Orexin Research Articles

Original Research: Orexins A and B stimulate proliferation and differentiation of porcine preadipocytes.

Orexin A (OXA) and B (OXB) are neuropeptides which regulate appetite, energy expenditure, and arousal via G-protein coupled receptors termed as OXR1 and OXR2. The aim of this study was to characterize the effects of OXA and OXB on proliferation and differentiation of porcine preadipocytes. Porcine preadipocytes express both OXRs. OXA and OXB enhance porcine preadipocyte proliferation by 54.8% or 63.2 %, respectively. OXA and OXB potentiate differentiation of porcine preadipocytes, as judged by the increased lipid accumulation and expression of proadipogenic genes. Cellular lipid content after exposure of preadipocytes for six days to 100 nM OXA or OXB increased by 82.2% or 59.2%, respectively. OXA and OXB suppressed glycerol release by 23.9% or 24.9% in preadipocytes differentiated for six days. OXA (100 nM) increased peroxisome proliferator-activated receptor gamma (PPARγ) expression in cells differentiated for 24 h by 100.5%. PPARγ expression was also stimulated in preadipocytes differentiated in the presence of 10 nM (58.3%) or 100 nM OXA (50.6%) for three days. OXB potentiated PPARγ mRNA expression at 1 nM (59%), 10 nM (53.2%), and 100 nM (73.9%) in cells differentiated for three days. OXA increased CCAAT/enhancer binding protein alpha expression in preadipocytes differentiated for six days by 65%. OXB stimulated CCAAT/enhancer binding protein beta expression in preadipocytes differentiated for three days at 10 nM (149.5%) as well as 100 nM (207.2%). Lipoprotein lipase mRNA expression increased in cells treated with 10 nM OXA by 152.6% and 100 nM OXA by 162%. Lipoprotein lipase expression increased by 134% at 100 nM OXB. Furthermore, OXA (100 nM) and OXB (100 nM) increased leptin mRNA expression in preadipocytes differentiated for three days by 49.9% or 71.3%, respectively. These data indicate that orexin receptors may be relevant in the context of white adipose tissue formation.

Orexin A attenuates palmitic acid-induced hypothalamic cell death.

Palmitic acid (PA), an abundant dietary saturated fatty acid, contributes to obesity and hypothalamic dysregulation in part through increase in oxidative stress, insulin resistance, and neuroinflammation. Increased production of reactive oxygen species (ROS) as a result of PA exposure contributes to the onset of neuronal apoptosis. Additionally, high fat diets lead to changes in hypothalamic gene expression profiles including suppression of the anti-apoptotic protein B cell lymphoma 2 (Bcl-2) and upregulation of the pro-apoptotic protein B cell lymphoma 2 associated X protein (Bax). Orexin A (OXA), a hypothalamic peptide important in obesity resistance, also contributes to neuroprotection. Prior studies have demonstrated that OXA attenuates oxidative stress induced cell death. We hypothesized that OXA would be neuroprotective against PA induced cell death. To test this, we treated an immortalized hypothalamic cell line (designated mHypoA-1/2) with OXA and PA. We demonstrate that OXA attenuates PA-induced hypothalamic cell death via reduced caspase-3/7 apoptosis, stabilization of Bcl-2 gene expression, and reduced Bax/Bcl-2 gene expression ratio. We also found that OXA inhibits ROS production after PA exposure. Finally, we show that PA exposure in mHypoA-1/2 cells significantly reduces basal respiration, maximum respiration, ATP production, and reserve capacity. However, OXA treatment reverses PA-induced changes in intracellular metabolism, increasing basal respiration, maximum respiration, ATP production, and reserve capacity. Collectively, these results support that OXA protects against PA-induced hypothalamic dysregulation, and may represent one mechanism through which OXA can ameliorate effects of obesogenic diet on brain health.

Formation of OX-1R/CB1R heteromeric complexes in embryonic mouse hypothalamic cells: Effect on intracellular calcium, 2-arachidonoyl-glycerol biosynthesis and ERK phosphorylation

