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.     

Localization of neuropeptide Y mRNA and peptide in the chicken hypothalamus and their alterations after food deprivation, dehydration, and castration.

Localization of neuropeptide Y (NPY) mRNA in the hypothalamus of chickens was studied by in situ hybridization with digoxigenin-labeled chicken NPY cRNA probe. The largest number of perikarya-expressing NPY mRNA was found within the mediobasal hypothalamus, including the infundibular nucleus, inferior hypothalamic nucleus, and median eminence. Many NPY perikarya were noted to surround the nucleus rotundus and to be present in the supraoptic nucleus. Moreover, some perikarya were detected in the nucleus of basal optic root, bed nucleus pallial commissure, and nucleus striae terminalis close to the lateral forebrain bundle. NPY-immunoreactive nerve fibers were densely distributed in these regions containing the NPY mRNA-expressing perikarya. Following food deprivation for four days, perikarya-expressing NPY mRNA and peptide were markedly increased in the mediobasal hypothalamus and particularly so in the infundibular nucleus. No changes, however, were detected in other regions containing NPY-positive perikarya. Water deprivation induced less increase in NPY-positive perikarya in the mediobasal hypothalamus compared to food deprivation. After gonadectomy, the number of NPY-positive perikarya in the mediobasal hypothalamus was unaltered. Northern blot analysis with (32)P-labeled chicken NPY cDNA probe demonstrated that a 2.7-fold increase of NPY mRNA was induced by starvation and a 1.5-fold increase was induced by dehydration, whereas the NPY mRNA band remained unchanged after gonadectomy. Thus, it seems that NPY neurons located in the mediobasal hypothalamus are involved in feeding behavior but not reproductive activity.     

Localisation of mRNA encoding the protein precursor of galanin in the monkey hypothalamus and basal forebrain

The hypothalamic and basal forebrain sites of synthesis of preprogalanin mRNA were identified in three adult monkeys (Macaca fascicularis) by in situ hybridisation performed with a radiolabelled cRNA probe transcribed from human preprogalanin cDNA. With stringent hybridisation conditions, the cRNA probe was hybridised to free-floating sections containing structures contiguous with the rostral hypothalamus through to the caudal limit of the hypothalamus as defined by the mammillary bodies. Specific hybridisation of the preprogalanin cRNA probe occurred throughout the hypothalamus but was particularly intense in the arcuate, paraventricular (parvicellular and magnocellular portions), and dorsomedial nuclei. Moderate hybridisation was found in the periventricular nucleus and scattered hybridisation in the medial preoptic nucleus. The medial preoptic area and the anterior and lateral hypothalmic areas showed moderate to intense hybridisation in scattered cells. A few cells in the tuberal portion and dorsal cap of the anterior portion of the supraoptic nucleus were labelled. Isolated cells were also labelled in the zona incerta. There was little labelling in the dorsal hypothalamic area but moderate labelling in the posterior hypothalamic area. Structures contiguous with the rostral hypothalamus including the diagonal band of Broca, bed nucleus of stria terminalis, substantia innominata, and basal nucleus of Meynert showed intense hybridisation. These data indicate a widespread distribution of preprogalanin mRNA in the monkey hypothalamus. A comparison with the previously reported distribution of preprogalanin mRNA in the rat, as well as with the distribution of galanin-like immunoreactivity in the rat and human, suggests some important species differences. Of particular interest were differences in the supraoptic, suprachiasmatic, and dorsomedial nuclei. The intense hybridisation throughout the paraventricular nucleus and in the rostral arcuate nucleus suggests that galanin may play a role in the regulation of both posterior and anterior pituitary function. © 1993 Wiley-Liss, Inc.     

