Adrenalectomy decreases neuropeptide Y mRNA levels in the arcuate nucleus

Recent studies suggest that glucocorticoids may increase NPY and NPY mRNA levels. To determine if endogenous corticosterone affects the level of NPY mRNA in areas that control NPY levels in the paraventricular nucleus, we examined the effects of adrenalectomy and corticosterone replacement on NPY mRNA levels in the arcuate nucleus and brainstem. Rats were either adrenalectomized, adrenalectomized and corticosterone replaced, or sham-operated. The arcuate nucleus, hypothalamus (excluding arcuate nucleus), and brainstem were collected and the RNA isolated. Dot blots were made of each tissue and the NPY mRNA quantitated by densitometry. Adrenalectomy significantly reduced NPY mRNA levels in the arcuate nucleus, while corticosterone replacement restored the NPY mRNA levels. NPY mRNA levels in the remainder of the hypothalamus were not affected by adrenalectomy. Adrenalectomy also had no affect on NPY mRNA levels in the brainstem. These data suggest that the paraventricular nucleus may be affected by glucocorticoids via an NPY pathway and that the two major afferent pathways of NPY-containing neurons to the paraventricular nucleus may be regulated by different mechanisms.     

Effect of Sustained Physiological Hyperinsulinaemia on Hypothalamic Neuropeptide Y and NPY mRNA Levels in the Rat

Neuropeptide Y (NPY) synthesized in the arcuato-paraventricular projection in the rat hypothalamus is thought to play an important role in controlling energy homeostasis. The factors that regulate hypothalamic NPY are not known but, amongst others, insulin has been postulated as an inhibitory modulatory agent. To test this hypothesis, normal male rats were given either insulin (2 units/day) or saline via subcutaneous osmotic minipumps for 3 days. Euglycaemia was maintained by a concomitant glucose infusion in insulin-infused rats which had peripheral insulin levels 5–8 times higher than saline-infused controls. Hyperinsulinaernic rats ate 42% less than controls, but their total energy intake (food intake plus glucose infusion) was higher than that of controls, and they gained more weight than controls during the experimental period. Hyperinsulinaemia had no significant effect on hypothalamic NPY mRNA or NPY levels in the arcuate nucleus. NPY concentrations in the paraventricular nucleus were, however, significantly increased by 73% in hyperinsulinaemic rats, but were closely similar to controls in all other areas. Insulin may act as a satiety factor in that hyperinsulinaemic rats ate less, but the fact that these animals had increased total energy intake and gained excessive weight suggests that insulin may not function as an overall regulator of energy balance. In addition, physiological hyperinsulinaemia does not apparently inhibit NPY gene expression in the arcuate nucleus. Due to the lack of effect of hyperinsulinaemia on NPY synthesis in the arcuate nucleus, the elevated NPY concentrations in the paraventricular nucleus could result from a reduction of its release, which would be in keeping with the reduction in food intake.     

Effects of monosodiuml-glutamate administration on neuropeptide Y-containing neurons in the rat hypothalamus

Immunocytochemical localization of neuropeptide Y (NPY) was performed in the hypothalamus of rats of which the arcuate nucleus had been destroyed with monosodiuml-glutamate in the neonatal period. The treatment produced a disappearance of most of the NPY cell bodies normally found in the arcuate nucleus. The concentration of fibers was decreased in the paraventricular nucleus, but not in the other hypothalamic nuclei. The treatment also induced the appearance of a large number of immunoreactive cell bodies in the paraventricular nucleus. These results strongly suggest that arcuate NPY neurons are projecting to the paraventricular nucleus and that the arcuate nucleus probably exerts some inhibitory tonic influence on NPY paraventricular neurons.     

Effects of monosodium L-glutamate administration on neuropeptide Y-containing neurons in the rat hypothalamus

Immunocytochemical localization of neuropeptide Y (NPY) was performed in the hypothalamus of rats of which the arcuate nucleus had been destroyed with monosodium L-glutamate in the neonatal period. The treatment produced a disappearance of most of the NPY cell bodies normally found in the arcuate nucleus. The concentration of fibers was decreased in the paraventricular nucleus, but not in the other hypothalamic nuclei. The treatment also induced the appearance of a large number of immunoreactive cell bodies in the paraventricular nucleus. These results strongly suggest that arcuate NPY neurons are projecting to the paraventricular nucleus and that the arcuate nucleus probably exerts some inhibitory tonic influence on NPY paraventricular neurons.     