Orexin 1 (OX-1R) and cannabinoid receptor (CB1R) belong to the superfamily of G-protein-coupled receptors (GPCRs) and are mostly coupled to Gq and Gi/o proteins, respectively. In vitro studies in host cells over-expressing OX-1R and CB1R revealed a functional interaction between these receptors, through either their ability to form heteromers or the property for OX-1R to trigger the biosynthesis of 2-arachidonoylglycerol (2-AG), an endogenous CB1R ligand. Since: i) OX-1R and CB1R co-espression has been described at postsynaptc sites in hypothalamic circuits involved the regulation of energy homeostasis, and ii) increased orexin-A (OX-A) and 2-AG levels occur in hypothalamic neurons during obesity, we sought here to investigate the OX-1R/CB1R interaction in embryonic mouse hypothalamic NPY/AgRP mHypoE-N41 neurons which express, constitutively, both receptors. Treatment of mHypoE-N41 cells with OX-A (0.1-0.3μM), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3μM), transiently elevated [Ca2+]i. Incubation with a subeffective dose of OX-A (0.1μM)+ACEA (0.1μM) led to stronger and longer lasting elevation of [Ca2+]i, antagonized by OX-1R or CB1R antagonism with SB-334867 or AM251, respectively. FRET and co-immunoprecipitation experiments showed the formation of OX-1R/CB1R heteromers after incubation with OX-A (0.2μM), or OX-A (0.1μM)+ACEA (0.1μM), but not after ACEA (0.2μM), in a manner antagonized by SB-334867 or AM251. OX-A (0.2μM) or OX-A (0.1μM)+ACEA (0.1μM) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2Thr202/185 phosphorylation in comparison to basal or each agonist alone (0.1-0.2μM), was induced by incubation with OX-A (0.1μM)+ACEA (0.1μM), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca2+]i, or in combination with ACEA, at subeffective concentrations, triggers intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by 2-AG.

Abstract 4581: Combination treatment of orexin-A and NAB-paclitaxel in pancreas cancer: in vitro and in vivo studies

Abstract Orexin-A (OxA) and orexin-B (OxB) are hypothalamic peptides involved in the sleep/wake control which interact with two class A GPCR, OX1R and OX2R. We have demonstrated that OX1R was highly expressed in digestive cancers including cancer of colon1, pancreas2 and liver. In these cancers, orexin-A induces a mitochondrial apoptosis and a strong inhibition of tumor growth in nude mice xenografted with digestive cancer cell lines1,2. In the present work, we have compared and combined the effect of OxA and NAB-paclitaxel which represents the “gold standard” reference in the chemotherapeutic treatment of pancreas cancer, on their anti-tumoral properties. The incubation of AsPC-1 pancreatic cancer cell line which expressed OX1R, with 0.1μM OxA or 0.1 μM NAB-paclitaxel reveals a cell growth inhibition of 36% and 51%, respectively. The addition of 0.1 μM OxA and 0.1μM NAB-paclitaxel on AsPC-1 cells reveals a significantly cell growth inhibition of 70% suggesting that the double treatment was more efficient than individual treatment. Moreover, the addition of 0.1μM OxA and 0.1 μM NAB-paclitaxel induces 25% of cell apoptosis determined by annexin-V labeling, as compared to single treatment with 0.1μM OxA (18%) or 0.1 μM NAB-paclitaxel (12%). Additionally, we explore the sequential treatment by OxA and NAB-paclitaxel on cell growth of AsPC-1 cells. Our results evidenced than 48h treatment by OxA followed by 48h treatment of NAB-paclitaxel induced an inhibition of 35% of cell growth. In contrast, the reverse treatment (48H NAB-paclitaxel followed by 48h OxA) induces an inhibition of cell growth of 60%. OxA intraperitoneal injection (2 injections/week of 1.12 μmoles/kg OxA and/or NAB-paclitaxel) in nude mice xenografted with AsPC-1 cells, shows that OxA and NAB-paclitaxel induces an inhibition of tumoral volume of 60% and 62%, respectively. Moreover, injection of OxA plus NAB-paclitaxel induces an inhibition of tumoral volume of 70%. Sequential treatments of xenografted tumors in mice with OxA and NAB-paclitaxel was investigated and revealed 72% tumor growth inhibition when mice were treated 30 days with OxA followed by 30 days with NAB-paclitaxel and 83% tumor growth inhibition when they were treated 30 days with NAB-paclitaxel followed by 30 days with OxA. These results indicate that: 1) the addition of OxA and NAB-paclitaxel improves the effect of individual treatment; 2) the sequential treatment consisting of first OxA treatment followed by NAB-paclitaxel treatment was more efficient than reverse treatment. In conclusion, OxA was close to NAB-paclitaxel treatment in term of response and suggest that combined treatment OxA/ NAB-paclitaxel represents a new promising pancreas cancer therapy 1Voisin et al., Cancer research 2011, 71:3341-51 2Speisky et al., AACR annual meeting, 2014, San Diego, USA Citation Format: Thierry Voisin, Dina Plaut, Stephanie Dayot, Maxime Bergere, Valerie Gratio, Pascal Nicole, Anne Couvelard, Alain Couvineau. Combination treatment of orexin-A and NAB-paclitaxel in pancreas cancer: in vitro and in vivo studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4581.

