Modulation of respiration and hypothalamus.

Prader‐Willi Syndrome (PWS) is a rare and incurable congenital neurodevelopmental disorder, resulting from the absence of expression of a group of genes on the paternally acquired chromosome 15q11‐q13. Phenotypical characteristics of PWS include infantile hypotonia, short stature, incomplete pubertal development, hyperphagia and morbid obesity. Hypothalamic dysfunction in controlling body weight and food intake is a hallmark of PWS. Neuroimaging studies have demonstrated that PWS subjects have abnormal neurocircuitry engaged in the hedonic and physiological control of feeding behavior. This is translated into diminished production of hypothalamic effector peptides which are responsible for the coordination of energy homeostasis and satiety. So far, studies with animal models for PWS and with human post‐mortem hypothalamic specimens demonstrated changes particularly in the infundibular and the paraventricular nuclei of the hypothalamus, both in orexigenic and anorexigenic neural populations. Moreover, many PWS patients have a severe endocrine dysfunction, e.g. central hypogonadism and/or growth hormone deficiency, which may contribute to the development of increased fat mass, especially if left untreated. Additionally, the role of non‐neuronal cells, such as astrocytes and microglia in the hypothalamic dysregulation in PWS is yet to be determined. Notably, microglial activation is persistently present in non‐genetic obesity. To what extent microglia, and other glial cells, are affected in PWS is poorly understood. The elucidation of the hypothalamic dysfunction in PWS could prove to be a key feature of rational therapeutic management in this syndrome. This review aims to examine the evidence for hypothalamic dysfunction, both at the neuropeptidergic and circuitry levels, and its correlation with the pathophysiology of PWS.

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.

Background/Aims: Nesfatin-1, processed from nucleobindin-2 (NUCB2), is a potent anorexigenic peptide being expressed in rodent hypothalamic nuclei and involved in the regulation of feeding behavior and body weight in animals. The present study aimed to investigate NUCB2/nesfatin-1 protein expression in the human hypothalamus as well as its correlation with body weight. Methods: Sections of hypothalamus and adjacent cholinergic basal forebrain nuclei, including the nucleus basalis of Meynert (NBM) and the diagonal band of Broca (DBB), from 25 autopsy cases (17 males, 8 females; 8 lean, 9 overweight, 8 obese) were examined using immunohistochemistry and double immunofluorescence labeling. Results: Prominent NUCB2/nesfatin-1 immunoexpression was detected in supraoptic, paraventricular, and infundibular nuclei, lateral hypothalamic area (LHA)/perifornical region, and NBM/DBB. NUCB2/nesfatin-1 was found to extensively colocalize with (a) oxytocin and vasopressin in paraventricular and supraoptic nuclei, (b) melanin-concentrating hormone in the LHA, and (c) cocaine- and amphetamine-regulated transcript in infundibular and paraventricular nuclei and LHA. Interestingly, in the LHA, NUCB2/nesfatin-1 protein expression was significantly decreased in obese, compared with lean (p < 0.01) and overweight (p < 0.05) subjects. Conclusions: The findings of the present study are suggestive of a potential role for NUCB2/nesfatin-1 as an integral regulator of food intake and energy homeostasis in the human hypothalamus. In the LHA, an appetite- and reward-related brain area, reduced NUCB2/nesfatin-1 immunoexpression may contribute to dysregulation of homeostatic and/or hedonic feeding behavior and obesity. NUCB2/nesfatin-1 localization in NBM/DBB might imply its participation in the neuronal circuitry controlling cognitive influences on food intake and give impetus towards unraveling additional biological actions of NUCB2/nesfatin-1 in human neuronal networks.

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 monotremes are an intriguing group of mammals that have major differences in their reproductive physiology and lactation from therian mammals. Monotreme young hatch from leathery skinned eggs and are nourished by milk secreted onto areolae rather than through nipples. Parturition and lactation are in part controlled through the paraventricular and supraoptic nuclei of the hypothalamus. We have used Nissl staining, enzyme histochemistry, immunohistochemistry for tyrosine hydroxylase, calbindin, oxytocin, neurophysin and non-phosphorylated neurofilament protein, and carbocyanine dye tracing techniques to examine the supraoptic and paraventricular nuclei and the course of the hypothalamo-neurohypophysial tract in two monotremes: the short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus). In both monotremes, the supraoptic nucleus consisted of loosely packed neurons, mainly in the retrochiasmatic position. In the echidna, the paraventricular nucleus was quite small, but had similar chemoarchitectural features to therians. In the platypus, the paraventricular nucleus was larger and appeared to be part of a stream of magnocellular neurons extending from the paraventricular nucleus to the retrochiasmatic supraoptic nucleus. Immunohistochemistry for non-phosphorylated neurofilament protein and carbocyanine dye tracing suggested that hypothalamo-neurohypophysial tract neurons in the echidna lie mainly in the retrochiasmatic supraoptic and lateral hypothalamic regions, but most neurophysin and oxytocin immunoreactive neurons in the echidna were found in the paraventricular, lateral hypothalamus and supraoptic nuclei and most oxytocinergic neurons in the platypus were distributed in a band from the paraventricular nucleus to the retrochiasmatic supraoptic nucleus. The small size of the supraoptic nucleus in the two monotremes might reflect functional aspects of monotreme lactation.