Decreased NPY innervation of the hypothalamic nuclei in rats with cancer anorexia

Whether the decrease in food intake that occurs at the onset of anorexia in tumor bearing (TB) rats is related to a change in the hypothalamic neuropeptide Y (NPY) system was tested by comparing NPY expression in sham operated Fischer Control and anorectic TB rats. Coronal cryocut sections of their fixed brain were processed by the peroxidase-antiperoxidase method with NPY polyclonal antibodies. NPY-immunoreactive fibers were widely distributed throughout the forebrain, but were most prominent in the hypothalamic paraventricular, suprachiasmatic, arcuate and periventricular nuclei. NPY-immunoreactive neurons were visualized in Control and anorectic TB rats in the preoptic region, the arcuate nucleus, and occasionally in the lateral hypothalamus. Semiquantitative image analysis showed a significant decrease in the NPY immunostaining in some hypothalamic nuclei of the anorectic TB rats, most prominently in the supraoptic nucleus, the parvocellular portion of the paraventricular nucleus, and, to a lesser extent, the suprachiasmatic and arcuate nuclei. No changes in NPY innervation were seen in the ventromedial nucleus and the lateral hypothalamus. The data support the hypothesis of an altered hypothalamic NPY system at the onset of anorexia in TB rats and also reveal the hypothalamic nuclei through which NPY influences food intake.     

Evidence that hypothalamic neuropeptide Y gene expression and NPY levels in the paraventricular nucleus increase before the onset of hyperphagia in experimental diabetes

Neuropeptide Y (NPY) is the most potent endogenous orexigenic signal. Several lines of evidence indicate that the site of NPY action in transducing feeding signal may reside in the paraventricular nucleus (PVN) and neighboring sites in the hypothalamus. To test the hypothesis that an increase in NPY activity in the ARC-PVN pathway precedes the onset of diabetic hyperphagia, we evaluated NPY levels in seven hypothalamic nuclei and NPY gene expression in the hypothalamus at 48, 72 or 96 h after streptozotocin (STZ) treatment in rat. In STZ-treated diabetic rats, NPY gene expression in the hypothalamus and NPY levels only in the PVN significantly elevated at 48 h, while hyperphagia occurred sometimes after 48 h post-injection. These results show that augmentation in NPY neuronal activity in the ARC-PVN axis precedes the onset of increased food intake produced by STZ-induced insulinopenia. These findings affirm the hypothesis that increased NPY neurosecretion in the PVN may underlie the diabetes-induced hyperphagia.     

Directional cues for arcuate NPY projections are present in the adult brain

Arcuate nucleus neuropeptide Y (NPY) neurons project within the hypothalamus and to several extrahypothalamic brain areas. Plasticity in the formation of arcuate NPY projections established postnatally may underlie the phenotypic characteristics of food intake and body weight. In this work we determined if directional cues for axonal outgrowth of NPY arcuate neurons exist in the adult brain. For this purpose, an embryonic (E15) arcuate nucleus of WT mice was grafted into the third ventricle of 2-week- and 2-month-old NPY knockout (KO) mice. One month after the transplantation, the distribution of NPY-positive terminals in the brains of NPY-KO mice was studied using immunohistochemistry. NPY-positive terminals were found inside of the grafted tissue as well as in the host hypothalamus, including the arcuate nucleus, the paraventricular and periventricular nuclei, the lateral hypothalamic and preoptic areas, and in extrahypothalamic areas such as the amygdala and the thalamic paraventricular nucleus. This pattern of distribution of NPY fibers was found in both groups of grafted mice. The brain areas reinnervated by NPY-positive terminals in the NPY-KO mice closely corresponded to the normal targets for the arcuate NPY neurons as revealed by the distribution of agouti gene-related protein immunoreactivity. Our data show that directional cues for NPY arcuate nucleus projections are present in the adult brain, suggesting their involvement in the formation of normal arcuate NPY connections and a possibility for their functional reconstruction.     

