Coronary vasoconstriction during stimulation in hypothalamic defense region

Orexins/hypocretins are newly discovered neuropeptides synthesized by neurones located mainly in the lateral hypothalamus. They were originally believed to have an important role in the regulation of appetite. However, nerve fibres from orexin/hypocretin neurones are widely distributed in the brain suggesting that orexins/hypocretins have multiple functions. In particular, these peptides have now been implicated as important regulators of sleep, arousal and locomotor activity. Animal studies suggest these peptides are also involved in the regulation of pituitary hormone secretion and autonomic nervous system activity. The aim of this review is to describe how these fascinating peptides were discovered, what their structures are, the available evidence on their putative actions and their possible relevance to clinical medicine. Orexins (A and B), or hypocretins (1 and 2), have recently been discovered as a result of two independent modern approaches to the identification of neuropeptides. The hypocretins were discovered, using directional tag PCR subtraction, in an effort to determine abundant messenger RNAs that are selectively expressed in the rat hypothalamus (de Lecea et al., 1998). In this study, one clone (clone 35) was found to be particularly abundant in the dorsolateral hypothalamus and resulted in the identification of the prepro-hypocretin and its possible peptide products (hypocretin 1 and 2). These peptides were named 'hypocretins' (hypothalamic incretins) because of some amino acid sequence homology to the peptide hormone secretin. The orexins were discovered through the reverse pharmacology approach in search of endogenous ligands for 'orphan' G protein coupled receptors (GPCRs; Sakurai et al., 1998). Transfectant cell lines, expressing the orphan GCPR HFGAN72 (now orexin-A/hypocretin-1 receptor, OX1R/Hcrt1R) cDNA, were challenged with chromatographic fractions derived from rat brain extracts (Fig. 1). The fractions that elicited an increase in cytoplasmic Ca2+ levels in cells were further purified and analysed to identify and isolate the peptide orexin A. A smaller activity peak was identified as orexin B and led to a search for a different orphan receptor now known as the orexin-2/hypocretin-2 receptor (OX2R/Hcrt2R). The orexins ('stimulators of appetite') were named because orexin neuronal cell bodies were found to be largely confined to the lateral hypothalamus, an area classically described as the 'feeding' centre, and based on observations that when administered into the cerebral ventricles (intracerebroventricular; ICV), orexins potently increased food intake in rats (Sakurai et al., 1998). The 'reverse' pharmacology approach to identification of ligands for orphan G protein-coupled receptors. In the discovery of orexins, transfectant cells (HEK-293) expressing the orphan receptor (HFGAN72) were challenged with chromatographic fractions derived from rat brain extracts. The observed response was a rise in cytoplasmic calcium. Hypocretin-1 and -2 are essentially the same peptides as orexin A and B, respectively (Fig. 2) and the prepro-orexin and prepro-hypocretin genes are the same. Much confusion has occurred because the structure of biologically active hypocretin-1 was not fully determined when the hypocretins were first described. Thus, hypocretin-1, as originally reported (Fig. 2), is longer than orexin A, lacks the intrachain disulphide bonds and carboxy-terminal amidation, and is inactive at the orexin/hypocretin receptors (Smart et al. 2000). It is not surprising that in the original description (de Lecea et al., 1998) only hypocretin-2 was reported to have neuroexcitatory activity since its structure (only different from orexin B by one amino acid), unlike hypocretin-1, was the biologically active form. Whether we should use the name 'hypocretins' or 'orexins' for these neuropeptides is a matter of debate. These peptides have only weak homology to secretin and it is unlikely that they belong to the secretin family of peptides; therefore the name 'hypocretins' may be inappropriate. On the other hand, these peptides are now known to have multiple functions beside the regulation of appetite (Fig. 3) thus making the name 'orexins' confusing. In this review, we will refer to the biologically active peptide products of prepro-orexin/hypocretin as orexin A/Hcrt-1 and Orexin B/Hcrt-2. Schematic representation of the structure of the prepro-orexin/hypocretin gene, mRNA and its prepro-peptide product, and the amino acid structures of orexins (A and B) and hypocretins (1 and 2; as originally described). The amino acids underlined are shared between orexin A and B (and between hypocretin 1 and 2). The main reported actions of orexins/hypocretins. The human prepro-orexin/hypocretin gene is located on chromosome 17q21 (Sakurai et al., 1998). The gene, spanning 1432 bp, consists of 2 exons and 1 intron (Fig. 2; Sakurai et al., 1999). Orexins/hypocretins are derived from a 131 amino acid human (130 amino acids in the rat) precursor prepro-orexin/hypocretin. The first 33 amino acids of prepro-orexin/hypocretin have the characteristics of a signal sequence. Orexin A/Hcrt-1 is a 33 amino acid carboxy-amidated peptide of 3562 Da with an N-terminal pyroglutamyl residue and two intrachain disulphide bonds (Fig. 2). The human orexin A/Hcrt-1 sequence is identical to the mouse, rat, bovine and porcine orexin A/Hcrt-1. Orexin