Effect of 2-mercaptoacetate and 2-deoxy-D-glucose administration on the expression of NPY, AGRP, POMC, MCH and hypocretin/orexin in the rat hypothalamus

Using in situ hybridization, the mRNA levels encoding neuropeptide Y (NPY), agouti gene-related protein (AGRP), proopiomelanocortin (POMC), melanin-concentrating hormone (MCH) and hypocretin/orexin (HC/ORX) were investigated in the rat arcuate nucleus (Arc) and lateral hypothalamic area (LHA) 2 h after a single dose of the glucose antimetabolite 2-deoxy-D-glucose (2-DG; 600 mg/kg) or of the fatty acid oxidation inhibitor mercaptoacetate (MA; 600 mumol/kg). Two hours after 2-DG or MA injection food intake was significantly increased. NPY and AGRP mRNA levels in the Arc were increased by 2-DG but not affected by MA, and MCH mRNA levels in the LHA were increased by both antimetabolites. These results suggest that Arc neurons expressing NPY and AGRP are regulated by changes in glucose, but not fatty acid availability, whereas both factors affect MCH neurons in the LHA.     

Integration of the regulation of reproductive function and energy balance: lactation as a model

Lactation is a physiological model for studying how the hypothalamus integrates peripheral signals, such as sensory signals (suckling stimulus) and those denoting energy balance (leptin), to alter hypothalamic function regulating food intake/energy balance and reproduction. The characteristics of food intake/energy balance during lactation are extreme hyperphagia, coupled with negative energy balance. The arcuate nucleus Neuropeptide Y (ARH-NPY) system is activated by: (1) brainstem projections specifically activated by the suckling stimulus, and (2) the decrease in leptin in response to the metabolic drain of milk production. NPY neurons from the ARH make direct contact with GnRH neurons and with CRH neurons in the PVH. NPY neurons also make contact with orexin and MCH neurons in the LHA, which, in turn, make contacts with GnRH neurons. Thus, the ARH-NPY system provides a neuroanatomical framework by which to integrate changes in food intake/energy with the regulation of cyclic reproductive function.     

Distribution of orexin neurons in the adult rat brain

Orexin (ORX)-A and -B are recently identified neuropeptides, which are specifically localized in neurons within and around the lateral hypothalamic area (LHA) and dorsomedial hypothalamic nucleus (DMH), the regions classically implicated in feeding behavior. Here, we report a further study of the distribution of ORX-containing neurons in the adult rat brain to provide a general overview of the ORX neuronal system. Immunohistochemical study using anti-ORX antiserum showed ORX-immunoreactive (ir) neurons specifically localized within the hypothalamus, including the perifornical nucleus, LHA, DMH, and posterior hypothalamic area. ORX-ir axons and their varicose terminals showed a widespread distribution throughout the adult rat brain. ORX-ir nerve terminals were observed throughout the hypothalamus, including the arcuate nucleus and paraventricular hypothalamic nucleus, regions implicated in the regulation of feeding behavior. We also observed strong staining of ORX-ir varicose terminals in areas outside the hypothalamus, including the cerebral cortex, medial groups of the thalamus, circumventricular organs (subfornical organ and area postrema), limbic system (hippocampus, amygdala, and indusium griseum), and brain stem (locus coeruleus and raphe nuclei). These results indicate that the ORX system provides a link between the hypothalamus and other brain regions, and that ORX-containing LHA and DMH neurons play important roles in integrating the complex physiology underlying feeding behavior.     

Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area

Recent studies have identified several neuropeptide systems in the hypothalamus that are critical in the regulation of body weight. The lateral hypothalamic area (LHA) has long been considered essential in regulating food intake and body weight. Two neuropeptides, melanin-concentrating hormone (MCH) and the orexins (ORX), are localized in the LHA and provide diffuse innervation of the neuraxis, including monosynaptic projections to the cerebral cortex and autonomic preganglionic neurons. Therefore, MCH and ORX neurons may regulate both cognitive and autonomic aspects of food intake and body weight regulation. The arcuate nucleus also is critical in the regulation of body weight, because it contains neurons that express leptin receptors, neuropeptide Y (NPY), α-melanin-stimulating hormone (α-MSH), and agouti-related peptide (AgRP). In this study, we examined the relationships of these peptidergic systems by using dual-label immunohistochemistry or in situ hybridization in rat, mouse, and human brains. In the normal rat, mouse, and human brain, ORX and MCH neurons make up segregated populations. In addition, we found that AgRP- and NPY-immunoreactive neurons are present in the medial division of the human arcuate nucleus, whereas α-MSH-immunoreactive neurons are found in the lateral arcuate nucleus. In humans, AgRP projections were widespread in the hypothalamus, but they were especially dense in the paraventricular nucleus and the perifornical area. Moreover, in both rat and human, MCH and ORX neurons receive innervation from NPY-, AgRP-, and α-MSH-immunoreactive fibers. Projections from populations of leptin-responsive neurons in the mediobasal hypothalamus to MCH and ORX cells in the LHA may link peripheral metabolic cues with the cortical mantle and may play a critical role in the regulation of feeding behavior and body weight. J. Comp. Neurol. 402:442–459, 1998. © 1998 Wiley-Liss, Inc.     

Neuroanatomy of melanocortin‐4 receptor pathway in the lateral hypothalamic area

The central melanocortin system regulates body energy homeostasis including the melanocortin‐4 receptor (MC4R). The lateral hypothalamic area (LHA) receives dense melanocortinergic inputs from the arcuate nucleus of the hypothalamus and regulates multiple processes including food intake, reward behaviors, and autonomic function. By using a mouse line in which green fluorescent protein (GFP) is expressed under control of the MC4R gene promoter, we systemically investigated MC4R signaling in the LHA by combining double immunohistochemistry, electrophysiology, and retrograde tracing techniques. We found that LHA MC4R‐GFP neurons coexpress neurotensin as well as the leptin receptor but do not coexpress other peptide neurotransmitters found in the LHA including orexin, melanin‐concentrating hormone, and nesfatin‐1. Furthermore, electrophysiological recording demonstrated that leptin, but not the MC4R agonist melanotan II, hyperpolarizes the majority of LHA MC4R‐GFP neurons in an ATP‐ sensitive potassium channel–dependent manner. Retrograde tracing revealed that LHA MC4R‐GFP neurons do not project to the ventral tegmental area, dorsal raphe nucleus, nucleus accumbens, and spinal cord, and only limited number of neurons project to the nucleus of the solitary tract and parabrachial nucleus. Our findings provide new insights into MC4R signaling in the LHA and its potential implications in homeostatic regulation of body energy balance. J. Comp. Neurol. 4168–4183, 2012. © 2012 Wiley Periodcicals, Inc.     

Neuronal Activation of the Hypothalamic Magnocellular System in Response to Oropharyngeal Stimuli in the Rat

The present study was designed to delineate the neuronal site, the nature, and the gastrointestinal origin of the stimulation of the hypothalamic magnocellular system induced by the ingestion of sweetened condensed milk. Concomitant localization of the c- fos protein (Fos) with either arginine-vasopressin (AVP) mRNA or oxytocin (OT) mRNA in the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON) revealed that the hypothalamic neurons containing AVP and OT were activated following ingestion of sweetened condensed milk. Expression of c- fos mRNA was also determined in rats implanted with a gastric cannula that allowed for real, sham, and gastric feeding of sweetened condensed milk. The results provide evidence that the stimulation of the PVH and SON induced by sweetened condensed milk originate from oropharyngeal stimuli. Indeed, in real-and sham-fed rats, the postprandial levels of c- fos mRNA in the PVH and SON were significantly higher than the preprandial values, whereas there was no early postprandial rise in c- fos mRNA levels within the magnocellular division of the PVH and SON after gastric feeding. The results of this study also suggested that the stimulation of the PVH and SON induced by sweetened condensed milk was related to the hypertonicity of the milk. Indeed, ingestion of an hypertonic solution of sucrose with a carbohydrate content close to that of sweetened condensed milk led to a stimulation of the PVH and SON that was comparable to that induced by the milk, whereas ingestion of an isotonic solution of sucrose did not trigger any significant activation of the PVH and SON. Taken together, the present results indicate that magnocellular neurosecretory neurons are sensitive to oropharyngeal stimuli and further support the view of the existence of oropharyngeal osmoreceptors.     

