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This study examined the effect of leptin and orexin-A on autonomic baroreflex control in conscious Wistar rats exposed to high-fat (45% fat) or normal (3.4%) diet for 4 weeks. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were monitored during the generation of baroreflex gain curves and acute volume expansion (VEP). Intracerebroventricular (ICV) leptin (1 μg/min) increased RSNA in the normal diet group (0.31 ± 0.04 vs 0.23 ± 0.03 mV/s) and MAP in the high-fat diet group (115 ± 5 vs 105 ± 5 mm Hg, P < .05). Orexin-A (50 ng/min) increased RSNA, HR and MAP in the high-fat diet group (0.26 ± 0.03 vs 0.22 ± 0.02 mV/s, 454 ± 8 vs 417 ± 12 beats/min, 117 ± 1 vs 108 ± 1 mm Hg) and the normal diet group (0.18 ± 0.05 vs 0.17 ± 0.05 mV/s, 465 ± 10 vs 426 ± 6 beats/min, 116 ± 2 vs 104 ± 3 mm Hg). Baroreflex sensitivity for RSNA was increased during ICV leptin by 50% in the normal diet group, compared to 14% in the high-fat diet group (P < .05). Similarly, orexin-A increased baroreflex sensitivity by 56% and 50% in the high-fat and normal diet groups, respectively (all P < .05). During ICV saline, VEP decreased RSNA by 31 ± 5% (P < .05) after 10 minutes and the magnitude of this response was blunted during ICV infusion of leptin (17 ± 2%, P < .05) but not orexin-A in the normal diet group. RSNA response to VEP was not changed during ICV leptin or orexin-A in the high-fat diet group. These findings indicate possible central roles for leptin and orexin-A in modulating the baroreflexes under normal or increased fat intake in conscious rats and potential therapeutic approaches for obesity associated hypertension., (© 2020 John Wiley & Sons Australia, Ltd.)

New Findings: • What is the central question of this study? We hypothesized that central inflammatory processes that involve activation of microglia and astrocytes contribute to the development of Gαi 2 protein-dependent, salt-sensitive hypertension. • What is the main finding and its importance? The main finding is that PVN-specific inflammatory processes, driven by microglial activation, appear to be linked to the development of Gαi 2 protein-dependent, salt-sensitive hypertension in Sprague-Dawley rats. This finding might reveal new mechanistic targets in the treatment of hypertension., Abstract: The central mechanisms underlying salt-sensitive hypertension, a significant public health issue, remain to be established. Researchers in our laboratory have reported that hypothalamic paraventricular nucleus (PVN) Gαi 2 proteins mediate the sympathoinhibitory and normotensive responses to high sodium intake in salt-resistant rats. Given the recent evidence of central inflammation in animal models of hypertension, we hypothesized that PVN inflammation contributes to Gαi 2 protein-dependent, salt-sensitive hypertension. Male Sprague-Dawley rats received chronic intracerebroventricular infusions of a targeted Gαi 2 or control scrambled oligodeoxynucleotide (ODN) and were maintained for 7 days on a normal-salt (NS; 0.6% NaCl) or high-salt (HS; 4% NaCl) diet; in subgroups on HS, intracerebroventricular minocycline (microglial inhibitor) was co-infused with ODNs. Radiotelemetry was used in subgroups of rats to measure mean arterial pressure (MAP) chronically. In a separate group of rats, plasma noradrenaline, plasma renin activity, urinary angiotensinogen and mRNA levels of the PVN pro-inflammatory cytokines TNFα, IL-1β and IL-6 and the anti-inflammatory cytokine IL-10 were assessed. In additional groups, immunohistochemistry was performed for markers of PVN and subfornical organ microglial activation and cytokine levels and PVN astrocyte activation. High salt intake evoked salt-sensitive hypertension, increased plasma noradrenaline, PVN pro-inflammatory cytokine mRNA upregulation, anti-inflammatory cytokine mRNA downregulation and PVN-specific microglial activation in rats receiving a targeted Gαi 2 but not scrambled ODN. Minocycline co-infusion significantly attenuated the increase in MAP and abolished the increase in plasma noradrenaline and inflammation in Gαi 2 ODN-infused animals on HS. Our data suggest that central Gαi 2 protein prevents microglial-mediated PVN inflammation and the development of salt-sensitive hypertension., (© 2019 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