Increased neuropeptide Y content in the arcuato-paraventricular hypothalamic neuronal system in both insulin-dependent and non-insulin-dependent diabetic rats

Neuropeptide Y (NPY) concentration was determined by radioimmunoassay in selected hypothalamic regions microdissected from fresh brain slices of different types of diabetic rats. In spontaneously diabetic (BB) rats and streptozotocin (STZ)-induced diabetic rats, models of insulin-dependent diabetes mellitus, an elevated concentration of NPY was detected in the paraventricular nucleus (PVN) and arcuate nucleus (ARH) of the hypothalamus. In Wistar fatty rats, a model of non-insulin-dependent diabetes mellitus, NPY concentration was also high in the PVN as compared to controls. When STZ-induced diabetic rats were treated with insulin, elevated NPY content in the PVN returned to the normal level. These findings, together with our previous finding of increased secretion of insulin after microinjection of NPY into the PVN, suggest a crucial role of NPYergic neuronal system in the ARH-PVN area in controlling endocrine pancreas and glucose homeostasis.     

Neuropeptide Y in the arcuato-paraventricular pathway and diet selection in the vasopressin-deficient Brattleboro rat

Neuropeptide Y (NPY) is one of the most important brain peptides involved in feeding behavior. It influences both food choice and fluid homeostasis. The paraventricular and arcuate nuclei belong to the main pathway through which NPY stimulates carbohydrate intake. In this study, we measured NPY in various hypothalamic microdissected areas in Brattleboro di/di rats, a rat model of diabetes insipidus with specific dietary preferences. We confirmed that this rat is characterized by an increased fat intake (+10%; p < 0.001) and a decreased carbohydrate intake (−10%; p < 0.001) leading to a completely different dietary profile than that of di/+ controls. This profile was associated with a decrease in NPY in the paraventricular nucleus (−33%; p < 0.005) and in the ventromedial nucleus (−24%; p < 0.002). Intake of carbohydrate was negatively correlated with the gradient of NPY concentration between the arcuate and paraventricular nuclei. NPY could therefore contribute to the qualitative changes of feeding behavior in the Brattleboro rat through altered transport/release of the peptide and participate in the balance of neuropeptides that determines food choice in this strain of rat.     

Orexin-A regulates growth hormone-releasing hormone mRNA content in a nucleus-specific manner and somatostatin mRNA content in a growth hormone-dependent fashion in the rat hypothalamus

The orexins or hypocretins are two neuropeptides involved in the regulation of diverse biological processes such as feeding, sleep and neuroendocrine function. Recent findings suggest a possible functional interaction between orexins, somatostatin and growth hormone-releasing hormone (GHRH) in the rat hypothalamus. In order to understand the possible functional linkage between orexins and these neuropeptides, we determined the effects of intracerebroventricular orexin-A administration on hypothalamic somatostatin and GHRH mRNA levels. Furthermore, we examined whether growth hormone (GH) mediates these interactions by using two animal models that showed GH deficiency: hypophysectomized rats and dwarf Lewis rats. Using in situ hybridization, our data showed that GHRH mRNA levels in the paraventricular nucleus of the hypothalamus are decreased after orexin-A treatment, without changes in the arcuate nucleus of the hypothalamus. On the other hand, orexin-A treatment induces a GH-dependent stimulatory effect on somatostatin mRNA content in the periventricular nucleus of the hypothalamus. Finally, we demonstrated, for the first time, that hypophysectomized rats and dwarf Lewis rats, two classical models of GH deficiency with alterations in sleep patterns, showed a marked reduction in the GHRH mRNA levels in the paraventricular nucleus of the hypothalamus. These data improve our understanding of the interactions among the different systems involved in the control and pathophysiology of food intake, sleep and GH secretion.     