B/Hcrt-2 is also C-terminally amidated, but is a linear peptide of 28 amino acids with a molecular weight of 2937 Da. Human orexin B/Hcrt-2 has two amino acid substitutions compared with rodent orexin B/Hcrt-2 and one substitution compared to porcine orexin B/Hcrt-2. There is 46% amino acid sequence identity between orexin B/Hcrt-2 and orexin A/Hcrt-1 (Fig. 2). Orexin A/Hcrt-1, but not orexin B/Hcrt-2, is lipophilic and crosses the blood–brain barrier (Kastin & Akerstrom, 1999). The OX1R/Hcrt1R receptor has structural homology to several neuropeptide receptors including the NPY Y2 receptor, TRH receptor, CCK-A receptor and NK2 neurokinin receptor. Orexin A/Hcrt-1 has two to three times greater affinity than orexin B/Hcrt-2 for the human OX1R/Hcrt1R receptor. Both orexin A/Hcrt-1 and orexin B/Hcrt-2 have similar affinities for the human OX2R/Hcrt2R receptor. Using oligonucleotide probes, OX1R/Hcrt1R receptor mRNA has been reported to be most abundant in the ventromedial hypothalamus, but has also been detected in the tenia tecta, hippocampus, dorsal raphe and locus coeruleus (Trivedi et al., 1998). OX2R/Hcrt2R receptor mRNA is expressed in the paraventricular hypothalamic nucleus, the subthalamic and thalamic nuclei, the septum, the cerebral cortex, nucleus accumbens, anterior pretectal nucleus and several regions in the medulla oblongata. The subcellular location of these orexin/hypocretin receptors is interesting. Immunohistochemical studies have shown that the location of OX1R/Hcrt1R is mainly cytosolic while that of OX2R/Hcrt2R is mainly nuclear (G. Bewick, unpublished data). OX1R/Hcrt1R and OX2R/Hcrt2R have been detected in the pituitary gland (Date et al. 2000) while OX2R/Hcrt2R mRNA has also been detected in the adrenal medulla (Lopez et al., 1999), where it may alter catecholamine secretion, but the physiological relevance of these observations is at present unclear. Orexins are not found in plasma. However, it is possible that they may be present in nerve endings, in sufficiently low density to be undetectable by radioimmunoassay, in peripheral tissues. Alternatively, peripheral orexin receptors may function as receptors for another ligand. Curiously, orexin/hypocretin neurones appear to be labelled by an antiserum raised against ovine prolactin (Risold et al., 1999). A small proportion of orexin/hypocretin neurones are immunopositive for galanin (Hakansson et al., 1999). Orexin A/Hcrt-1 and Orexin B/Hcrt-2 are found in secretory vesicles at neuronal synapses and are neuroexcitatory (de Lecea et al., 1998; van Den Pol et al., 1998). These peptides cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx in vitro (Smart et al., 1999; Lund et al. 2000). Orexin/hypocretin immunoreactivity and immunoreactive fibres are widely distributed throughout the central nervous system (Peyron et al., 1998; Mondal et al., 1999; Nambu et al., 1999; Taheri et al., 1999), but have only occasionally been reported in peripheral tissues (Kirchgessner & Liu, 1999; Date et al. 2000). Orexin/hypocretin immunoreactive fibres are densely distributed in the hypothalamus, septum, thalamus, brainstem and spinal cord (Peyron et al., 1998; Cutler et al., 1999; Date et al., 1999; Nambu et al., 1999). Orexin fibres innervating neurones in the locus coeruleus appear to represent the greatest peptidergic input into this pontine region (Horvath et al., 1999a). The locus coeruleus is a major monoaminergic site within the central nervous system involved in the regulation of arousal. Elevated c-fos (a measure of neuronal activation) immunoreactivity in response to orexins (Date et al., 1999; Edwards et al., 1999; Mullett et al. 2000) has been detected in hypothalamic areas involved in food intake, neuroendocrine regulation and sleep and circadian rhythms (the lateral hypothalamus, the posterior and dorsomedial hypothalamus, anterior hypothalamus, the perifornical, arcuate and paraventricular nuclei), in the lateral septal area, the central nucleus of the amygdala, the shell of the nucleus accumbens, the bed nucleus of the stria terminalis, and the nucleus of the solitary tract (involved in autonomic and visceral regulation). Orexin neurones in the lateral hypothalamus have reciprocal connections with neuropeptide Y (NPY) and agouti-related peptide (AgRP) neurones in the arcuate nucleus (Broberger et al., 1998; Horvath et al., 1999b). The lateral hypothalamus has classically been considered to be the 'feeding' centre with lesions of this area being associated with a marked decrease in food intake. The ventromedial hypothalamus is known as the 'satiety' centre since lesions of this area result in increased food intake and obesity. These early experiments have been clarified by the discovery of neuropeptide mediators of food intake (Table 1; Kalra et al., 1999). Several neuropeptides synthesized in the arcuate nucleus of the mediobasal hypothalamus are mediators of food intake including NPY, the pro-opiomelanocortin derivative α-melanocyte-stimulating hormone (α-MSH), AgRP and the peptide product of cocaine and amphetamine-regulated transcript (CART). When administered ICV to rats, NPY and AgRP stimulate food intake, while α-MSH and CART reduce food intake. These neuropeptides are regulated by leptin, a cytokine that signals the extent of fat stores to the central nervous system (Meister 2000). The lateral hypothalamus contains neurones, distinct from orexin/hypocretin neurones, expressing melanin-concentrating hormone (MCH), a central stimulator