We determined the immunohistochemical distributions of orexin-A and orexin-B, hypothalamic peptides that function in the regulation of feeding behavior and energy homeostasis. Orexin-A and -B neurons were restricted to the lateral and posterior hypothalamus, whereas both orexin-A and -B nerve fibers projected widely into the olfactory bulb, cerebral cortex, thalamus, hypothalamus, and brainstem. Dense populations of orexin-containing fibers were present in the paraventricular thalamic nucleus, central gray, raphe nuclei, and locus coeruleus. Moderate numbers of these fibers were found in the olfactory bulb, insular, infralimbic and prelimbic cortex, amygdala, ventral, and dorsolateral parts of the suprachiasmatic nucleus, paraventricular nucleus except the lateral magnocellular division, arcuate nucleus, supramammillary nucleus, nucleus of the solitary tract, and dorsal motor nucleus of the vagus. Small numbers of orexin fibers were present in the perirhinal, motor and sensory cortex, hippocampus, and supraoptic nucleus, and a very small number in the lateral magnocellular division of the paraventricular nucleus. Intracerebroventricular injections of orexins induced c-fos expression in the paraventricular thalamic nucleus, locus coeruleus, arcuate nucleus, central gray, raphe nuclei, nucleus of the solitary tract, dorsal motor nucleus of the vagus, suprachiasmatic nucleus, supraoptic nucleus, and paraventricular nucleus except the lateral magnocellular division. The unique neuronal distribution of orexins and their functional activation of neural circuits suggest specific complex roles of the peptides in autonomic and neuroendocrine control.

Galanin has previously been reported to elicit feeding in satiated animals when injected into the hypothalamic paraventricular nucleus. It is not known, however, 1) whether this action is due to activation of feeding signals or suppression of satiety signals or both or 2) whether other hypothalamic regions such as the lateral hypothalamus (LH) or the ventromedial hypothalamus (VMH) are involved in this action. The effects of galanin on food intake were therefore examined in satiated and in fasted rats both after intracerebroventricular injection (0.1, 1, and 10 micrograms/10 microliters) and after microinjection (1 and 5 micrograms/0.5 microliters) into the LH and VMH. Twenty minutes after intracerebroventricular injection, galanin significantly and dose dependently augmented food intake by up to sevenfold in freely feeding rats and by up to 79% in fasted animals. The galanin-induced augmentation of cumulative food intake up to 2 h after injection was due to the initial increase in food consumption during the 0 to 20-min interval. This suggests that galanin acts by activation of feeding behavior and not by suppression of satiety signals in these fasted animals, in which satiety signals are presumably not initially operative. Twenty minutes after intrahypothalamic injections into both the LH and VMH, galanin (5 micrograms) significantly increased food consumption, fivefold in freely feeding rats and 30-35% in fasted rats. Thus stimulation of feeding by centrally injected galanin also involves loci within the LH and VMH.

Could deep brain stimulation be a possible solution for acquired hypothalamic obesity?

The connections between the cerebellum and the hypothalamus have been well documented. However, the specific cerebellar peduncle through which the hypothalamo-cerebellar and cerebello-hypothalamic connections pass has not been demonstrated. The present study aims to define the specific cerebellar peduncle through which connects the cerebellum to specific hypothalamic nuclei. Seventeen male albino rats received 20-50-nl pressure injections of either Fluoro-Gold (FG) or biotinylated dextran amine (BDA) tracer into the superior (SCP), middle (MCP), and inferior (ICP) cerebellar peduncle. Following 7-10days of survival period, the animals were processed according to the appropriate protocol for the two tracers used. Labeled cells and axons were documented using light or fluorescence microscopy. The present study showed connections between the hypothalamus and the cerebellum via both the SCP and the MCP but not the ICP. The hypothalamo-cerebellar connections via the SCP were from the lateral, dorsomedial, paraventricular, and posterior hypothalamic nuclei, and cerebello-hypothalamic connections were to the preoptic and lateral hypothalamic nuclei. The hypothalamo-cerebellar connections via the MCP were from the lateral, dorsomedial, ventromedial, and mammillary hypothalamic nuclei; and cerebello-hypothalamic connections were to the posterior, arcuate, and ventromedial hypothalamic nuclei. The hypothlamo-cerebellar connections were denser compared to the cerebello-hypothlamic connections via both the SCP and the MCP. The connection between the cerebellum and the hypothalamus was more prominent via the SCP than MCP. Both the hypothlamo-cerebellar and cerebello-hypothalamic connections were bilateral, with ipsilateral preponderance. Reciprocal connections were with the lateral hypothalamic nucleus via the SCP and the ventromedial nucleus via the MCP were observed. Cerebellum takes part in the higher order brain functions via its extensive connections. The knowledge of hypothalamo-cerebellar and cerebello-hypothalamic connections conveyed within the SCP and MCP can be important for the lesions involving the MCP and SCP. These connections can also change the conceptual architecture of the cerebellar circuitry and deepen current understanding.