Rapid and localized alterations of neuropeptide Y in discrete hypothalamic nuclei with feeding status

Neuropeptide Y (NPY) is believed to regulate the normal eating behavior and body weight in rats via central mechanisms. We have investigated whether NPY, which stimulates food intake, may in turn be modified by the nutritional state of the animals. Thus the impact of food deprivation (FD) (48 h) and subsequent refeeding on the levels of NPY in discrete hypothalamic areas was examined in this study. The results showed site specific change in only 3 of 7 hypothalamic sites. A 5-fold increment in NPY was reported in the paraventricular nucleus (PVN) and a 10-fold increase was observed in the arcuate nucleus-median eminence (ARC-ME). While subsequent refeeding for 6 h reversed the effect of FD in the ARC-ME, the levels of NPY in the PVN remained high in the refed rats. The perifornical lateral hypothalamus displayed a different pattern, namely, a significant increase in NPY content in refed as compared to satiated and deprived rats. The NPY levels in 4 other hypothalamic sites, namely, the dorsomedian, ventromedian, supraoptic and suprachiasmatic nuclei, and two extrahypothalamic sites, namely caudate nucleus and nucleus accumbens, showed total resistance to any change following deprivation and refeeding. These data emphasize the important and specific role of the paraventricular and arcuate nuclei in NPY's regulation of food intake and provide support for the idea that the variations of hypothalamic NPY after food deprivation reflect a specific physiological response of feeding regulatory system to alterations in the animal nutritional state and body weight.     

Evidence that NPY-containing neurons in the brainstem project into selected hypothalamic nuclei: implication in feeding behavior

Recent studies show that bilateral neural transections (NT) at the level of dorsal tegmentum in the mesencephalon significantly increase food intake and decrease latency to onset of feeding behavior in response to neuropeptide Y (NPY). The increased responsiveness to NPY may be due to denervation-induced hypersensitivity to NPY in hypothalamic sites that mediate feeding behavior in rats. To test this hypothesis we have studied the effect of NT on NPY concentrations in 7 neural sites of male rats. Two weeks after NT, NPY levels in 3 hypothalamic nuclei—suprachiasmatic nucleus, arcuate nucleus and ventromedial hypothalamus—were not altered by NT thereby suggesting that NPY innervations in these nuclei may be derived mainly from NPY perikarya in the ARC and elsewhere in the diencephalon. On the other hand, NPY concentrations were markedly decreased (50–60%) in the medial preoptic area, paraventricular nucleus, median eminence and dorsomedial nucleus indicating that a substantial number of neurons in the brainstem, which show coexistence of NPY and adrenergic transmitters, project into these 4 diencephalic nuclei. These findings indicate that NPY-containing neurons in the brainstem may project into selected hypothalamic sites and the reduction in the NT rats of NPY levels, especially in the paraventricular nucleus, may be responsible for the reported increase in sensitivity of the NPY-induced feeding response.     

The effects of acute phencyclidine treatment on neuropeptide Y (NPY) neuronal system in the rat arcuate nucleus studied by immunocytochemistry and in situ hybridization

Summary Phencyclidine (PCP) is a dissociative drug and an antagonist of N-methyl-D-aspartate (NMDA) receptor. The effects of PCP treatment on neuropeptide Y (NPY) system in the arcuate nucleus of the rat hypothalamus were examined both by immunocytochemistry and in situ hybridization. In acute PCP-treated rats, the NPY-immunoreactive perikarya appeared in the arcuate nucleus but no perikarya were detected in controls, without colchicine pretreatment. The signals of NPY mRNA by in situ hybridization increased in the PCP-treated rats than those of controls. These results suggest that the NPY system in the arcuate nucleus might be partly controlled by glutamatergic neurons.     

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.     

