocalization, and ependymal cells of third ventricle exhibited weak SSTand CB1 receptor-like immunoreactivity with poor colocalization (Fig. 2f). In addition, neuronal populations expressing either CB1 receptor- or SST-like immunoreactivity were also present in PeVN (Fig. 2f). In contrast, sparsely distributed SST-positive neurons showing colocalization with CB1 receptor or neuronal population displaying only CB1 receptor-like immunoreactivity were seen in VMH (Fig. 2g– i). In AN, neurons identified exhibit strong to moderate colocalization between SST and CB1 receptors (Fig. 2j–l). In median eminence (ME), SST- and CB1 receptor-like immunoreactivity was mostly seen in nerve fibers (Fig. 2m–o). Dorsomedial hypothalamic nucleus was devoid of SST- and CB1 receptor-like immunoreactivity (data not shown). Quantitative analysis of SST and CB1 colocalization revealed significantly distinct pattern of coexpression in different hypothalamic nuclei. As shown in Fig. 3, in PVN, 59.38 % neurons exhibited colocalization, whereas in PeVN and

CB1 Receptor and nNOS Are Coexpressed in the Hypothalamus Using double-labeled immunofluorescence immunocytochemistry, three different populations exhibiting either nNOS- or CB1 receptor-like immunoreactivity and colocalization between nNOS and CB1 receptor were observed. As shown in Fig. 4, mild to strong colocalization between nNOS and CB1 receptor was present in most nuclei of the hypothalamus, including PVN (a–c), PeVN (d–f), VMH (g–i), and AN (j–l). Neuronal populations devoid of colocalization but positive to either nNOS or CB1 receptor were frequently seen in most regions except AN (Fig. 4l). Intensely innervated nerve fibers positive to nNOS and CB1 receptor in ME were devoid of colocalization (Fig. 4m–o). In addition to neuronal cells, processes as well as interconnected nerve fibers displaying variable patterns of immunoreactivity, mostly positive to CB1 receptor, were seen throughout the hypothalamus. The ependymal cells of the third ventricle exhibited weak nNOS- and CB1 receptor-like immunoreactivity (Fig. 4d–f). We next determined the percentage distribution of neurons displaying colocalization between nNOS and CB1. As shown in Fig. 5, in PVN, PeVN, and VMH, 60, 85, and 30 % of total neurons exhibited colocalization, respectively, whereas remaining neurons displayed either CB1- or nNOS-like immunoreactivity. Colocalization of SST and nNOS in Hypothalamic, Cortical, and Striatal Brain Regions Having seen the region-specific expression of nNOS and SST with CB1 receptor, we next determined whether nNOS and SST also exhibited distinct and region-specific colocalization in the hypothalamus. As shown in Fig. 6, SST- and nNOS-positive neurons exhibited variable

Fig. 5 Representative histograms showing quantitative analysis of neurons coexpressing CB1 receptor and nNOS in PeVN, PVN, and VMH. Neurons positive to CB1 receptor and/or nNOS were counted from selective hypothalamic regions, and results were presented as the percentage of neurons coexpressing CB1 receptor and nNOS. Data were collected from five rat brains, and a total number of 250~300 neurons were analyzed. Bars present the mean±SE (n=5)

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6 Representative confocal photomicrographs showing colocalization between SST and nNOS in the rat brain hypothalamus, cortex, and striatum. SST immunoreactivity was identified by FITC fluorescence (green) while nNOS was localized by Cy3 fluorescence (red). Colocalization of SST with nNOS in merged image is indicated by arrows. Neurons positive to only SST or nNOS are indicated by arrowheads and titled arrows, respectively. Neuronal processes and nerve fibers showing colocalization are indicated by asterisks. PVN was enriched with nNOS-positive neurons which displayed weak colocalization with SST (arrows, c). Large population of nNOSpositive neurons in PVN was devoid of colocalization (tilted arrows, c). In contrast, SST-positive neurons in PeVN displayed selective and specific colocalization (arrows, f). Note that not all SST-positive neurons in PeVN exhibited colocalization (arrowheads, f). In contrast, comparatively strong colocalization was seen between SST and nNOS in VMH (arrows, i). In both of cortex (l) and striatum (o), all nNOS-positive neurons displayed colocalization with SST, whereas some cortical neurons showed only SST-like immunoreactivity (arrowheads, l). In cortex and striatum, strong immunoreactivity was also seen in neuronal processes and nerve fibers. Scale bar=40 μm