Nucleus incertus Orexin2 receptors mediate alcohol seeking in rats

Alcoholism is a chronic relapsing disorder and a major global health problem. Stress is a key precipitant of relapse in human alcoholics and in animal models of alcohol seeking. The brainstem nucleus incertus (NI) contains a population of relaxin-3 neurons that are highly responsive to psychological stressors; and the ascending NI relaxin-3/RXFP3 signalling system is implicated in stress-induced reinstatement of alcohol seeking. The NI receives orexinergic innervation and expresses orexin1 (OX1) and orexin2 (OX2) receptor mRNA. In alcohol-preferring (iP) rats, we examined the impact of yohimbine-induced reinstatement of alcohol seeking on orexin neuronal activation, and the effect of bilateral injections into NI of the OX1 receptor antagonist, SB-334867 (n = 16) or the OX2 receptor antagonist, TCS-OX2-29 (n = 8) on stress-induced reinstatement of alcohol seeking. We also assessed the effects of orexin-A on NI neuronal activity and the involvement of OX1 and OX2 receptors using whole cell patch-clamp recordings in rat brain slices. Yohimbine-induced reinstatement of alcohol seeking activated orexin neurons. Bilateral NI injections of TCS-OX2-29 attenuated yohimbine-induced reinstatement of alcohol seeking. In contrast, intra-NI injection of SB-334867 had no significant effect. In line with these data, orexin-A (600 nM) depolarized a majority of NI neurons recorded in coronal brain slices (18/28 cells), effects prevented by bath application of TCS-OX2-29 (10 μM), but not SB-334867 (10 μM). These data suggest an excitatory orexinergic input to NI contributes to yohimbine-induced reinstatement of alcohol seeking, predominantly via OX2 receptor signalling.

Localization and expression of Orexin A and its receptor in mouse testis during different stages of postnatal development

Orexin A (OXA), a hypothalamic neuropeptide, is involved in regulation of various biological functions and its actions are mediated through G-protein-coupled receptor, OX1R. This neuropeptide has emerged as a central neuroendocrine modulator of reproductive functions. Both OXA and OX1R have been shown to be expressed in peripheral organs such as gastrointestinal and genital tracts. In the present study, localization and expression of OXA and OX1R in mouse testis during different stages of postnatal development have been investigated. Immunohistochemical results demonstrated localization of OXA and OX1R in both the interstitial and the tubular compartments of the testis throughout the period of postnatal development. In testicular sections on 0day postpartum (dpp), gonocytes, Sertoli cells and foetal Leydig cells showed OXA and OX1R-immunopositive signals. At 10dpp, Sertoli cells, spermatogonia, early spermatocytes and Leydig cells showed immunopositive signals for both, the ligand and the receptor. On 30 and 90dpp, the spermatogonia, Sertoli cells, spermatocytes, spermatids and Leydig cells showed the OXA and OX1R-immunopositive signals. At 90dpp, strong OXA-positive signals were seen in Leydig cells, primary spermatocytes and spermatogonia, while OX1R-immunopositive intense signals were observed in Leydig cells and elongated spermatids. Further, semiquantitative RT-PCR and immunoblot analyses showed that OXA and OX1R were expressed in the testis both at transcript and protein levels during different stages of postnatal development. The expression of OXA and OX1R increased progressively from day of birth (0dpp) until adulthood (90dpp), with maximal expression at 90 dpp. The results suggest that OXA and OX1R are expressed in the testis and that they may help in proliferation and development of germ cells, Leydig cells and Sertoli cells, and in the spermatogenic process and steroidogenesis.

Changes in orexinergic immunoreactivity of the piglet hypothalamus and pons after exposure to chronic postnatal nicotine and intermittent hypercapnic hypoxia.

We recently showed that orexin expression in sudden infant death syndrome (SIDS) infants was reduced by 21% in the hypothalamus and by 40-50% in the pons as compared with controls. Orexin maintains wakefulness/sleeping states, arousal, and rapid eye movement sleep, abnormalities of which have been reported in SIDS. This study examined the effects of two prominent risk factors for SIDS, intermittent hypercapnic hypoxia (IHH) (prone-sleeping) and chronic nicotine exposure (cigarette-smoking), on orexin A (OxA) and orexin B (OxB) expression in piglets. Piglets were randomly assigned to five groups: saline control (n=7), air control (n=7), nicotine [2mg/kg per day (14 days)] (n=7), IHH (6min of 7% O2 /8% CO2 alternating with 6-min periods of breathing air, for four cycles) (n=7), and the combination of nicotine and IHH (N+IHH) (n=7). OxA/OxB expression was quantified in the central tuberal hypothalamus [dorsal medial hypothalamus (DMH), perifornical area (PeF), and lateral hypothalamus], and the dorsal raphe, locus coeruleus of the pons. Nicotine and N+IHH exposures significantly increased: (i) orexin expression in the hypothalamus and pons; and (ii) the total number of neurons in the DMH and PeF. IHH decreased orexin expression in the hypothalamus and pons without changing neuronal numbers. Linear relationships existed between the percentage of orexin-positive neurons and the area of pontine orexin immunoreactivity of control and exposure piglets. These results demonstrate that postnatal nicotine exposure increases the proportion of orexin-positive neurons in the hypothalamus and fibre expression in the pons, and that IHH exposure does not prevent the nicotine-induced increase. Thus, although both nicotine and IHH are risk factors for SIDS, it appears they have opposing effects on OxA and OxB expression, with the IHH exposure closely mimicking what we recently found in SIDS.