Intraperitoneal injection of ghrelin induces Fos expression in the paraventricular nucleus of the hypothalamus in rats

Ghrelin is a 28-amino acid peptide hormone secreted from the stomach that acts as a gut-brain peptide with potent stimulatory effects on food intake. The aim of the present study was to investigate the effects of peripheral ghrelin (1 and 10 nmol/rat) injected intraperitoneally (i.p.) on food intake and neuronal activity in the hypothalamus and brain stem, as assessed by c-Fos-like-immunoreactivity (c-FLI), using a confocal laser scanning microscope (cLSM) as a sensitive microscopic technique to detect c-FLI-positive neurons. Cumulative food intake was significantly increased 5.3- and 3.7-fold for the 4-h period after i.p. injection of ghrelin at both doses. The number of c-FLI-positive neurons in the paraventricular nucleus of the hypothalamus (PVN) was significantly increased after peripheral administration of ghrelin (1 nmol i.p.; median: 41.8) compared with i.p. saline (median: 17.5). As described before, c-fos expression was increased in the arcuate nucleus of the hypothalamus (ARC). In the nucleus of the solitary tract (NTS) or the area postrema (AP), there was no significant change in the density of c-FLI-positive neurons. Our data suggest that an activation of the arcuate-paraventricular axis may be part of the brain circuits involved in the orexigenic effect of peripheral ghrelin.     

Ventromedial hypothalamic lesions produced by gold thioglucose do not impair induction of NPY mRNA in the arcuate nucleus by fasting

There is increasing evidence that neuropeptide Y (NPY) plays an important role in the regulation of food intake. Neuropeptide Y mRNA in the arcuate nucleus increases after fasting and it has been proposed that this increase in NPY activity occurs as a result of the decreased circulating levels of both insulin and glucose associated with a fast. Glucose-responsive neurons in the ventromedial nucleus (VMN) of the hypothalamus alter their activity in response to changes in circulating glucose levels and these neurons have been proposed to be involved in the regulation of feeding behavior and metabolism. However, it is not known if these glucose-responsive neurons are involved in the response of NPY mRNA in the arcuate nucleus to fasting. To address this relationship, mice were injected with either saline or gold thioglucose (GTG), which appears to act on glucose-responsive neurons, and killed 6 weeks later after a 72 h fast or under ad lib fed conditions. In situ hybridization histochemistry for NPY mRNA was performed on hypothalamic sections containing the arcuate nucleus. The number of labelled cells was counted and the density of autoradiographic silver grains overlying the cells was also quantified (i.e. pixels per cell). Fasting resulted in increased levels of total NPY mRNA (number of labelled cells multiplied by the pixels per cell) in the arcuate nucleus of both control and GTG-treated mice. In addition, the relative fasting-induced increase (i.e. the fasted to fed ratio) in number of cells detected, number of pixels per cell, and total NPY mRNA was similar in both the control and GTG-treated mice. These data suggest that GTG-sensitive VMN neurons play little role in the induction of NPY mRNA by fasting in the arcuate nucleus.     

The effect of neuropeptide Y on drinking in mice

Neuropeptide Y (NPY) when administered intracerebroventricularly is a potent stimulator of feeding and drinking in rats. In these studies we demonstrated that, in contrast, in mice NPY inhibits drinking induced by water deprivation and that associated with food intake. In addition, we found that mice failed to respond to the rat dipsogen angiotensin II. Old mice demonstrated hypodipsia compared to young mice and NPY failed to inhibit drinking in older mice. Monosodium glutamate (MSG) administered neonatally produces lesions of the arcuate nucleus, an area rich in NPY cell bodies. NPY inhibited drinking and enhanced feeding in MSG treated mice. NPY also significantly inhibited the intake of water flavored with 8% sucrose and 0.1% quinine. NPY failed to alter ingestion of 0.2% or 5% saline. These studies support the contention that marked species differences exist in the regulation of water intake between rats and mice.     

Diurnal rhythm of neuropeptide Y-like immunoreactivity in the suprachiasmatic, arcuate and paraventricular nuclei and other hypothalamic sites

The diurnal rhythm of neuropeptide Y (NPY)-like immunoreactivity was examined in 9 discrete hypothalamic sites of rats maintained on a 12:12 h light/dark cycle. Significant bimodal rhythms of NPY concentration were detected in the suprachiasmatic and arcuate nuclei, with significant peaks just prior to onset of the nocturnal period and also at onset of the light period. In the parvocellular division of the paraventricular nucleus, a unimodal NPY peak was observed prior to dark onset. No diurnal rhythm was seen in the magnocellular division of the paraventricular nucleus, nor in 5 other hypothalamic areas examined.