of food intake (Elias et al., 1998). The anatomical location of neurones expressing orexins/hypocretins, led to the hypothesis that they are important regulators of food intake. When originally administered into the lateral ventricle of rats, both orexin A/Hcrt-1 and orexin B/Hcrt-2 dose-dependently and potently stimulated food intake in rats and prepro-orexin/hypocretin mRNA was shown to be upregulated with fasting (Sakurai et al., 1998). The stimulation of food intake by orexins/hypocretins has been difficult to consistently repeat in several laboratories, particularly for orexin B/Hcrt-2 (Edwards et al., 1999). While orexin A/Hcrt-1 may increase food intake in the first 4 hours after ICV administration, it actually decreases food intake in the subsequent 20 hours (Taheri et al. 2000a). Furthermore, chronic administration of orexin A/Hcrt-1 does not result in obesity (Haynes et al., 1999; Yamanaka et al., 1999). It may be that orexins/hypocretins are not as important in food intake as originally believed, or that they may be important in food intake only in particular circumstances (e.g. in response to hypoglycaemia and/or in the regulation of circadian food intake). Orexin/hypocretin neurones have leptin receptors and are immunoreactive for STAT3, a transcription factor activated by leptin (Hakansson et al., 1999). However, the interaction of leptin with orexins/hypocretins is unlike the interaction observed with the potently orexigenic peptide NPY. NPY mRNA is upregulated in leptin-deficient ob/ob and leptin receptor deficient db/db mice, while prepro-orexin/hypocretin mRNA is downregulated, but increases with starvation in these animals (Yamamoto et al., 1999; Yamamoto et al. 2000). Although one group has recently reported moderately lower prepro-orexin/hypocretin mRNA in obese Zucker rats (Cai et al. 2000), which have defective leptin receptor action compared to their lean controls, we have noted no differences in prepro-orexin/hypocretin mRNA or orexin A/Hcrt-1 immunoreactivity in these strains (Taheri et al., in press). It is therefore unlikely that orexins/hypocretins are directly involved or are altered in the obesity of Zucker rats. These findings do not mean that orexins/hypocretins are not regulated by leptin since the effects of the other neuroendocrine abnormalities present in these animals may mask the effects of leptin deficiency on orexin/hypocretin neurones. Orexins/hypocretins appear to have an important role in the regulation of metabolic rate. Using indirect calorimetry, orexin A/Hcrt-1, but not orexin B/Hcrt-2, increased metabolic rate (Lubkin & Stricker-Krongrad, 1998). Mice overexpressing prepro-orexin/hypocretin have reduced body weight despite increased food intake since they also have a concomitant increase in metabolic rate (Inui 2000). Mice with targeted disruption of the prepro-orexin/hypocretin are hypophagic with reduced metabolic rate, but are prone to diet induced obesity, presumably due to their lower metabolic rate (Willie et al., in press). Orexin/hypocretin fibres innervate hypothalamic regions involved in the regulation of pituitary hormone release. Orexin A/Hcrt-1 releases several neuropeptides and releasing factors from mediobasal hypothalamic explants in vitro (Russell et al. 2000). Centrally administered orexin A/Hcrt-1 alters plasma prolactin, growth hormone and corticosterone. In the studies that originally reported these endocrine responses, orexin A/Hcrt-1 was shown to increase arousal and to activate neurones in the locus coeruleus (Hagan et al., 1999). The profound inhibition of plasma prolactin by ICV orexin A/Hcrt-1 is partially attenuated by the administration of the dopamine receptor antagonist domperidone suggesting that while in the presence of domperidone centrally administered TRH, neurotensin and vasopressin lose their prolactin lowering effect, the effect of orexin A/Hcrt-1 is partly independent of dopamine (Russell et al. 2000). Orexin A/Hcrt-1 increases plasma ACTH and corticosterone and there is increased c-fos mRNA in the paraventricular nucleus (PVN) of the hypothalamus where corticotrophin-releasing factor (CRF) neurones are located (Hagan et al., 1999; Ida et al. 2000; Kuru et al. 2000). It is therefore likely that orexin A/Hcrt-1 is an important regulator of the hypothalamo-pituitary-adrenal axis, which is consistent with its effect on increasing arousal. The mechanism through which orexin A/Hcrt-1 inhibits GH secretion remains to be determined, but may involve the release of somatostatin. Orexins/hypocretins may regulate LH secretion via GnRH neurones in the hypothalamus. In one study, the effects of orexin A/Hcrt-1 and orexin B/Hcrt-2 on LH secretion were evaluated in ovariectomized (ovx) and ovarian steroid-treated ovx rats (Pu et al., 1998). Central injection of orexin A/Hcrt-1 or orexin B/Hcrt-2 rapidly stimulated LH secretion in ovx animals pretreated with oestradiol and progesterone. However, these peptides inhibited LH release in unprimed ovx rats. Interestingly, we have observed changes in orexin A/Hcrt-1 immunoreactivity in the rat CNS with the oestrous cycle (Taheri & Russell, unpublished data). The available evidence for the importance of orexins/hypocretins in the hypothalamic regulation of pituitary hormone release is derived from animal experiments in which these peptides have been administered into the cerebral ventricles. This experimental approach, however, will result in the activation of several circuits in which