intensity of immunoreactivity in PVN (a–c), PeVN (d–f), and VMH (g–i), respectively. In PVN, nNOS-positive neurons were relatively more in numbers than SST-positive neurons and displayed weak colocalization (Fig. 6a–c). Conversely, PeVN was enriched with SST-positive neurons (Fig. 6d–f). Large population of SST-positive neurons in PeVN was lacking colocalization, whereas majority of nNOS-positive neurons in PeVN displayed mild to strong colocalization (Fig. 6d–f). In contrast to PVN and PeVN, SST- and nNOSpositive neurons were comparable and exhibited strong colocalization in VMH (Fig. 6g–i). All SST-positive neurons showed colocalization with nNOS in addition to neurons stained only for nNOS (Fig. 6g–i). Interestingly, in agreement with several previous studies, all nNOS-positive neurons in cortex (Fig. 6j–l) and striatum (Fig. 6m–o) exhibited colocalization with SST. However, in cortical brain regions, we also noticed some neurons expressing only SST but not nNOS (Fig. 6l). Quantitative analysis of SST and nNOS in hypothalamic regions is displayed in Fig. 7. In PVN, 21 % neurons exhibited strong colocalization. SST and nNOS were colocalized in 55 % of total neurons positive to SST or nNOS in PeVN, whereas in VMH, 53 % neurons displayed colocalization.

Discussion In the present study, we describe the colocalization of CB1 receptor with SST and nNOS to establish a possible relationship in hypothalamic functions, specifically in the regulation of appetite. SST and nNOS are coexpressed with CB1 receptor in the hypothalamus in region-specific manner. Comparable colocalization of SST and nNOS with CB1 receptor might be associated with cannabinoid-induced stimulation of appetite

Fig. 7 Quantitative analysis of neurons coexpressing SST and nNOS in three hypothalamic nuclei. Neurons positive to SST and/or nNOS were counted from PeVN, PVN, and VMH, and data were presented as the percentage of neurons coexpressing SST and nNOS over neurons positive to either SST or nNOS. Data were collected from five rat brains, and a total number of 250~300 neurons were analyzed. Bars present the mean ±SE (n=5)

as well as other functions linked to CB1 receptor in the hypothalamus. To our knowledge, this is the first attempt to establish the physical association between CB1 receptor and neurons positive to SST and nNOS in the hypothalamus. Furthermore, almost 50 % SST positive neurons in the hypothalamus are devoid of nNOS-like immunoreactivity, distinct from cortical and striatal regions. The functional significance of our observations is of two folds: first, we uncovered that SST and nNOS colocalized with CB1 receptor; second, the colocalization of SST with nNOS, specifically in PeVN, was different from cortex and striatum. Colocalization of CB1 receptor with SST does not necessarily mean that SST is a physiological regulator of CB1 receptor in the hypothalamus; however, it does strengthen our speculation that CB1 receptor and SST might function in concert. SST-positive neurons are rich in PeVN and colocalize with CB1 receptor, suggesting that the release of SST may indirectly stimulate CB1 receptor-positive neurons in VMH or directly activate CB1 receptor-positive neurons in AN to stimulate appetite. It has been described previously that using intracerebroventricular administration of low SST doses in rats increases food intake whereas high doses resulted in loss of appetite (Danguir 1988; Aponte et al. 1984). Importantly, such effect was reversed in presence of SSTR2 antagonist, suggesting that SST-induced appetite might be linked to SSTR2 activation. Of the note, intraperitoneal injection had no similar effect, suggesting the central effect of SST (Stengel et al. 2010). Additionally, enhanced SST release in pituitary during food restriction may well account for the activated food drive in such condition (Mounier et al. 1989). Furthermore, studies have also suggested that CB1 receptor is required for α-melanocyte-stimulating hormone, the cleavage product of POMC, to modulate appetite (Verty et al. 2004). Activation of POMC neurons leads to an anorexigenic effect while antagonism causes obesity (Elias et al. 1999). Considering the

