Immunohistochemical localization of orexin/hypocretin-like immunoreactive peptides and melanin-concentrating hormone in the brain and pituitary of medaka

Orexin/hypocretin is a neuropeptide that is involved in the regulation of feeding behavior and the sleep–wakefulness cycle in mammals. Melanin-concentrating hormone (MCH) is believed to be another candidate involved in food intake in teleost fish as well. Thus, it is interesting to examine whether neural connections exist between the neurons producing these two hormones. We first examined the localization of orexin-like immunoreactivity (orexin-LI) in the brain of the medaka Oryzias latipes by using immunohistochemistry. We further examined the interaction between the orexin and MCH neurons in the medaka brain by performing double-staining immunohistochemistry. Orexin-LI cell bodies were located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-LI fibers were detected not only in the hypothalamus but also extensively throughout the brain. Some orexin-LI fibers were in close contact with the MCH-immunoreactive (ir) cell bodies in the hypothalamus, as revealed by double-staining immunohistochemistry. Moreover, a few MCH-ir fibers were in close contact with the orexin-LI cell bodies. These results suggest that in the medaka brain, orexin performs various functions, including neuromodulation, and that neural connections exist between the orexin and MCH neurons.     

Lateral hypothalamic acetylcholinesterase-immunoreactive neurons co-express either orexin or melanin concentrating hormone

The lateral hypothalamic area (LHA) contains a large population of neurons that express the enzyme acetylcholinesterase (AChE), but are not themselves cholinergic. Some of these neurons have been shown to contain melanin-concentrating hormone (MCH), a neuropeptide implicated in regulating feeding, but the identities of the remaining neurons are unknown. We now report that nearly all AChE-immunoreactive neurons in the LHA express immunoreactivity for either MCH or for orexin, a peptide implicated in regulating wakefulness. Furthermore, most orexin neurons and MCH neurons appear to contain AChE. AChE immunoreactivity appears to be a key feature of nearly all of the diffusely-projecting cortical systems.     

Three-dimensional visualization of the distribution of melanin-concentrating hormone producing neurons in the mouse hypothalamus

We present here a new procedure to represent the 3D distribution of neuronal cell bodies within the brain, using exclusively softwares free for research purposes.Our technique is based on digitalized photos of brain slices processed by immunohistochemical technique, and the 3D Slicer software. The technique presented enables transposition of immunohistochemical or in situ hybridization data to the stereotaxic mouse brain atlas (e.g. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. second ed. Academic Press, San Diego). By exporting the finalized models into a popular 3D design software (3DS Max) arbitrary environment and motion simulation can be created to improve the visual understanding of the area studied.Application of this technique provides the possibility to store, analyze and compare data – e.g. on the hypothalamic neuropeptides – across experimental techniques and laboratories.The method is exemplified by visualizing the distribution of immunohistochemically identified melanin-concetrating hormone (MCH) containing perikarya within the mouse hypothalamus.     

A role of the histaminergic system for the control of feeding by orexigenic peptides

A considerable number of neuropeptides are involved in the hypothalamic regulation of feeding behavior. We previously reported that leptin, the ob gene product, expressed its anorectic effect though the histaminergic system via histamine H(1) receptors. However, the interactions among the orexigenic neuropeptides, such as orexin-A, neuropeptide Y (NPY), and ghrelin, and the histaminergic system have not yet been clarified. In this study, we investigated the effect of the neuropeptides on the hypothalamic histamine release in rats, and on food intake and locomotor activity in H(1)-receptor knockout (H1R-KO) mice. Orexin-A increased the histamine release and locomotor activity, but not food intake, suggesting that the histaminergic system participates in arousal rather than feeding by orexin-A. NPY also increased histamine release, but its effect was not immediate. NPY-injected H1R-KO mice consumed more food than the wild-type mice; thus, the histaminergic system may act as a feedback factor downstream of NPY. Ghrelin did not affect histamine release, and it increased food intake, even in H1R-KO mice. Thus, ghrelin expresses its action in a histamine-independent manner.     