orexins/hypocretins are involved and any hormonal changes in the plasma will represent the net effect of the circuits activated. It is therefore not surprising that intracerebroventricular administration of orexins/hypocretins stimulates the HPA axis, while paradoxically inhibiting prolactin release. The neuronal circuitry through which orexins/hypocretins may regulate pituitary hormone release remain to be defined. Further, the physiological circumstances in which orexins/hypocretins regulate the neuroendocrine system and the existence of any negative or positive feedback loops remain to be determined. The distribution of orexin/hypocretin immunoreactive fibres within the CNS raised the possibility that they may be involved in the regulation of sleep, arousal and activity. Targeted disruption of the prepro-orexin gene in mice results in a phenotype with features similar to the human narcolepsy syndrome (Chemelli et al., 1999). In the same report, modafinil, a drug used in the treatment of narcolepsy through an unknown mechanism, was shown to activate orexin/hypocretin neurones. Narcolepsy is a debilitating sleep disorder characterized by excessive daytime sleepiness, cataplexy (loss of muscle tone in response to emotional stimuli) and disturbance of rapid eye movements (REM) sleep. Canine models of narcolepsy, which inherit narcolepsy in an autosomal recessive fashion with full penetrance through the canarc-1 gene, have been shown to have mutations in the OX2/Hcrt-2 receptor gene (Lin et al., 1999). In rats, ICV administration of orexin A/Hcrt-1 increases arousal while reducing REM sleep and prolonging the latency to the first occurrence of REM sleep (Piper et al. 2000). These studies suggested that orexins have a role in the regulation of arousal and the sleep-wake cycle. Indeed, orexin A/Hcrt-1 immunoreactivity shows diurnal variation in areas of the brain involved in the regulation of sleep, arousal and circadian hormone release (Taheri et al. 2000a). When orexin A/Hcrt-1 immunoreactivity was measured in the cerebrospinal fluid of patients with narcolepsy, it was shown that the majority of the patients examined had undetectable orexin A/Hcrt-1 compared to controls (Nishino et al. 2000; Taheri et al. 2000b). This is now further supported by undetectable prepro-orexin mRNA in autopsy specimens from patients with narcolepsy (Peyron et al. 2000). It is likely that in human narcolepsy, unlike canine narcolepsy, there is an abnormality in orexin production or prepro-orexin processing rather than mutations in the OX2/Hcrt-2 receptor gene. Since orexin A/Hcrt-1 abnormalities have not been detected in all patients diagnosed with narcolepsy suggests that there may be different subsets of patients with narcolepsy. Interestingly, little is known about endocrine abnormalities in both canine and human narcolepsy except a greater propensity towards type 2 diabetes mellitus in human narcolepsy (Honda et al., 1986). It is not surprising that orexins/hypocretins are involved in the regulation of hormones such as prolactin, GH and corticosterone, which are intimately associated with sleep and arousal. Orexins have been implicated in the CNS regulation of gastric acid secretion (vagally mediated), sympathetic activation, cardiovascular function and drinking behaviour. Both orexin A/Hcrt-1 and orexin B/Hcrt-2, when injected ICV increase blood pressure and heart rate in rats (Samson et al., 1999; Shirasaka et al., 1999). ICV orexin A/Hcrt-1 increases water intake, while prepro-orexin/hypocretin mRNA increases with water deprivation (Kunii et al., 1999). In the spinal cord, orexins/hypocretins may influence the sensory and autonomic nervous systems (van Den Pol, 1999). There are several reports on the effect of orexins/hypocretins on peripheral tissues such as the gastrointestinal tract, the pancreas and adrenal gland (Kirchgessner & Liu, 1999; Malendowicz et al., 1999; Nowak et al. 2000). Orexins/hypocretins are good examples of how modern molecular biology techniques have contributed to the discovery and greater understanding of the physiological actions of neuropeptides. It is remarkable how quickly these peptides have made the transition from basic scientific research to clinical medicine. Since the secretion of most pituitary hormones is intimately linked to sleep and arousal, it is likely that under basal conditions, orexins/hypocretins link sleep and arousal to pituitary hormone release. It now remains to study the orexin/hypocretin neuronal circuitry in conditions that disturb this basal state, for example, ageing, sleep loss, night or shift work, jet lag, affective disorders and endocrine diseases (e.g. Cushing's syndrome). Manipulation of the orexin/hypocretin ligand-receptor system may prove therapeutically useful not only in the treatment of sleep disorders such as narcolepsy and insomnia, but also in the treatment of several medical and psychiatric disorders associated with sleep disturbance. However, a greater understanding of the role of the individual peptides and receptors is required combined with an effort to tease out the neuronal circuits involved. Further study of these fascinating peptides may also clarify how hormone release is linked to sleep and arousal. Shahrad Taheri is funded by the Wellcome Trust. We would like to thank members of our laboratory who have contributed to the study of orexins/hypocretins and Dr Mignot, Dr Yanagisawa and Dr Chemelli for providing us with information regarding their work.