Distribution of prodynorphin mRNA and its interaction with the NPY system in the mouse brain

Using radioactive in situ hybridisation, the distribution of prodynorphin mRNA in the brains of C57Bl/6 mice was systemically investigated, and double-labelling in situ hybridisation was used to determine the extent to which neuropeptide Y (NPY) and prodynorphin mRNAs were co-expressed. Our results demonstrate that prodynorphin mRNA expression in the mouse brain is localised at specific subregions of the olfactory bulb, cortex, hippocampus, amygdala, basal ganglia, thalamus, hypothalamus, mesencephalon and myelencephalon. Among the regions displaying the most intense labelling were the olfactory tubercle, lateral septum (LS), caudate putamen (Cpu), central amygdaloid nucleus (Ce), paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SO), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), lateral reticular nucleus (LRt) and solitary tract nucleus (NTS). In the arcuate nucleus of the hypothalamus (Arc), double-labelling in situ hybridisation revealed that prodynorphin expressing neurons also contained NPY mRNA, with a co-localisation rate of approximately 88% in the lateral part of the Arc, and 79% in the dorsal part of the Arc, respectively, suggesting potential overlapping functions of these two neurotransmitters in feeding type behaviour.     

Effects of Cholecystokinin in the Supraoptic Nucleus and Paraventricular Nucleus are Negatively Modulated by Leptin in 24‐h Fasted Lean Male Rats

Cholecystokinin (CCK) and leptin are two important satiety factors that are considered to act in synergy to reduce meal size. Peripheral injection of CCK activates neurones in several hypothalamic nuclei, including the supraoptic (SON) and paraventricular (PVN) nuclei and neurones in the brainstem of fed rats. We investigated whether peripheral leptin would modulate the effects of CCK on neuronal activity in the hypothalamus and brainstem of fasted rats by investigating Fos expression in the PVN, SON, arcuate nucleus, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), area postrema (AP) and the nucleus tractus solitarii (NTS). Male rats, fasted for 24 h, received either one i.p. injection of vehicle, leptin or CCK‐8 alone, or received one injection of vehicle or leptin before an i.p. injection of CCK‐8. We found that CCK increased Fos expression in the PVN and SON as well as in the NTS and AP, but had no effect on Fos expression in the arcuate nucleus, VMH or DMH compared to vehicle. Leptin injected alone significantly increased Fos expression in the arcuate nucleus but had no effect on Fos expression in the VMH, DMH, SON, PVN, AP or NTS compared to vehicle. Fos expression was significantly increased in the AP in rats injected with both leptin and CCK compared to rats injected with vehicle and CCK. Unexpectedly, there was significantly less Fos expression in the PVN and SON of fasted rats injected with leptin and CCK than in rats injected with vehicle and CCK, suggesting that leptin attenuated CCK‐induced Fos expression in the SON and PVN. However, Fos expression in the NTS was similar in fasted rats injected with vehicle and CCK or with leptin and CCK. Taken together, these results suggest that leptin dampens the effects of CCK on Fos expression in the SON and PVN, independently from NTS pathways, and this may reflect a direct action on magnocellular neurones.     

Contribution of the vagus nerve and lamina terminalis to brain activation induced by refeeding