Heterodimerization of orexin 2 alpha and 2 beta receptor

Objective To explore the possibility of heterodimerization between orexin type 2α receptor (OX2αR) and orexin type 2β receptor (OX2βR). Methods Using confocal laser scanning microscope, enzyme linked immunosorbent assay (ELISA), fluorescence resonance energy transfer (FRET) and Bioluminescence resonance energy transfer (BRET) to study the interaction between OX2αR and OX2βR. Result Confocal laser scanning microscope and ELISA showed that OX2αR and OX2βR were both expressed in the cytoplasm.The FRET demonstrated that the signal of the experimental group (OX2αR-YFP+ OX2βR-CFP) was significantly stronger than that of control group (YFP+ OX2βR-CFP). The BRET value of the experimental group (OX2αR-YFP+ OX2βR-Rluc, mBRET ratio was 65±15) was higher than that of control group (YFP+ OX2βR-Rluc/ OX2αR-YFP+ Rluc, mBRET ratio was 10±5) (P<0.05). Conclusion There are heterodimerization between mOX2αR and mOX2βR. Key words: Orexin receptor; Fluorescence resonance energy transfer; Bioluminescence resonance energy transfer; Heterodimerization

Orexin-A represses satiety-inducing POMC neurons and contributes to obesity via stimulation of endocannabinoid signaling

In the hypothalamic arcuate nucleus (ARC), proopiomelanocortin (POMC) neurons and the POMC-derived peptide α-melanocyte-stimulating hormone (α-MSH) promote satiety. POMC neurons receive orexin-A (OX-A)-expressing inputs and express both OX-A receptor type 1 (OX-1R) and cannabinoid receptor type 1 (CB1R) on the plasma membrane. OX-A is crucial for the control of wakefulness and energy homeostasis and promotes, in OX-1R-expressing cells, the biosynthesis of the endogenous counterpart of marijuana's psychotropic and appetite-inducing component Δ(9)-tetrahydrocannabinol, i.e., the endocannabinoid 2-arachidonoylglycerol (2-AG), which acts at CB1R. We report that OX-A/OX-1R signaling at POMC neurons promotes 2-AG biosynthesis, hyperphagia, and weight gain by blunting α-MSH production via CB1R-induced and extracellular-signal-regulated kinase 1/2 activation- and STAT3 inhibition-mediated suppression of Pomc gene transcription. Because the systemic pharmacological blockade of OX-1R by SB334867 caused anorectic effects by reducing food intake and body weight, our results unravel a previously unsuspected role for OX-A in endocannabinoid-mediated promotion of appetite by combining OX-induced alertness with food seeking. Notably, increased OX-A trafficking was found in the fibers projecting to the ARC of obese mice (ob/ob and high-fat diet fed) concurrently with elevation of OX-A release in the cerebrospinal fluid and blood of mice. Furthermore, a negative correlation between OX-A and α-MSH serum levels was found in obese mice as well as in human obese subjects (body mass index > 40), in combination with elevation of alanine aminotransferase and γ-glutamyl transferase, two markers of fatty liver disease. These alterations were counteracted by antagonism of OX-1R, thus providing the basis for a therapeutic treatment of these diseases.

Central Exogenous Neuropeptide‐S Ameliorates Stress‐induced Impaired Gastric Emptying through Orexin‐mediated Pathway in Rats