Calcitonin gene-related peptide (CGRP) evokes anxiety-like responses when infused into the lateral ventricle of rats. Because the bed nucleus of the stria terminalis (BNST) lies immediately adjacent to the lateral ventricle, is rich in CGRP receptors, and has itself been implicated in anxiety, we evaluated the hypothesis that these effects are attributable to stimulation of CGRP receptors within the BNST itself. Bilateral intra-BNST, but not dorsal, CGRP infusions (0, 200, 400, 800 ng/side) dose-dependently enhanced startle amplitude, and produced an anxiety-like response on the elevated plus maze. Intra-BNST infusion of the CGRP antagonist, αCGRP8-37, blocked the effect of CGRP on startle, and also blocked startle potentiation produced by exposure to trimethylthiazoline (TMT – a component of fox feces that induces anxiety-like behavior in rats). Intra-BNST, but not dorsal, CGRP infusions also increased c-Fos immunoreactivity in a number of anxiety-related brain areas (central nucleus of the amygdala, locus coeruleus, ventrolateral septal nucleus, paraventricular hypothalamic nucleus, lateral hypothalamus, lateral parabrachial nucleus, dorsal raphe nucleus, and nucleus accumbens shell), all of which receive direct projections from the BNST. Together, the results indicate that the activation of BNST CGRP receptors is both necessary and sufficient for some anxiety responses and that these effects may be mediated by activation of a wider network of BNST efferent structures. If so, inhibition of CGRP receptors may be a clinically useful strategy for anxiety reduction.