Following refeeding, c-fos expression is induced in a particular set of brain regions that include the nucleus of the solitary tract (NTS), parabrachial nucleus (PB), central amygdala (CeA), paraventricular hypothalamic nucleus (PVH), supraoptic nucleus (SON) and the circumventricular organs. Within the PVH, the expression is particularly intense in the magnocellular division of the nucleus and it is as yet not clear how this activation occurs. The respective contribution of the vagus afferents and lamina terminalis, which conveys signals entering the brain through the forebrain circumventricular organs, has been investigated in rats subjected to a unilateral cervical vagotomy (UCV) or a unilateral lesion of the fibres running within the lamina terminalis (ULT) and projecting to the neuroendocrine hypothalamus. UCV significantly decreased postprandial c-fos expression in the NTS, PB, CeA and parvocellular division of the PVH. In contrast, ULT impaired postprandial activation of the magnocellular neurons in the PVH and SON. The present study also characterized the types of neurons activated in the PVH and SON during refeeding. In the magnocellular regions, arginine-vasopressin (AVP) neurons were activated upon refeeding whereas there was no apparent induction of Fos expression in oxytocin cells. In the parvocellular PVH, postprandial Fos was induced only in 30% of the corticotrophin-releasing factor (CRF) and AVP neurons. The results of the present study suggest that the postprandial activation of the brain requires the integrity of both the vagal- and lamina terminalis-associated pathways.     

Oxytocin neurons in the supraoptic nucleus receive excitatory inputs from the bilateral dorsomedial hypothalamic nuclei

To examine whether inputs from the dorsomedial hypothalamic nucleus (DMH) alter the discharge of putative oxytocin (OT) neurons with hypothesis that excitation of DMH neurons would increase the activity of OT neurons, electrical stimulation was applied to the DMH in both sides of the hypothalamus while electrical activity of single OT neurons in the supraoptic nucleus (SON) was recorded in urethane-anesthetized lactating rats. About half of the OT neurons showed orthodromic excitation or inhibition followed by excitation in response to electrical stimulation of the DMH on both sides. Continuous electrical stimulation of the DMH both ipsi- and contralateral to the recording side at 10-50 Hz for 30-60 s increased firing rate in 58% of OT neurons tested. Continuous electrical stimulation of the DMH not only excited spiking activity of single OT neurons but also increased intramammary pressure. The results may suggest that some of the projections from the DMH to the SON are bilateral and possibly contribute to coordinated bilateral activation of OT neurons in the hypothalamus during the milk-ejection reflex.     

CRFergic Innervation Of the Paraventricular Nucleus of the Rat Hypothalamus: A Tract-Tracing Study

It has been reported that corticotropin-releasing factor (CRF) may regulate its own biosynthesis in the paraventricular nucleus of the hypothalamus (PVH). Whether this CRF autoregulation is mediated by local circuitry or from extra-PVH CRF neuronal fibers terminating on CRF perikarya within the PVH is unknown. In the present study, we sought to determine the origin(s) of this CRF innervation using retrograde transport of wheat germ-conjugated-gold particles (WGA-apoHRP-Au) combined with immunohistochemistry for CRF. The rats also received colchicine (100 μg, icv) 5–7 days after tracer injection and were perfused 24 h later. Results of retrograde labeling with pressure injections of WGA-apoHRP-Au centered to PVH and subsequent immunohistochemical staining for CRF demonstrated numerous retrogradely labeled CRF neurons in the perifornical hypothalamic nucleus (PeF), the dorsolateral hypothalamic area (DA) (medial and lateral portions) and the dorsomedial nucleus of the hypothalamus (DMH). Smaller groups of CRF-ir neurons that were retrogradely labeled were found in the bed nuclei of the stria terminalis (BnST), the Barrington’s nucleus (Bar) and the dorsal raphé (DR). These CRFergic pathways to the PVH may represent an anatomical substrate underlying the function of the stress-integrative PVH neurons in the autonomic, behavioral and neuroendocrine regulation during the stress response, including CRF autoregulation.     

Glucocorticoids are required for meal-induced changes in the expression of hypothalamic neuropeptides