We have previously demonstrated that the mitochondria-derived cytoprotective peptide humanin (HN), when administered intratesticularly to rats, rescues germ cells from apoptosis secondary to testicular stress of hormonal deprivation induced by gonadotropin-releasing hormone antagonist (GnRH-A). To decipher the cellular mechanisms of HN action in the amelioration of GnRH-A-induced germ cell apoptosis, adult male rats received the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of GnRH-A on Day 1 and daily IT injection of saline; (iii) daily IT injection of synthetic HN; and (iv) GnRH-A injection on Day 1 and daily IT injection of HN (GnRH-A+HN). HN alone had no effect on germ cell apoptosis. GnRH-A increased germ cell apoptosis and BAX in the testicular mitochondrial fractions. Synthetic HN decreased germ cell apoptosis induced by GnRH-A and BAX in the mitochondria. We deduced that the cytoprotective action of synthetic HN on GnRH-A-induced germ cell apoptosis was mediated by attenuating p38 mitogen-activated protein kinase activity and increasing STAT3 phosphorylation. The effect of synthetic HN on the expression of endogenous rat HN in the testis was studied using rat HN specific antibody. GnRH-A treatment increased, but concomitant treatment with synthetic HN reduced endogenous rat HN expression in both cytosolic and mitochondrial fractions in testis. Co-immunoprecipitation experiments demonstrated that the increased rat HN was physically associated with BAX in the cytosolic testicular fractions after GnRH-A treatment. Double-immunofluorescence staining confirmed the co-localization of BAX and rat HN in the cytoplasm of Leydig cells and spermatocytes after GnRH-A treatment. We conclude that the cytoprotective effect of exogenously administered synthetic HN is mediated by interactions of endogenous rat HN with BAX in the cytoplasm preventing the entry of BAX to the mitochondria to govern the fate of germ cell survival or death during pro-apoptotic stress to the testis in rats., (© 2013 American Society of Andrology and European Academy of Andrology.)

Although orexin-A peptide was recently found to inhibit the brain reward system, the exact neural substrates for this phenomenon remain unclear. The aim of the present study was to investigate the role of orexin neurons in intra-cranial self-stimulation behavior and to clarify the pathways through which orexin-A inhibits the brain reward system. Immunohistochemical examination using Fos, a neuronal activation marker, revealed that the percentage of activated orexin cells was very low in the lateral hypothalamus even in the hemisphere ipsilateral to self-stimulation, suggesting that orexin neurons play only a small part, if any, in performing intra-cranial self-stimulation behavior. Intra-ventral tegmental area administration of orexin-A (1.0 nmol) significantly increased the intra-cranial self-stimulation threshold. Furthermore, the threshold-increasing effects of intra-ventral tegmental area or intracerebroventricular orexin-A were inhibited by administration of the nonspecific corticotropin-releasing factor receptor antagonist, d-Phe-CRF(12-41) (20 μg). Following intra-ventral tegmental area infusion of orexin-A, the percentage of cells double-labeled with corticotropin-releasing factor and Fos antibodies increased in the central nucleus of the amygdala but not in the bed nucleus of the stria terminalis, and brain microdialysis analyses indicated that dopamine efflux in both the central nucleus of the amygdala and bed nucleus of the stria terminalis were enhanced. Taken together, the present findings suggest that intra-ventral tegmental area or intracerebroventricular administration of orexin-A exerts its threshold-increasing effect via subsequent activation of the corticotropin-releasing factor system., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Studies of gastrointestinal physiology in humans and intact animals are usually conducted after overnight fast. We compared the effects of orexin-A, vasoactive intestinal polypeptide (VIP), melatonin, serotonin, uroguanylin, ghrelin and prostaglandin E(2) (PGE(2)) on duodenal bicarbonate secretion in fed and overnight fasted animals. This review is a summary of our findings. Secretagogues were administered by intra-arterial infusion or luminally (PGE(2)). Enterocyte intracellular calcium ([Ca(2+)](i)) signalling was studied by fluorescence imaging. Total RNA was extracted, reverse transcripted to cDNA and expression of orexin receptors measured by quantitative real-time PCR. Orexin-A stimulates the duodenal secretion in continuously fed animals but not in food-deprived animals. Similarly, short-term fasting causes a 100-fold decrease in the amount of the muscarinic agonist bethanechol required for stimulation of secretion. In contrast, fasting does not affect secretory responses to intra-arterial VIP, melatonin, serotonin, uroguanylin and ghrelin, or that to luminal PGE(2). Orexin-A induces [Ca(2+)](i) signalling in enterocytes from fed rats but no significant [Ca(2+)](i) responses occur in enterocytes from fasted animals. In addition, overnight fasting decreases the expression of mucosal orexin receptors. Short-term food deprivation thus decreases duodenal expression of orexin receptors and abolishes the secretory response to orexin-A as well as orexin-A-induced [Ca(2+)](i) signalling. Fasting, furthermore, decreases mucosal sensitivity to bethanechol. The absence of declines in secretory responses to other secretagogues tested strongly suggests that short-term fasting does not affect the secretory capacity of the duodenal mucosa in general. Studies of intestinal secretion require particular evaluation with respect to feeding status.