Peptide immunoreactive neurons in the amygdala and the bed nucleus of the stria terminalis project to the midbrain central gray in the rat

Systemic infection activates the hypothalamic-pituitary-adrenal (HPA) axis, and brainstem catecholamine cells have been shown to contribute to this response. However, recent work also suggests an important role for the central amygdala (CeA). Because direct connections between the CeA and the hypothalamic apex of the HPA axis are minimal, the present study investigated whether the bed nucleus of the stria terminalis (BNST) might act as a relay between them. This was done by using an animal model of acute systemic infection involving intravascular delivery of the proinflammatory cytokine interleukin-1beta (IL-1beta, 1 microg/kg). Unilateral ibotenic acid lesions encompassing the ventral BNST significantly reduced both IL-1beta-induced increases in Fos immunoreactivity in corticotropin-releasing factor (CRF) cells of the hypothalamic paraventricular nucleus (PVN) and corresponding increases in adrenocorticotropic hormone (ACTH) secretion. Similar lesions had no effect on CRF cell responses to physical restraint, suggesting that the effects of BNST lesions were not due to a nonspecific effect on stress responses. In further studies, we examined the functional connections between PVN, BNST, and CeA by combining retrograde tracing with mapping of IL-1beta-induced increases in Fos in BNST and CeA cells. In the case of the BNST, these studies showed that systemic IL-1beta administration recruits ventral BNST cells that project directly to the PVN. In the case of the CeA, the results obtained were consistent with an arrangement whereby lateral CeA cells recruited by systemic IL-1beta could regulate the activity of medial CeA cells projecting directly to the BNST. In conclusion, the present findings are consistent with the hypothesis that the BNST acts as a relay between the CeA and PVN, thereby contributing to CeA modulation of hypophysiotropic CRF cell responses to systemic administration of IL-1beta.

Amygdala Activity, Fear, and Anxiety: Modulation by Stress

An HRP study of the afferent connections to rat lateral hypothalamic region

Nonapeptides and the evolutionary patterning of sociality

Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor peptide. Controversy persists regarding the predominant ET receptor that mediates coronary vasoconstriction at pathophysiological concentrations. The aim of the present study was to test the hypothesis that ET mediates local coronary vasoconstriction via the ET-A receptor at low concentrations of exogenous ET-1 designed to mimic pathophysiological states compared with pharmacological concentrations. ET-1 (group 1, n = 5) or sarafotoxin, a specific ET-B receptor agonist (group 3, n = 6) (each at 2 ng/kg per minute), was infused into the left circumflex coronary artery in the anesthetized dog. In group 2 dogs (n = 5), the same dose of ET-1 was infused with 4 micrograms/kg per minute of the specific ET-A receptor antagonist FR-139317. In group 4 (n = 5), the same dose of sarafotoxin was infused with 50 micrograms/kg per minute of the specific inhibitor of nitric oxide formation, NG-monomethyl-L-arginine (L-NMMA). No difference in hemodynamics, coronary blood flow (CBF), coronary vascular resistance (CVR), or coronary artery diameter (CAD) was observed at baseline between the groups. In group 1, intracoronary ET-1 significantly decreased CBF and CAD in association with an increase in CVR. The percentage decrease in CBF and CAD in the group that received ET-1 and the ET-A receptor antagonist (group 2) was significantly less than that in the group that received ET-1 alone (group 1) (-12 +/- 3% versus -48 +/- 6% [P < .001] and -4.6 +/- 0.8 versus 1.0 +/- 0.3 [P < .05], respectively). The administration of the ET-A receptor antagonist (group 2) abolished the ET-mediated increase in CVR (7 +/- 5% versus 105 +/- 21%, P < .005). There was no significant effect on CBF, CVR, or CAD in the group receiving sarafotoxin alone (group 3). The administration of L-NMMA and sarafotoxin (group 3). The administration of L-NMMA and sarafotoxin (group 4) resulted in a significant percentage decrease in CBF compared with the group that received sarafotoxin alone (-28 +/- 7% versus -8 +/- 2% [P < .05]). The present study demonstrates that low concentrations of exogenous ET-1, which may mimic pathophysiological concentrations, result in coronary vasoconstriction mediated predominantly via the ET-A receptor because such vasoconstriction is significantly attenuated by blockade with FR-139317. The ET-B receptor may have a dual vasoconstrictive and vasodilatory effect.