Stress is widely believed to play a major role in developing functional gastrointestinal (GI) disorders. Acute restraint stress (ARS) stimulates corticotrophin releasing factor (CRF) in the amygdala and paraventricular nucleus (PVN) of the hypothalamus which in turn plays a dominant role to delay gastric emptying (GE) via peripheral autonomic neural pathways in rats. Neuropeptide‐S (NPS), composed of 20 amino acids, is the endogenous ligand for the NPS receptor (NPSR) which is highly conserved in vertebrates including human. Within central nervous system (CNS), the NPS‐producing neurons are found mainly in locus coeruleus (LC) and amygdala which is involved in coping with fear and stress. Activation of the hypothalamus by stress is likely to be mediated in part by amygdala, the key limbic structure involved in the stress‐induced effects on the GI functions. Furthermore, CRF has been shown to excite NPS neurons in LC through the CRF‐1 receptor. In rodents, centrally administered NPS has been shown to inhibit anxiety, stress behaviors and stress‐stimulated fecal output in the rats. Orexin‐A (OXA), a hypothalamic neuropeptide, regulates feeding activity, energy homeostasis and arousal. Orexin receptor type‐1 (OXR1) is highly expressed in the dorsal vagal motor nucleus and OXA was shown to stimulate postprandial gastric motor functions via central vagal pathways. On the other hand, it has been shown that centrally administered NPS activates orexin‐containing neurons in hypothalamus in rats suggesting that OXA may contribute to the NPS‐mediated alterations in gastric motor functions under stressed conditions. To perform neuropeptide microdialysis, a microdialysis probe with large pore membrane was unilaterally placed into the central amygdala and LC. Central administrations of the drugs were performed through an injection cannula stereotaxically placed in the right lateral ventricle. NPS concentrations in microdialysates were assayed using EIA method. Solid GE was measured following an overnight fasting. Compared to their basal levels, ARS significantly reduced the NPS concentrations both in LC (5564.8 ± 186.8 ng/ml vs 562.9 ± 86.8 ng/ml, p&lt;0.01) and amygdala (5197.7 ± 217.1 ng/ml vs 91.8 ± 12.18 ng/ml, p&lt;0.01). In control rats, GE was 61.2 ± 3.4%. ARS significantly delayed gastric emptying 24.4 ± 3.2%; whereas, central administration of NPS (8 nmol, icv) completely restored the delayed GE induced by ARS (62.5 ± 5.7%, p&lt;0.01). Pretreatment of selective OXA antagonist SB‐334867 (40 μg, icv) significantly (p&lt;0.05) abolished the NPS‐induced restoration of delayed GE (38.9 ± 2.7%, p&lt;0.05). It is suggested that stress may alter NPS expression within CNS. Exogenous administration of NPS restores stress‐induced gastric dysmotility which was abolished by OXA antagonist, suggesting that the effect of NPS on stress‐induced gastric motility is partly OXA‐dependent. Central NPS may be a novel candidate for the treatment of stress‐induced gastric motility disorders.

Relationship of the Chemokine, CXCL12, to Effects of Dietary Fat on Feeding-Related Behaviors and Hypothalamic Neuropeptide Systems.

The intake of a high fat diet (HFD), in addition to stimulating orexigenic neuropeptides in the hypothalamus while promoting overeating and reducing locomotor behavior, is known to increase inflammatory mediators that modulate neuronal systems in the brain. To understand the involvement of chemokines in the effects of a HFD, we examined in rats whether HFD intake affects a specific chemokine, CXCL12, and its receptors, CXCR4 and CXCR7, in the hypothalamus together with the neuropeptides and whether CXCL12 itself acts similarly to a HFD in stimulating the neuropeptides and altering ingestion and locomotor behavior. Compared to low-fat chow, a HFD for 5 days significantly increased the expression of CXCL12 and its receptors, in both the paraventricular nucleus (PVN) where the neuropeptides enkephalin (ENK) and galanin were also stimulated and the perifornical lateral hypothalamus (PFLH) where orexin (OX) and melanin-concentrating hormone (MCH) were increased. In contrast, the HFD had no impact on expression of CXCL12 or its receptors in the arcuate nucleus (ARC) where the carbohydrate-related peptide, neuropeptide Y (NPY), was suppressed. Analysis of protein levels revealed a similar stimulatory effect of a HFD on CXCL12 levels in the PVN and PFLH, as well as in blood, and an increase in the number of CXCR4-positive cells in the PVN. In the ARC, in contrast, levels of CXCL12 and number of CXCR4-positive cells were too low to measure. When centrally administered, CXCL12 was found to have similar effects to a HFD. Injection of CXCL12 into the third cerebral ventricle immediately anterior to the hypothalamus significantly stimulated the ingestion of a HFD, reduced novelty-induced locomotor activity, and increased expression of ENK in the PVN where the CXCR4 receptors were dense. It had no impact, however, on NPY in the ARC or on OX and MCH in the PFLH where the CXCR4 receptors were not detected. These results, showing CXCL12 in the hypothalamus to be stimulated by a HFD and to mimic the effects of the HFD where its receptors are located, suggest that this chemokine system may have a role in mediating both the neuronal and behavioral effects induced by a fat-rich diet.

Role of Leptin and Orexin-A Within the Suprachiasmatic Nucleus on Anxiety-Like Behaviors in Hamsters.