Aim: Orexin-producing neurones, located primarily in the perifornical region of the lateral hypothalamus, project to a wide spectrum of brain sites where they influence numerous behaviours as well as modulating the neuroendocrine and autonomic responses to stress. While some of the actions of orexin appear to be mediated via the type 1 receptor, some are not, including its action on the release of one stress hormone, prolactin. We describe here the ability of orexin to increase locomotor behaviours and identify the importance of both receptor subtypes in these actions., Methods: Rats were tested for their behavioural responses to the central activation of both the type 1 (OX(1)R) and type 2 (OX(2)R) receptor (ICV orexin A), compared to OX(2)R activation using a relatively selective OX(2)R agonist in the absence or presence of an orexin receptor antagonist that possesses highest affinity for OX(1)R., Results: Increases in locomotor activity were observed, effects which were expressed by not only orexin A, which binds to both the OX(1)R and the OX(2)R receptors, but also by the relatively selective OX(2)R agonist [(Ala(11), Leu(15))-orexin B]. Furthermore, the OX(1)R selective antagonist only partially blocked the action of orexin A on most locomotor behaviours and did not block the actions of [(Ala(11), Leu(15))-orexin B]., Conclusion: We conclude that orexin A exerts its effects on locomotor behaviour via both the OX(1)R and OX(2)R and that agonism or antagonism of only one of these receptors for therapeutic purposes (i.e. sleep disorders) would not provide selectivity in terms of associated behavioural side effects.

Hypocretinergic/orexinergic neurons, which are known to be implicated in narcolepsy, project to the pontine tegmentum areas involved in the control of rapid eye movement (REM) sleep. Here, we report the effects on sleep-wakefulness produced by low-volume microinjections of hypocretin (Hcrt)1 (20-30 nL, 100, 500 and 1000 microm) and carbachol (20-30 nL, 0.1 m) delivered in two areas of the oral pontine tegmentum of free-moving cats with electrodes for chronic sleep recordings: in the dorsal oral pontine tegmentum (DOPT) and in the ventral part of the oral pontine reticular nucleus (vRPO). Carbachol in the DOPT produced dissociate polygraphic states, with some but not all REM sleep signs. In contrast, carbachol in the vRPO produced a shift with short latency from wakefulness (W) to REM sleep with all of its polygraphic and behavioral signs. Hcrt-1 in the DOPT increased W and decreased both slow-wave sleep (SWS) and REM sleep during the first 3 h post-drug. The same doses of Hcr-1 in the vRPO produced a significant suppression of REM sleep without a definitive trend for changes in the other states. Both groups showed significant decreases in the number of transitions from SWS to REM sleep. Thus, Hcrt-1 produced distinct effects in cholinoceptive areas of the oral pontine tegmentum; in the DOPT it promoted W, suppressed SWS and probably defacilitated REM sleep, and in the vRPO it directly inhibited REM sleep. Hypocretinergic/orexinergic signaling is lost in narcoleptics and this absence would mean that pontine defacilitation/inhibition of REM sleep would also be absent, explaining why these patients can fall directly into REM sleep from W.