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presence of SST in hypothalamic POMC neurons, it is also highly possible that SST may indirectly exert inhibitory effect on POMC-positive neurons and stimulate appetite. In the hypothalamus, PVN and VMH are known to be the centers of satiety, and inhibition of PVN neural activity stimulates appetite and weight gain (Grandison and Guidotti 1977). The administration of γ-aminobutyric acid (GABA) in PVN and VMH leads to increased food intake (Kelly et al. 1977). Consistent with previous studies, GABAergic neurons express CB1 receptor in VMH (Reguero et al. 2011). Moreover, SST and CB1 receptors are known to inhibit GABA release (Wilson and Nicoll 2002; Leresche et al. 2000). However, studies have also revealed the inhibitory effect of SST and CB1 receptors on glutamatergic neurons, whereas the activation of excitatory neurons in the lateral hypothalamus stimulates appetite (Stanley et al. 2011). In this direction, we predict that the role of SST and CB1 receptors in food intake is differentially regulated by different nuclei in the hypothalamus by modulating GABAergic and glutamatergic neurotransmission. In the CNS and PNS, NO is recognized as a neurotransmitter. NO has been shown to exert a profound role in mediating the release of hypothalamic peptides through neurons containing nNOS. Neuroendocrine function of nNOS is supported by the fact that these neurons in the hypothalamus contain oxytocin, vasopressin (VP), and CRF (Yamada et al. 1996). In addition, NOergic neurons actively participate in controlling the release of CRF, prolactin, VP, GHRH, and SST (Aguila 1994; Karanth et al. 1993; Ota et al. 1993; Rettori et al. 1994). Similar interaction between CB1 receptor and several important hypothalamic hormones, such as leptin, CRF, and NPY, has been well documented (Bellocchio et al. 2008). Di and colleagues described that the release of glucocorticoids in PVN leads to activation of presynaptic CB1 receptor in glutamatergic neurons and postsynaptic nNOS in GABAergic neurons, consequently leading to the reduction of glucocorticoid-induced hormone release, such as VP and oxytocin (Di et al. 2003, 2009). These results clearly indicate the existence of two distinct populations of neurons expressing CB1 receptor and nNOS in PVN and their differential regulation in energy control. Taken into consideration that glucocorticoids increase food intake by activating presynaptic CB1 receptor in PVN, whether nNOS exert any plausible role in concert with CB1 receptor in the regulation of appetite is not known and future studies are needed (Malcher-Lopes et al. 2006). Moreover, CB1 receptor has been shown to interact with nNOS in several in vivo and in vitro models. CB1 receptor-mediated nNOS inhibition has been observed in rat neurogenesis zones as well as cultured rat cerebellar granule cells, supporting their interaction in other areas (Hillard et al. 1999; Kim et al. 2006). Having seen the colocalization of CB1 receptor with SST and nNOS, respectively, it will be interesting to know

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whether SST and nNOS modulate CB1 receptor activity together to affect appetite and energy control. Ghrelininduced hyperphagic effect is abolished by CB1 receptor antagonist and in CB1 receptor knockout mice (Kola et al. 2008). The central administration of ghrelin and NPY has been shown to increase nNOS activity (Gaskin et al. 2003; Morley et al. 1999). Intracerebroventricular injection of ghrelin increases NPY mRNA expression in AN in both fed and fasted rats (Seoane et al. 2003). NPY, on the other hand, is also increased by CB1 receptor agonist while decreased by CB1 receptor antagonist (Gamber et al. 2005). We have previously shown that SST, nNOS, and NPY are coexpressed in striatum and cortex (Dawson et al. 1991). In contrast, in PeVN, two populations of SSTpositive neurons either expressing SST alone or exhibiting colocalization with nNOS were seen in present study. Whether these SST-positive neurons express NPY needs to be determined. In the hypothalamus, SST and nNOS are coexpressed, suggesting a possible regulatory role of NPY in the function of SST (Hisano et al. 1990). Together, these observations support the model that CB1 receptor, SST, and nNOS might regulate appetite and energy control through direct interaction or indirect crosstalk with other hormones in the hypothalamus. In conclusion, we anticipate that SST and nNOS in combination with cannabinoids may participate in regulation of physiological responses of the hypothalamus. It would be interesting to determine whether SSTR subtypes colocalize with CB1 receptor in rat brain and further studies are in progress in this direction. Whether ablation of SST and nNOS in general or conditional knocking down in PeVN and VMH in particular will perturb that the role of CB1 receptor is not known and needs to be determined to derive any direct pharmacological significance. Most importantly, our results emphasize that SST/ nNOS-positive neurons, which are selectively preserved in excitotoxicity and afford neuroprotection in cortex and striatum, may also function as a physiological regulator of appetite in the hypothalamus. The quantitative analysis of SST and CB1 receptor colocalization in region-specific manner is an indication of selective preference of SSTR subtypes. Accordingly to elucidate the preponderance of SST14 and SST28 selective role, the colocalization of SSTR subtypes with CB1 receptor is warranted for receptor specificity. Taken together, results presented here uncovered a potential therapeutic avenue for the development of new drugs in inducing food intake in cancer patients. Acknowledgments This work was supported by grants from Canadian Institute of Health Research (MOP 74465) and NSERC, Canada to UK. UK is a senior scholar of Michael Smith Foundation for Health Research.

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Colocalization of cannabinoid receptor 1 with somatostatin and neuronal nitric oxide synthase in rat brain hypothalamus.

Despite several overlapping functions of cannabinoid receptor 1 (CB1 receptor), somatostatin (SST), and neuronal nitric oxide synthase (nNOS) in the h...
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