It is well established that the maintenance of energy expenditure is linked to active hypothalamic neural mechanisms controlling adaptive stimuli such as food intake. Variations of glucose levels and hormonal (leptin plus orexin-A) parameters, which are involved with energy homeostasis during different behavioral states, have not yet been fully defined. In this first study, behavioral analyses of an unpredictable stress model dealing with the actions of a sub-chronic administration of orexin-A (ORX-A) and the anti-hunger neuropeptide, i.e., leptin (LEP) within the hypothalamic suprachiasmatic (SCH) nucleus, were conducted on the valuable hibernating rodent (hamster; Mesocricetus auratus) model noted for its distinct depression and anxiety states. Treatment with LEP accounted for a notable reduction (p < 0.01) of body weight in stressed hamsters that not only executed very evident (p < 0.001) movements to and from elevated plus maze (EPM) but also spent less time in the dark area of the light-dark box test (LDT). Conversely, ORX-A predominantly evoked anxiogenic effects that were inverted by LEP. Interestingly, the anti-hunger neuropeptide accounted for both down-regulated NPY1 transcripts in mostly lateral-posterior hypothalamic areas while up-regulated levels were detected in the parietal cerebral cortex, hippocampus, and amygdala, which largely behaved in an opposite manner to ORX-A-dependent effects. Overall, the present findings corroborate a predominating LEPergic effect of the SCH toward the reduction of hamster anxiety-like behaviors with respect to that of ORX-A signaling, which may constitute useful therapeutic targets for stress-related obesity states.

Ketamine and propofol have opposite effects on postanesthetic sleep architecture in rats: relevance to the endogenous sleep-wakefulness substances orexin and melanin-concentrating hormone.

Anesthesia and surgery disturb sleep. Disturbed sleep adversely affects postoperative complications involving the cardiovascular system, diabetes, and infection. General anesthetics share neuronal mechanisms involving endogenous sleep-wakefulness-related substances, such as orexin (OX) and melanin-concentrating hormone (MCH). We evaluated changes in sleep architecture and the concentration of OX and MCH during the peri-anesthetic period. To examine sleep architecture, male Sprague-Dawley rats weighing 350-450g received ketamine 100mg/kg (n=9) or propofol 80mg/kg (n=6) by intraperitoneal injection. Electroencephalography was recorded from 2days pre- to 5days postanesthesia. To quantify levels of OX and MCH, 144 similar rats received the same doses of ketamine (n=80) or propofol (n=64). Brain concentrations of these substances were determined at 0, 20, 60, and 120min after anesthetic administration. Ketamine decreased OX content in the hypothalamus during the anesthesia period. OX content was restored to pre-anesthesia levels in the hypothalamus and pons. Both anesthetics increased brain MCH content in the postanesthetic period, with the degree of increase being greater with propofol. Ketamine enhanced wakefulness and inhibited non-rapid eye movement sleep (NREMS) immediately after anesthesia. Conversely, propofol inhibited wakefulness and enhanced NREMS in that period. Ketamine inhibited wakefulness and enhanced NREMS during the dark phase on the first postanesthesia day. Anesthetics affect various endogenous sleep-wakefulness-related substances; however, the modulation pattern may depend on the type of anesthetic. The process of postanesthetic sleep disturbance was agent specific. Our results provide fundamental evidence to treat anesthetic-related sleep disturbance.

Involvement of orexin-1 and orexin-2 receptors within the dentate gyrus of the hippocampus in the acquisition, expression and extinction of lateral hypothalamic-induced conditioned place preference in the rats

Orexinergic projections originating from the lateral hypothalamus (LH) have an important role in the acquisition of the LH-stimulation conditioned place preference (CPP). Among the brain areas associated with reward processing, LH orexinergic neurons send projections to the dentate gyrus (DG) region of the hippocampus, and it has been shown that orexin receptors are expressed in the DG. In this study, we investigated the role of intra-DG orexin-1 (OX1) and orexin-2 (OX2) receptors on acquisition, expression and extinction of CPP induced by stimulation of the LH. Rats were unilaterally implanted by two separate cannulae into the LH and DG. The CPP paradigm was done; conditioning scores and locomotor activities were recorded by Ethovision software. The results showed that intra-DG administration of SB334867, a selective OX1r antagonist, and TCS OX2 29, a selective OX2r antagonist, (0.5, 5, 12.5 and 50nM/0.5µl DMSO) before carbachol microinjection (250nM; effective dose) during the 3-days conditioning phase, dose-dependently inhibited the development of LH stimulation-induced CPP in the rats. However, this reduction in OX1r antagonist treated groups was more than that in OX2r antagonist treated animals. In addition, these antagonists decreased the expression of LH-induced CPP. Moreover, OX1r but not OX2r antagonist could shorten the extinction duration of place preference. We conclude that the orexinergic projections from the LH to DG are involved in the development, expression and extinction of CPP induced by LH stimulation.