Development of metabolic systems

In the normal adult rodent and primate, arcuate nucleus (ARH) neurons function as conduits for transmitting metabolic hormonal signals into the hypothalamic circuitry that modulates feeding and energy expenditure. We and others have shown that ARH projections do not fully develop until the 3rd postnatal week in the rodent. This is in stark contrast to the nonhuman primate (NHP) in which ARH projections develop during the 3rd trimester of pregnancy. This species difference suggests that maternal diet and health are likely key factors for the development of ARH projections in the primate, whereas the postnatal environment (i.e., diet) would be more important in the rodent. Furthermore, pertubations in these circuits during critical periods of development may have long-term consequences on feeding behavior and body weight management. Our group has used a rat model of overfeeding and underfeeding specifically during the postnatal period to begin to investigate the metabolic adaptions that may cause developmental abnormalities in the hypothalamic circuitry. While the overfed animals become obese as adults and the underfed maintain a lean phenotype, both models display low basal insulin and IGFII levels as adults. Furthermore, both models have abnormal expression of several key genes in peripheral metabolic tissue that are suggestive of changes in sympathetic outflow. Human studies show that gestational diabetes can also contribute to the development of obesity and diabetes in children; however, the mechanism is unknown. Since the critical periods for the development of hypothalamic circuits are different between rodents and primates our group has begun studies using NHP model to determine if maternal obesity/diabetes causes abnormalities in the development of metabolic systems, including the brain, in the offspring. To do this we have placed female NHPs on either a control diet or a high fat/calorie diet to induce obesity and diabetes. We have characterized the onset of insulin resistance and hyperleptinemia in these animals over the last 2(1/2) years and have collected offspring. Ongoing studies will investigate the metabolic abnormalities in these offspring.     

Co-existence of neuropeptides and differential inhibition of vasodilator responses by neuropeptide Y in guinea pig uterine arteries

The co-localization of substance P (SP) with calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP) with neuropeptide Y (NPY) of the guinea pig uterine artery were investigated with immunocytochemistry. The SP/CGRP fibre population was distinct from the VIP/NPY fibre population. Both types of fibres ran in the medial-adventitial border, and appeared as coarsed and fine varicosed. Uterine arterial dilatation was evoked by acetylcholine (ACh), SP, CGRP, and VIP in precontracted arteries as examined by a sensitive in vitro method. Strong relaxations were seen by ACh, CGRP and VIP. NPY had no relaxant effect per se but was found to be a potent inhibitor of vasodilation induced by ACh and SP, while relaxations induced by VIP and CGRP were unaffected. The functional significance of NPY in the uterine artery may to a large extent be to increase tension not only by potentiation of contraction but additionally by inhibiting vasodilator responses.     

Megestrol acetate increases short-term food intake in zinc-deficient rats

Rats offered a zinc-deficient (-Zn) diet voluntarily reduce their food intake within 3-4 days. Megestrol acetate (MA) is an appetite-stimulating drug used to treat cachexia of chronic diseases. In previous work, we found MA administration to male rats increased consumption of a -Zn diet. This approach would provide a useful tool for nutritional studies in which nutrient intake, except for zinc, would be maintained. The present study further examined the use of MA to increase consumption of a -Zn diet over a longer time period in both male and female rats. Rats were fed either a -Zn or a zinc-adequate (+Zn) diet. In Experiment 1, rats were treated orally with 0, 20, 50 or 100 mg MA/kg BW in corn oil for 21 days. MA stimulated intake of the -Zn diet in a linear manner. In Experiments 2 and 3, male and female rats, respectively, were fed the -Zn or +Zn diets and treated with 100 mg MA/kg BW for 21 days. In both experiments, MA administration increased intake of the -Zn diet to levels similar to the +Zn diet through Day 14. MA increased the hypothalamic neuropeptide Y (NPY) concentration in male rats, but did not affect serum IGF-I. MA administration improved growth of female but not male rats fed the -Zn diet. In females, serum IGF-I was not lower in zinc-deficient rats, which may have allowed the improved growth response with MA. Hence, MA administration may be a useful tool to increase consumption of a -Zn diet in short-term studies.     