We hypothesized that brain regions showing neuronal activation after refeeding comprise major nodes in a satiety network, and tested this hypothesis with two sets of experiments. Detailed c-Fos mapping comparing fasted and refed rats was performed to identify candidate nodes of the satiety network. In addition to well-known feeding-related brain regions such as the arcuate, dorsomedial, and paraventricular hypothalamic nuclei, lateral hypothalamic area, parabrachial nucleus (PB), nucleus of the solitary tract and central amygdalar nucleus, other refeeding activated regions were also identified, such as the parastrial and parasubthalamic nuclei. To begin to understand the connectivity of the satiety network, the interconnectivity of PB with other refeeding-activated neuronal groups was studied following administration of anterograde or retrograde tracers into the PB. After allowing for tracer transport time, the animals were fasted and then refed before sacrifice. Refeeding-activated neurons that project to the PB were found in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamic area; arcuate, paraventricular, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; parasubthalamic nucleus; central amygdalar nucleus; area postrema; and nucleus of the solitary tract. Axons originating from the PB were observed to closely associate with refeeding-activated neurons in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamus; paraventricular, arcuate, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; central amygdalar nucleus; parasubthalamic nucleus; ventral posterior thalamic nucleus; area postrema; and nucleus of the solitary tract. These data indicate that the PB has bidirectional connections with most refeeding-activated neuronal groups, suggesting that short-loop feedback circuits exist in this satiety network. J. Comp. Neurol. 524:2803-2827, 2016. © 2016 Wiley Periodicals, Inc.

The coronary vasoconstrictor properties of digitals were evaluated in 61 anesthetized, openchest dogs after coronary sinus cannulation and under conditions of a constant heart rate (atrioventricular pacing) and near-constant blood pressure. The contribution of alpha adrenergic receptor stimulation to the digitalis-induced increase in coronary vascular resistance (CVR) was examined. With Na pentobarbital anesthesia (16 dogs), intravenous acetylstrophanthidin (0.5 mg) caused a significant (P<0.05) rise in CVR from 1 through 9 min after injection. The peak increase was +11+/-2% SE of the control of 1.8+/-0.2 mm Hg/cm(3)/min. The mean time to peak effect was 3 min, and to recovery was 21 min. Prior alpha adrenergic receptor blockade with phenoxybenzamine in 11 animals reduced (P<0.05) the acetylstrophanthidin-induced peak of CVR and substantially decreased (P<0.05) the time to recovery (5 min). Intravenous digoxin (1.0 mg) with Na pentobarbital anesthesia (five dogs) had no significant effect on CVR. However, with chloralose and urethane anesthesia (nine dogs) the same dose of digoxin produced a significant rise in CVR from 3 through 30 min. The peak increase was +20+/-3% of control (1.4+/-0.1 mm Hg/cm(3)/min). One-third the dose of intravenous digoxin (0.35 mg) produced a 9.5+/-1.0% increase in CVR (five additional dogs). Myocardial oxygen consumption did not change significantly in nine dogs after intravenous digoxin. In 10 additional dogs pretreated with phenoxy-benzamine and in 7 dogs pretreated with mecamylamine, the increase in CVR did not occur after 1.0 mg of intravenous digoxin. Thus there is a coronary vasoconstrictor effect of intravenous acetylstrophanthidin and digoxin, of rapid onset, which is mediated through alpha adrenergic receptor stimulation.