Developmental Responses of the Lateral Hypothalamus to Leptin in Neonatal Rats, and its Implications for the Development of Functional Connections with the Ventral Tegmental Area

Food intake is regulated by a close communication between the hypothalamus and the mesocorticolimbic pathways, which are still developing during the perinatal period in the rat, and are known targets for peripheral metabolic hormones such as leptin. A key region for this communication is the lateral hypothalamus (LH), although the onset of leptin responsiveness in the LH is unknown. We examined the activation of cellular signalling molecules in identified LH neurones on postnatal day (PND)10 and 16 and determined whether leptin directly targets orexin A (ORX-A) or neurotensin (NT) LH neurones through the detection of leptin receptors (ObRb) mRNA on these neurones. Next, using retrograde labelling in PND6 pups, we tested whether phenotypically identified neurones of the LH that respond to leptin project to ventral tegmental area (VTA) neurones. Leptin significantly induced phosphorylated extracellular signal-regulated kinase (pERK)1/2 and phosphorylated signal transducer activator of transcription (pSTAT)3 in the LH on PND16, whereas, on PND10, modest pERK1/2- and sparse pSTAT3-positive cells were identified. On PND16, most pERK1/2-activated neurones contain ORX-A and leptin-induced pSTAT3 was observed in other unidentified neurones. Afferents to the VTA were observed on PND6, including a large input from the LH, which contained both ORX-A-positive and non-ORX-A neurones, with some of these ORX-A neurones being activated by leptin treatment. Leptin receptor (ObRb) mRNA in the LH did not colocalise with ORX-A neurones on PND10, and only a few NT-positive neurones displayed ObRb mRNA expression. Thus, functional responsiveness to leptin in LH neurones is only partially achieved prior to the onset of independent feeding on PND16, and ORX-A neurones are indirectly activated by leptin. The presence of anatomical connections between the LH and the VTA in the first week of life, prior to the development of leptin responsiveness in both structures, suggests that tissue responsiveness to leptin, rather than the maturation of neuronal connections, critically regulates the onset of independent feeding.

Orexin/hypocretin neuron activation is correlated with alcohol seeking and preference in a topographically specific manner.

Orexin (ORX) (also known as hypocretin) neurons are located exclusively in the posterior hypothalamus, and are involved in a wide range of behaviours, including motivation for drugs of abuse such as alcohol. Hypothalamic subregions contain functionally distinct populations of ORX neurons that may play different roles in regulating drug-motivated and alcohol-motivated behaviours. To investigate the role of ORX neurons in ethanol (EtOH) seeking, we measured Fos activation of ORX neurons in rats following three different measures of EtOH seeking and preference: (i) context-induced reinstatement, or ABA renewal; (ii) cue-induced reinstatement of extinguished responding for EtOH; and (iii) a home cage task in which preference for EtOH (vs. water) was measured in the absence of either reinforcer. We found significant activation of ORX neurons in multiple subregions across all three behavioural tests. Notably, ORX neuron activation in the lateral hypothalamus correlated with the degree of seeking in context reinstatement and the degree of preference in home cage preference testing. In addition, Fos activation in ORX neurons in the dorsomedial hypothalamic and perifornical areas was correlated with context and home cage seeking/preference, respectively. Surprisingly, we found no relationship between the degree of cue-induced reinstatement and ORX neuron activation in any region, despite robust activation overall during reinstatement. These results demonstrate a strong relationship between ORX neuron activation and EtOH seeking/preference, but one that is differentially expressed across ORX field subregions, depending on reinstatement modality.

ID 32 – Mild network activation by orexin - A avoids hyper excitability in input deprived cortical networks

Objective Input deprived (sub)networks of cortical neurons may become hyper excitable, possibly causing certain types of epilepsy. Electrical stimulation and application of the mildly excitatory neurotransmitter ghrelin have been shown to reduce this hyper excitability, suggesting that it is a generic feature of mild excitation. Therefore, we hypothesize that another mildly excitatory agent, orexin-A (OXA), should also reduce excitability. Methods We used cultures of cortical neurons on multi electrode arrays to measure network activity. If left in isolation, activity patterns typically contain bursts that reflect network hyper excitability. The relative contribution of bursts to the total amount of spikes was used as a measure for excitability and assessed by the burstiness index (BI = 1 → burst dominated; BI = 0 → no bursts). Results Acute application of 1 μM OXA significantly increased the level of activity from 1500 ± 650(SEM) to 3200 ± 1300 spikes/min. Burstiness index dropped from 0.75 ± 0.09 to 0.58 ± 0.09 indicating more dispersed firing. Conclusions Like electrical stimulation or ghrelin, OXA mildly activates cultured networks of cortical neurons and reduces burstiness. Key message Besides cellular hyper excitability, networks may also become hyper excitable in case of insufficient ongoing activation. This may be treated by mild activation, rather than the commonly used anti-epileptic drugs that aim to reduce activity.