PACAP deficient mice display reduced carbohydrate intake and PACAP activates NPY-containing neurons in the rat hypothalamic arcuate nucleus

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates both insulin release from islets and insulin action in adipocytes. Therefore, this peptide is considered a regulator of glucose homeostasis. PACAP and its receptors are localized not only in the peripheral tissues but in the central nervous system. The present study examined whether PACAP regulates the feeding behavior and the activity of neurons in the hypothalamic arcuate nucleus (ARC), a feeding center. Food intake was measured in the PACAP knock-out mice. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in single neurons isolated from the ARC of rats was measured by fura-2 microfluorometry, followed by immunocytochemical staining with anti-NPY antiserum. PACAP knock-out mice showed a decrease in the intake of high carbohydrate, but not high fat, food. PACAP increased [Ca²⁺]_i in NPY neurons of the ARC that are implicated in the feeding, particularly the carbohydrate ingestion. Agonists of PACAP receptors, PAC1-R and VPAC2-R, also increased [Ca²⁺]_i. The present study, by demonstrating that PACAP directly reacts with the ARC NPY neurons to increase [Ca²⁺]_i and that ingestion of the carbohydrate-rich food is reduced in PACAP-deficiency, suggests a facilitative role for PACAP in the carbohydrate intake.     

Central alpha-melanocyte stimulating hormone attenuates behavioral effects of neuropeptide Y in chicks

This experiment was conducted to determine the effects of central alpha-melanocyte stimulating hormone (alpha-MSH) and its interaction with neuropeptide Y (NPY) on ingestive and non-ingestive behaviors in chicks. Chicks received intracerebroventricular injections of either 0, 0.12 nM alpha-MSH, 0.06 nM NPY, or 0.12 nM alpha-MSH+0.06 nM NPY. Immediately following injection, chicks were placed in an observation arena and the number of steps, jumps, feed pecks, drinks, exploratory pecks, escape attempts, the total distance traveled, and the amount of time spent standing, sitting, sleeping, and preening were monitored for 60 min. Chicks treated with NPY consumed 69% more feed than controls whereas alpha-MSH-treated chicks consumed 71% less. Feed intake of the NPY+alpha-MSH groups was similar to alpha-MSH-treated chicks at 66% less than aCSF-treated chicks. Differences in pecking were found and followed a similar pattern as feed intake. All treatments increased the amount of time chicks were in a sitting posture, and the alpha-MSH+NPY group spent more time sitting than alpha-MSH and NPY alone. The sitting response after alpha-MSH+NPY treatment was similar to the alpha-MSH group but not the NPY group. Other behaviors were not affected by treatment. Thus, we conclude that alpha-MSH, at a concentration that causes a similar magnitude decrease in feed intake as NPY increases feed intake, is a more potent appetite-related signal than NPY. alpha-MSH causes behavioral effects that may secondarily affect feed intake at a low magnitude and may modulate the behavioral effects of NPY in chicks, contributing to the overall effect on feed intake.     

Orexins increase mRNA expressions of neurotrophin-3 in rat primary cortical neuron cultures

Orexins and melanin-concentrating hormone (MCH) as orexigenic neuropeptides are present in the lateral hypothalamus, and their receptors are distributed in the cerebral cortex and hippocampus. In the present study, the regulatory effects of orexin-A, orexin-B and MCH on neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) expressions were examined in primary cortical neuron cultures using quantitative real-time PCR. Both orexin-A and orexin-B on 6-day exposure significantly increased the NT-3 mRNA at concentrations of 0.01, 0.1 and 1 μM. Orexin-A and B at 1 μM led to an increase of twofold or more over the control. However, no such NT-s mRNA increase occurred with exposure to MCH at the same concentrations as orexins. The mRNA expression of BDNF was significantly increased only by orexin-B at 1 μM. These findings suggest that orexins, but not MCH, may be an inducer of NT-3 in the cerebral cortex.     

Metabolic sensors: viewing glucosensing neurons from a broader perspective

Glucose is a critical substrate for brain and organ function. Specialized glucosensing neurons, which are involved in the control of energy homeostasis and neuroendocrine function, are located in specific anatomic locations in the brain. Glucose-excited neurons increase their firing rate when ambient glucose levels rise. This glucosensing capacity appears to be regulated by a combination of glucokinase and an ATP-sensitive K(+) (K(ATP)) channel whose activity is regulated by ATP derived from glucose metabolism. Glucose inhibited neurons decrease their firing rate when glucose levels rise, although it is unclear what mechanism is used to control this function. Neuropeptide Y and proopiomelanocortin neurons in the hypothalamic arcuate nucleus are examples of neurons that are capable of sensing both glucose and a host of other peripheral metabolic signals, possibly by their actions on the K(ATP) channel. These metabolic sensing neurons are intimately involved in energy homeostasis, and it is postulated that glucose is only one of several peripheral metabolic signals involved in this process under physiologic conditions. However, when glucose supply is severely limited, glucose appears to assume primacy as a stimulant of glucosensing in order to activate the counterregulatory and ingestive processes necessary to restore the vital supply of glucose. Thus, the role of glucosensing is postulated to be a relative one that is dependent upon the supply of peripheral glucose.     