Introduction: Prader-Willi syndrome (PWS) is a complex genetic syndrome in which hypothalamic dysfunction leads to extreme appetite (hyperphagia) and pituitary hormone deficiencies (PHD), among others. Compared with the ‘general endocrine population’, the population of adults with PWS is characterized by a high prevalence of intellectual disability (ID) and frequent use of psychotropic drugs. Due to hypotonia and the low muscle mass associated with the syndrome, adults with PWS have a low basal metabolic rate (BMR) and a high risk of developing obesity. Therefore, exercising is extremely important. However, PHD like hypothyroidism can cause fatigue and exercise intolerance. If left untreated, hypothyroidism can lead to a further decrease in BMR, an increase in Body Mass Index (BMI) and increased cardiovascular risk. As mortality in PWS is high (3% yearly) and often related to cardiovascular problems and obesity, it is of utmost importance to optimize thyroid function and other factors affecting BMR. Methods: In 122 adults with PWS (median age 29 y [IQR 21-39], median BMI 29 kg/m2 [IQR 26-36]), we measured TSH, free T4 and T3 (the active form of thyroid hormone) and searched the medical history for use of medication and any pre-existing diagnosis of hypothyroidism. Moreover, we performed an extensive literature search and summarized the current literature on hypothyroidism, T3 and T4 levels in adults with PWS. Results: Hypothyroidism was present in 17% and more prevalent in females (23%) than in males (10%), even though this was not statistically significant (P=0.06). Although within the reference range, serum T3 levels were relatively high compared to free T4 levels. T3 levels were significantly lower in patients that used psychotropic medication (n=45) than in patients that did not (median 1.7 [IQR 1.5-2.0] vs 2.1 [IQR 1.7-2.3], P=0.013). Conclusion: We found a prevalence of hypothyroidism of 17% (compared to 3% in the non-PWS population). T3 levels were relatively high, which might be explained by increased peripheral conversion of T4 to T3 by deiodinase type 2. Levels of the active thyroid hormone T3 were significantly lower in patients using psychotropic medication. Based on our findings, we recommend 1) yearly screening of thyroid hormone levels in adults with PWS to avoid negative effects of untreated hypothyroidism on BMR, BMI and cardiovascular risk and 2) extra monitoring of the active thyroid hormone T3 in patients using psychotropic drugs.

Previous studies have identified a site in lateral hypothalamus (LH) in which electrical stimulation elicits coronary vasoconstriction. We injected the retrogradely transported tracer Fast Blue to determine which brain regions project to LH. Projections to or through LH were found from the paraventricular nucleus (PVN) of the hypothalamus, bed nucleus of the stria terminalis (BNST), and dorsal raphe nucleus (DRN). In chloralose-anesthetized cats, electrical stimulation in DRN and BNST failed to increase coronary vascular resistance (CVR). However, stimulation lateral to PVN in the anterior hypothalamic area (AHA), a region not labeled by the tracer, caused a transient decrease in coronary blood flow similar to that elicited from LH. The increase in CVR was accompanied by hemodynamic changes that are characteristic of the defense reaction including a cholinergically mediated decrease in hindquarter vascular resistance. This response is likely due to activation of fibers of passage and not cell bodies, since cell bodies in the region were not retrogradely labeled and coronary vasoconstriction was not seen following microinjection of several excitatory amino acids into AHA. These data suggest that coronary vasoconstriction may be a component of the defense reaction elicited by electrical activation of AHA.