The effects of cocaine on the coronary circulation were examined in conscious dogs chronically instrumented to measure arterial and left ventricular pressures, coronary blood flow, and arterial and coronary sinus oxygen content. With heart rate held constant, the peak effects of cocaine (1 mg/kg i.v.) occurred within 2 minutes, when mean arterial pressure increased by 42 +/- 5 mm Hg, coronary blood flow increased by 13 +/- 3%, and coronary vascular resistance increased by 24 +/- 3%. The arterial oxygen content increased significantly (by 2.8 +/- 0.3 vol%), the arterial-coronary sinus oxygen difference increased by 2.5 +/- 0.6 vol%, and myocardial oxygen consumption increased by 41 +/- 9%. The increase in coronary vascular resistance induced by cocaine was attenuated (p < 0.05) in the presence of cholinergic blockade (12 +/- 3%) despite a similar increase in MVO2 (49 +/- 8%). The increase in coronary vascular resistance was enhanced (p < 0.05) in the presence of beta-adrenergic receptor blockade (46 +/- 8%), whereas the MVO2 response was less (28 +/- 3%). Again, the addition of cholinergic blockade to beta-blockade attenuated the increase in coronary vascular resistance (23 +/- 6%) without affecting the increase in MVO2 (25 +/- 4%). Combined alpha-, beta-, and cholinergic blockades abolished the systemic hemodynamic and coronary vasoconstrictor response to cocaine. In conscious dogs, cocaine induces coronary vasoconstriction, which competes with coronary vasodilator responses to increases in myocardial oxygen consumption. The mechanisms of cocaine's coronary vascular effects are mediated via adrenergic stimulation, and the intensity of the vasoconstrictor effects was reduced significantly by cholinergic blockade, in both the presence and absence of beta-adrenergic receptor blockade.

Hypocretin I (also referred to as orexin A) administered into the lateral hypothalamus (LH) stimulates feeding in rats. We undertook the present study to determine the brain regions activated by LH administration of hypocretin I. Hypocretin I administered into the LH significantly elevated cFos-immunoreactivity in the lateral septal area, the central nucleus of the amygdala, the shell of the nucleus accumbens, the bed nucleus of the stria terminalis, the LH, the posterior and dorsomedial hypothalamus, the perifornical, arcuate and paraventricular nuclei and the nucleus of the solitary tract. These data indicate that LH hypocretin I communicates with other key energy regulatory sites within the hypothalamus, the limbic region and the hindbrain, and suggest that these areas are important in the feeding-stimulatory actions of hypocretin I.

The neuropeptide oxytocin is released not only into the blood, but also within the brain in response to various stressors. Accumulating evidence suggests that central oxytocin may play a major role in the regulation of neuroendocrine responses to stress. In the present study, using the oxytocin knockout mouse model, we tested whether oxytocin might act to attenuate stress-induced up-regulation of corticotropin-releasing hormone (CRH) mRNA expression in the brain. The expression of CRH mRNA in the paraventricular nucleus (PVN) after 4 h of restraint stress was examined in oxytocin gene-deficient (OTKO), wild-type and heterozygous male mice using in situ hybridization histochemistry. We found that basal levels of CRH mRNA were not different among the three genotypes. Although restraint stress resulted in a significant increase of CRH mRNA expression in the PVN regardless of genotype, the degree of stress induced-up-regulation was significantly higher in OTKO mice than in wild-type mice. The effects of restraint stress on the expression of the arginine vasopressin (AVP) and the oxytocin genes were also examined. Unlike CRH mRNA, basal expression (in nonstressed control groups) of AVP mRNA in OTKO mice, as well as oxytocin mRNA in heterozygous mice, was significantly lower in the PVN and the supraoptic nucleus than in wild-type mice. After restraint stress, the expression of AVP mRNA was significantly increased in the PVN of OTKO mice compared to the nonstressed control group, whereas the expression of both AVP and oxytocin mRNA were unchanged in the PVN and the supraoptic nucleus of wild-type and heterozygous mice. Finally, in a separate set of mice, restraint stress-induced Fos expression was also examined in several brain regions involved in stress response, including the lateral septum, the bed nucleus of the stria terminalis (BNST), the medial preoptic area, the PVN, the medial and central amygdala using immunohistochemistry. After 90 min of restraint stress, the number of Fos-expressing cells significantly increased in all brain regions examined regardless of genotype. However, the number of stress-induced Fos-expressing cells in the BNST and the medial amygdala of OTKO mice was significantly lower than in wild-type mice. Collectively, the findings in the present study suggest that oxytocin may regulate stress-induced CRH gene expression in the PVN. Furthermore, neuronal activity in the BNST and the medial amygdala may be involved in this neuroendocrine regulatory system.

The anatomy of neuropeptide-y-containing neurons in rat brain

Transneuronal labelling of nerve cells in the CNS of female rat from the mammary gland by viral tracing technique