The dorsomedial hypothalamic nucleus and its role in ingestive behavior and body weight regulation: lessons learned from lesioning studies

This review article discusses the well-established role of the dorsomedial hypothalamic nucleus (DMN) in feeding, drinking and body weight (BW) regulation. DMN lesions (L) in both weanling and mature rats of both sexes produce hypophagia, hypodipsia and reduced ponderal and linear growth in the presence of normal body composition. The growth reduction is not due to a deficient secretion of growth hormone, insulin-like growth factor-1, thyroxine, triiodothyronine or insulin. DMNL rats actively defend their lower BW (BW settling point) by becoming either hyper- or hypophagic, depending on the experimental manipulation, thereby defending both lean and fat mass. They also regulate their 24-h caloric intake, but they may overeat during the first hour of refeeding following a fast, possibly due to a reduced ability to monitor blood glucose or to respond to cholecystokinin (CCK). 2-Deoxy-D-glucose (2DG) increases c-fos expression in orexin-A neurons in the DMN, and DMNL eliminated the orexigenic effect of 2DG. DMNL rats on high-fat diets do not get as obese as controls, which may be due to a reduction of DMN neuropeptide Y (NPY). Rats lacking DMN CCK-A receptors are obese and have increased expression of NPY in the DMN, supporting earlier data that CCK may act at the DMN to suppress food intake. Excitotoxin studies showed that loss of DMN cell somata, and not fibers of passage, is important in the development of the DMNL syndrome. The DMN is a site where opioids increase food intake and knife-cut studies have shown that fibers traveling to/from the DMN are important in this response. An interaction of glucose and opioids in DMN may also be involved in the control of food intake. DMN knife cuts interrupting fibers in the posterior and ventral directions additively produce the hypophagia and reduced linear and ponderal growth observed after DMNL. Ventral cuts may interrupt important connections with the arcuate nucleus. Lateral and posterior DMN cuts additively produce the hypodipsic effect seen after DMNL, but DMNL rats respond normally to all water-regulatory challenges, i.e., the hypophagia is not due to a primary hypodipsia. The DMN has been shown to be involved in the rat's feeding response to an imbalanced amino acid diet. These data show the DMN has an important role in many processes that control both food intake and BW regulation.     

TRH decreases food intake and increases water intake and body temperature in rats

Thyrotropin-releasing hormone (TRH) is a key regulator of the hypothalamus-pituitary-thyroid axis, which plays an important role in energy homeostasis and is involved in the regulation of feeding behavior. In the present study, we investigated the effects of acute and chronic TRH treatment on water intake, body temperature and feeding behavior in rats. TRH (0, 4, 16 and 64 mg/kg) was injected subcutaneously twice a day (06:00 and 18:00 h) in rats fed ad libitum. TRH decreased food and water intake in the first few hours (P < .05). There was a small reduction in food intake over the 24-h period, but body weight was not affected (P < .05). When TRH was injected at a dose of 32 mg/kg twice a day (06:00 and 18:00 h) for 5 days, T(3) and T(4) concentrations were increased (P < .05). TRH increased body temperature for 2 h after injection. Water intake was markedly increased (P < .05), but there was no effect on food intake or body weight. These results show that whereas a single injection of TRH decreases short-term food and water intake in rats, repeated daily treatments stimulate water intake but not food intake, and, thus, the increase in water consumption is mediated independently of food intake under these conditions.     