Introduction: Prader-Willi syndrome (PWS) is a complex hypothalamic disorder, combining hypotonia, intellectual disability (ID), pituitary hormone deficiencies and hyperphagia. In PWS, up to 3% of patients die every year. In half of the patients, the cause of death is obesity related and / or of cardiovascular (CV) origin.Obesity is caused by hyperphagia combined with a low energy expenditure. Untreated hormone deficiencies like hypogonadism and hypothyroidism can cause low muscle mass and low basal rest metabolism (BRM) leading to this low energy expenditure. Patients with PWS should exercise one hour daily to compensate for their low BRM. However, hormone deficiencies usually cause fatigue, leading to exercise intolerance. Musculoskeletal and / or behavioral problems can also cause reduced physical activity. The subsequent sedentary lifestyle can induce CV risk factors like hypertension, hypercholesterolemia and diabetes mellitus (DM).Another risk factor often present in PWS is sleep apnea, which can be central (CSA), obstructive (OSA) or both. Both CSA and OSA can lead to pulmonary hypertension and a further increase in obesity.The above mentioned health problems often remain unnoticed and untreated, which is partly due to the behavioral phenotype of PWS (patients seldomly report pain and hardly ever complain about physical problems). However, if left untreated, these risk factors can cause CV complications leading to hospital admission or even death. To reveal yet undiagnosed health problems, we performed a systematic health screening among adults with PWS.Methods: We systematically screened 115 adults with PWS (mean age 31.4 ± 12.1 y, mean BMI 31.8 ± 9,5 kg/m2) for the presence of undiagnosed health problems and cardiovascular risk factors. Based on a medical questionnaire, medical file search, extensive interview, thorough physical examination and biochemical measurements we made an overview of the undiagnosed health problems in adults with PWS. If possible, we performed polygraphy to test for sleep apnea.Results: Undiagnosed health problems (hypertension, DM, hypercholesterolemia, sleep apnea, hypothyroidism and hypogonadism) were present in 50% of the patients. 10% had multiple undiagnosed health problems simultaneously. All males and 94% of females had hypogonadism and 15% had hypothyroidism. Hypertension and / or hypercholesterolemia were present in 20% and DM was present in 16%. One third of patients was not on a diet and 22% exercised less than 30 minutes a day. Sleep apnea was present in 17 of 26 patients tested.Conclusion: We detected a striking number of undiagnosed health problems among adults with PWS which, if left untreated, can pose a serious health threat. Systematic screening is needed to detect these problems in an early phase. This will prevent burdensome and expensive complications and might even reduce mortality in this vulnerable patient population.

Distribution of orexin neurons in the adult rat brain

The central representation of neurogenically mediated coronary vasoconstriction produced by activation of the sympathetic nervous system was examined in anesthetized cats instrumented for continuous recording of coronary and femoral blood flows, arterial pressure, and heart rate. Electrical stimulation in a small region of perifornical lateral hypothalamus increased arterial pressure, heart rate, and coronary blood flow; following the administration of propranolol, a transient coronary vasoconstrictor response was unmasked. The response was mediated over the sympathetic nervous system since it was blocked by stellate ganglionectomy and by the alpha 1-adrenergic receptor antagonist prazosin. Projections to and from the lateral hypothalamic site were identified by using anterograde and retrograde pathway-tracing techniques. Paraventricular nucleus projected to lateral hypothalamus, which in turn made connections in periaqueductal gray with projections terminating in lateral reticular formation of medulla. Coronary vasoconstrictor responses qualitatively identical to those produced by hypothalamic stimulation were found with activation of paraventricular nucleus and lateral reticular formation. Interruption of neuronal transmission in the medullary site blocked the response produced by activation of hypothalamic site. These data demonstrate that coronary vasoconstriction mediated over the sympathetic nervous system can be elicited from an interconnected pathway that links sympathoexcitatory sites in forebrain and brainstem.

Nociceptin/orphanin FQ (N/OFQ), an endogenous peptide agonist of the opioid N/OFQ receptor, has been implicated in the regulation of energy balance. In the present study, we have used immunohistochemistry to investigate the cellular localisation and colocalisation of N/OFQ-immunoreactive cell bodies in hypothalamic regions containing neurones producing orexigenic or anorexigenic transmitters. In colchicine-treated rats, N/OFQ immunoreactivity was demonstrated in many cell bodies of the arcuate nucleus (Arc), paraventricular nucleus (PVN) and lateral hypothalamic area (LHA). Double-labelling revealed that N/OFQ was present in some neurones located in the ventrolateral part of the Arc producing pro-opiomelanocortin, as shown by the presence of the anorexigenic peptides alpha-melanocyte-stimulating hormone (alpha-MSH) and cocaine- and amphetamine-regulated transcript and, occasionally, in single neurones of the ventrolateral Arc producing orexigenic agouti-related peptide, but not neuropeptide Y. N/OFQ immunoreactivity was also demonstrated in a few tyrosine hydroxylase- or dynorphin (DYN)-containing neurones in the dorsomedial part of the Arc. In the parvocellular PVN, N/OFQ was demonstrated in some thyrotrophin-releasing hormone- or DYN-, but not corticotrophin-releasing hormone-containing neurones. Most N/OFQ-immunoreactive neurones in the LHA contained orexin- and DYN, but not melanin-concentrating hormone. The results obtained, demonstrating the presence of N/OFQ in some alpha-MSH- and in many orexin-containing neurones, suggest a functional relationship between these neuropeptides and N/OFQ in the control of feeding behaviour and body weight.