Dual projections of single orexin- or CART-immunoreactive, lateral hypothalamic neurons to the paraventricular thalamic nucleus and nucleus accumbens shell in the rat: Light microscopic study

The paraventricular thalamic nucleus (PVT) is a major relay station to the limbic forebrain areas such as the nucleus accumbens shell (AcbSh). Both PVT and AcbSh are known to receive feeding/arousal-related peptidergic fibers including orexin (ORX) and cocaine- and amphetamine-regulated transcript (CART) peptide. In the first series of experiments, we examined the peptidergic fiber distribution in the AcbSh; the density of ORX (or CART) fibers in the AcbSh was substantially lower than that in the PVT. At the light microscopic level, ORX (or CART) terminals formed close appositions to choline acetyltransferase (ChAT)-, glutamate decarboxylase (GAD)-, or enkephalin (Enk)-immunoreactive neuronal elements in the AcbSh. In the second series of experiments, we addressed the question of whether single ORX (or CART) cells in the hypothalamus provided divergent axon collaterals to the PVT and AcbSh. ORX neurons with dual projections were found in the medial, central, and lateral subdivisions of the lateral hypothalamus (LH), which amounted to an average of 1.6% of total ORX cells. CART neurons with divergent axon collaterals were observed in the LH, zona incerta, dorsal hypothalamic area, and retrochiasmatic nucleus, which represented a mean of 2.5% of total CART cells. None of arcuate CART cells sent dual projections. These data suggested that a portion of ORX (or CART) neurons in the hypothalamus, via divergent axon collaterals, might concurrently modulate the activity of PVT and AcbSh cells to affect feeding and drug-seeking behaviors.

Role of orexin-A in experimental autoimmune encephalomyelitis

The aim of this study was to evaluate the effects of orexin-A (OX-A) on behavioral and pathological parameters and on gene expression of some multiple sclerosis-related peptides in a model of experimental autoimmune encephalomyelitis (EAE). EAE was induced by subcutaneous administration of MOG 35–55. Following immunization, the treatment was initiated by using SB.334867 (orexin-1 receptor antagonist) and/or OX-A. Locomotor activity and exploratory behaviors were monitored using open field and T-maze continuous alternation task (T-CAT) respectively. Pain sensitivity was assessed by hot-plate test. Histopathological assessments were performed by H&E staining. The expression of TGF-β, MBP, MMP-9, IL-12, iNOS and MCP-1 were measured using real-time PCR method in lumbar spinal cord. OX-A administration in EAE mice remarkably attenuated the clinical symptoms, increased latency response in hot plate test, inhibited infiltration of inflammatory cells, up-regulated mRNA expression of TGF-β as well as MBP and down-regulated mRNA expression of iNOS, MMP-9 and IL-12. In contrast SB.334867 administration in EAE mice deteriorated the clinical symptoms, decreased the alternation in T-CAT, increased infiltration of inflammatory cells, down-regulated mRNA expression of TGF-β and MBP and up-regulated mRNA expression of iNOS. Results of this study suggest that the orexinergic system might be involved in pathological development of EAE. These findings suggest orexinergic system as a potential target for treatment of multiple sclerosis.

Effects of embryonic ethanol exposure at low doses on neuronal development, voluntary ethanol consumption and related behaviors in larval and adult zebrafish: Role of hypothalamic orexigenic peptides

Embryonic exposure to ethanol is known to affect neurochemical systems in rodents and increase alcohol drinking and related behaviors in humans and rodents. With zebrafish emerging as a powerful tool for uncovering neural mechanisms of numerous diseases and exhibiting similarities to rodents, the present report building on our rat studies examined in zebrafish the effects of embryonic ethanol exposure on hypothalamic neurogenesis, expression of orexigenic neuropeptides, and voluntary ethanol consumption and locomotor behaviors in larval and adult zebrafish, and also effects of central neuropeptide injections on these behaviors affected by ethanol. At 24h post-fertilization, zebrafish embryos were exposed for 2h to ethanol, at low concentrations of 0.25% and 0.5%, in the tank water. Embryonic ethanol compared to control dose-dependently increased hypothalamic neurogenesis and the proliferation and expression of the orexigenic peptides, galanin (GAL) and orexin (OX), in the anterior hypothalamus. These changes in hypothalamic peptide neurons were accompanied by an increase in voluntary consumption of 10% ethanol–gelatin and in novelty-induced locomotor and exploratory behavior in adult zebrafish and locomotor activity in larvae. After intracerebroventricular injection, these peptides compared to vehicle had specific effects on these behaviors altered by ethanol, with GAL stimulating consumption of 10% ethanol–gelatin more than plain gelatin food and OX stimulating novelty-induced locomotor behavior while increasing intake of food and ethanol equally. These results, similar to those obtained in rats, suggest that the ethanol-induced increase in genesis and expression of these hypothalamic peptide neurons contribute to the behavioral changes induced by embryonic exposure to ethanol.