神经肽Y放免分析法的建立

本文在自制了特异性神经肽Y(NPY)抗血清基础上,建立了血浆、脑组织NPY放免分析法。竞争抑制分析证明,与相关神经肽无交叉反应,最小检测范围为150～200 pg/ml,血浆和组织平均回收率为85～90%,说明该法特异性、灵敏性良好。反相高压液相层析表明所测血浆和脑组织NPY—Li的层析特性与标准NPY基本一致。     

Obesity in neurobiology

Obesity reflects an imbalance between energy uptake and expenditure that is mediated by behavior. Obesity is a growing epidemic and a major risk factor for neurobiological diseases like stroke, dementia, intracranial hypertension and sleep disorders. Conversely, obesity can also be induced by neurobiological disorders and drugs. The etiology of obesity is complex and includes biology, behavior and environment. Physicians are faced with the need to manage obesity while strategies for prevention and sustained weight reduction are limited. Present treatment options comprise lifestyle modification, diet, pharmacotherapy and bariatric surgery. Considerable headway has been made into elucidating the neurobiological underpinnings of obesogenic behavior. There is now a growing understanding of the metabolic, hormonal and behavioral circuitries that contribute to the complex and redundant system for energy balance. Changing the net balance of this system to prevent or reduce obesity requires multimodal and long-term interventions.     

Alertness and feeding behaviors in ADHD: Does the hypocretin/orexin system play a role?

Increasing evidence has suggested that patients with attention-deficit/hyperactivity disorder (ADHD) may present with a deficit of alertness and sleep disturbances. Recent studies have also pointed out a previously underestimated association between ADHD and abnormal eating behaviors, including binge eating. Since sleep/alertness disturbances and eating disorders may significantly increase the functional impairment of ADHD, gaining insight into their pathophysiology as well as into their treatment is of relevance to provide a better clinical management of patients suffering from ADHD. The hypocretin/orexin system comprises two distinct peptides, located in the hypothalamus, which are involved in several homeostatic functions. In particular, it has been suggested that hypocretin/orexin neurons located in perifornical and dorsomedial hypothalamic nuclei increase arousal, whereas those located in the lateral hypothalamus are primarily implicated in reward processing, stimulating feeding and other reward seeking behaviors. Given the involvement of the hypocretin/orexin system in the control of alertness and reward seeking (including feeding), we hypothesize that hypocretin/orexin neurons located in perifornical and dorsomedial hypothalamic areas are hypoactivated, while those located in the lateral hypothalamus are overactivated in patients with ADHD. If confirmed by further neurophysiological, imaging, and genetics studies, our hypothesis may help us progress in the understanding of the complex pathophysiology of ADHD. This might set the basis for the study of novel molecules, acting on the hypocretin/orexin system, aimed at increasing wakefulness and reducing binge eating and other abnormal reward seeking behaviors in patients with ADHD. We also suggest future studies on the potential therapeutic role of other molecules which have a complex interplay with the hypocretin/orexin system, such as the histamine H(1) receptor agonists, the histamine H(3) receptor antagonists, and the neuropeptide Y receptor antagonists. All this body of research would provide a tremendous opportunity to improve the quality of life of patients with ADHD by means of pathophysiologically oriented treatment.     

失血性休克患者血浆神经肽Y的含量变化

采用放射免疫分析法测定了３０例健康人、３１例轻度休克及３０例中度休克患者血浆神经肽Ｙ的含量。结果显示，轻度休克患者血浆ＮＰＹ含量明显高于正常对照组（ｐ〈０．０１）；中度休克患者血浆ＮＰＹ含量明显高于正常对照组（ｐ〈０．００１），也明显高于轻度休克组（ｐ〈０．０１）。血浆神经肽Ｙ含量增高与失血性休克的严重程度密切相关，提示神经肽Ｙ在休克的发病机理中可能起重要作用。     

瘦素（leptin）与肥胖

Obesity is a severely public health problem the whole society faces, and it is correlated closely with many diseases, such as diabetesⅡ, hypertension, coronary heart disease,gallqtone, and so on.Therefore it threatens people‘ s survival quality severely. Obesity is a multiple - factor disease including genetic, metabolic and behavioral factor, and the gene is the main determining factor. With the development of molecular biology technique, people have founded several genes involved in obesity. Among these genes, the research on obese gene is the most profound. The protein leptin is the expression product of the obese gene.This review elucidates the structure, the main biological function, the mechanism of leptin and it‘‘s relationship with obesity.     

Anabolic hormone levels in immunized rats

Primary immune response of rats to sheep red blood cells (SRBC) was accompanied by elevation of insulin and growth hormone (GH) levels in serum, and decrease of thyroxine T₄. No material change in proclatin level was observed.