Glucocorticoid deficiency is associated with a decrease of food intake. Orexigenic peptides, neuropeptide Y (NPY) and agouti related protein (AgRP), and the anorexigenic peptide proopiomelanocortin (POMC), expressed in the arcuate nucleus of the hypothalamus (ARC), are regulated by meal-induced signals. Orexigenic neuropeptides, melanin-concentrating hormone (MCH) and orexin, expressed in the lateral hypothalamic area (LHA), also control food intake. Thus, the present study was designed to test the hypothesis that glucocorticoids are required for changes in the expression of hypothalamic neuropeptides induced by feeding. Male Wistar rats (230-280 g) were subjected to ADX or sham surgery. ADX animals received 0.9% NaCl in the drinking water, and half of them received corticosterone in the drinking water (B: 25 mg/L, ADX+B). Six days after surgery, animals were fasted for 16 h and they were decapitated before or 2 h after refeeding for brain tissue and blood collections. Adrenalectomy decreased NPY/AgRP and POMC expression in the ARC in fasted and refed animals, respectively. Refeeding decreased NPY/AgRP and increased POMC mRNA expression in the ARC of sham and ADX+B groups, with no effects in ADX animals. The expression of MCH and orexin mRNA expression in the LHA was increased in ADX and ADX+B groups in fasted condition, however there was no effect of refeeding on the expression of MCH and orexin in the LHA in the three experimental groups. Refeeding increased plasma leptin and insulin levels in sham and ADX+B animals, with no changes in leptin concentrations in ADX group, and insulin response to feeding was lower in this group. Taken together, these data demonstrated that circulating glucocorticoids are required for meal-induced changes in NPY, AgRP and POMC mRNA expression in the ARC. The lower leptin and insulin responses to feeding may contribute to the altered hypothalamic neuropeptide expression after adrenalectomy.     

Novel hypothalamic and preoptic sites of prepro-melanin-concentrating hormone messenger ribonucleic Acid and Peptide expression in lactating rats

The deduced structure of the rat melanin-concentrating hormone (MCH) precursor predicted the existence of at least two peptides that may be processed from it, one similar to teleost MCH and a second novel neuropeptide, NEI. Cellular localization studies confirmed that prepro-MCH (ppMCH) mRNA and the MCH and NEI peptides are expressed predominantly in cells in the zona incerta and caudal lateral hypothalamic area with minor contingents seen in the olfactory tubercle and pons. A moderate MCH-and NEI-immunoreactive axonal projection to the median eminence and, particularly, to oxytocin-rich regions of the posterior pituitary suggested some anatomical heterogeneity of ppMCH-expressing neurons in the hypothalamus, and an involvement in neuroendocrine function. In the present study, immunohistochemical and hybridization histochemical methods were used to follow MCH gene and peptide expression as a function of reproductive status in female rats. Nursing dams sacrificed after 8 to 21 days of lactation consistently displayed ppMCH mRNA and MCH and NEI immunoreactivity in a discrete and contiguous band, encompassing the ventral aspect of the medial part of the medial preoptic nucleus, the periventricular preoptic nucleus, and the most rostral aspects of the paraventricular nucleus of the hypothalamus (PVH). Combined immunohistochemical (for oxytocin) and hybridization histochemical (for ppMCH mRNA) staining failed to reveal a significant degree of congruence in the two chemically-specified cell populations in the PVH of lactating dams. The apparent induction of ppMCH-derived peptides and mRNA in the preoptic area and PVH was not apparent in animals sacrificed 4 to 8 days after weaning, during late pregnancy, or at any point in the estrous cycle. Moreover, no frank alterations in ppMCH mRNA were evident in the principal sites at which ppMCH is expressed at constitutively high levels, i.e. in the lateral hypothalamic area and zona incerta, as a function of reproductive status. The loci and apparent state-dependency of the induction of ppMCH mRNA and peptide expression suggests a role for these gene products in the control of lactation.     

Metabolic alterations in the hypothalamus of the Brattleboro rat demonstrated with cytochrome oxidase histochemistry

Metabolic activity in the hypothalamus of homozygous and heterozygous Brattleboro rats and Long-Evans control rats was studied using cytochrome oxidase histochemistry. Increased metabolic activity was observed in the paraventricular nucleus (PVH), supraoptic nucleus (SON) and nucleus circularis (NC) of homozygous Brattleboro rats, and in the PVH of heterozygous rats. These results suggest that the metabolic activity of PVH and SON neurons is altered because of the inability of magnocellular neurosecretory neurons to produce vasopressin. In addition, the hyperactivity of neurons in the NC is probably related to the chronic dehydration present in these animals.