Effects of Long-Term Treatment with Antidepressant Drugs on Proopiomelanocortin and Neuropeptide Y mRNA Expression in the Hypothalamic Arcuate Nucleus of Rats

Antidepressant drugs have in common a delayed onset of clinical efficacy. In rats, long-term, daily administration of four different types of clinically effective antidepressant drugs results in decreased corticotropin releasing hormone (CRH) mRNA expression levels in the hypothalamic paraventricular nucleus (PVN). Because a subpopulation of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (Arc) projects to the PVN, we measured NPY and POMC mRNA expression in the Arc using in situ hybridization histochemistry at several time points following daily administration of four different antidepressant drugs. After 14 and 56 days of imipramine treatment, Arc NPY mRNA levels are decreased to 85% and 75% of control levels, but are unchanged compared to control after one or five days of treatment. Arc POMC mRNA levels are unchanged compared to controls at 1, 5, 14, or 56 days following imipramine treatment. Unlike after imipramine, Arc NPY and POMC mRNA levels are increased significantly to 134–172% of control following 56-day treatment with the antidepressant drugs fluoxetine, phenelzine, or idazoxan. The divergent effects of imipramine vs the other 3 antidepressant drugs on Arc NPY mRNA expression are similar to the pattern of changes in tyrosine hydroxylase (TH) mRNA expression levels in the locus coeruleus (LC) using the same experimental paradigm, but are different from the unidirectional depressive effects of all four drugs on CRH mRNA expression in the PVN. Thus, the Arc NPY and LC noradrenergic systems may act coordinately in mediating antidepressant effects. The present data are consistent with the delayed onset of clinical efficacy for antidepressant drugs, and suggest that Arc NPY and POMC neurotransmitter systems play a role in the pathophysiology of depression.     

Comparative distribution of mRNA encoding the growth hormone secretagogue-receptor (GHS-R) in Microcebus murinus (Primate, lemurian) and rat forebrain and pituitary

The forebrain and pituitary sites of synthesis of growth hormone secretagogue-receptor mRNA were identified in four adult lemurs (Microcebus murinus) by in situ hybridisation performed with a radiolabeled cRNA probe transcribed from human Growth Hormone Secretagogue-Receptor cDNA. The cRNA sense and antisense probes were hybridised to cryostat sections containing structures extending from the rostral hypothalamus to its caudal limit as defined by the mammillary bodies. The pituitary gland and areas adjacent to the hypothalamus were also analyzed. For comparative purposes, sections from five adult rats containing these structures were hybridised with the same probes. The results point to a widespread distribution of Growth Hormone Secretagogue-Receptor mRNA in the hypothalamus, hippocampal formation, and cerebellar cortex of both lemurs and rats. As in the rat, specific hybridisation was particularly dense in the arcuate nucleus. Significant species differences were observed in the periventricular nucleus, the ventromedial nucleus, the lateral hypothalamic area, and the pituitary gland. In contrast to the rat, the lemur exhibited marked labelling in the infundibular nucleus, the periventricular nucleus and the pars tuberalis of the pituitary gland, whereas no labeling was detectable in the ventromedial nucleus and the lateral hypothalamic area. These results are discussed in terms of difference between the control of growth hormone secretion, feeding behaviour and seasonal rhythmicity among murine species and primates.     

Mapping of hypothalamic sites involved in the effects of NPY on body temperature and food intake

The objective of the present study was to identify hypothalamic sites that might be implicated in the effects of neuropeptide Y (NPY) on both body temperature and food intake. For this purpose, the effects of direct microinjections of NPY in several doses (0.156–20 μg) into discrete hypothalamic nuclei on body temperature were examined in rats. To examine specificity of effects, food consumption of animals following injections was also measured. Results indicate that the influence of NPY on body temperature varies with the hypothalamic region where the peptide is administered. NPY had no effect on temperature after administration into the ventromedial (VMH) and the perifornical hypothalamus (PeF). However, a significant hypothermia was seen following administration into the preoptic (POA) and arcuate nucleus (Arc), and hyperthermia was seen after injection into the paraventricular nucleus (PVN). Finally, a biphasic effect was observed after injection into the lateral hypothalamus (LH): hyperthermia with relatively small doses and hypothermia with higher doses. Similar effects were obtained when administred into the third ventricle (3V) but in an inverted dose-related fashion: hypothermia at low and hyperthermia at higher doses. For feeding, NPY consistently increased food intake in all regions examined, with the strongest effect obtained after administration into the PeF. The present results clearly dissociate the effects of NPY on food intake and body temperature, and demonstrate that these effects are related to specific hypothalamic nuclei.     

Oestrogenic regulation of pro-opiomelanocortin, neuropeptide Y and corticotrophin-releasing hormone mRNAs in mouse hypothalamus

It is well documented that oestrogen suppresses food intake by an action at the hypothalamic level. Using in situ hybridisation, we studied the effect of castration (CX) and short-term administration of oestradiol (E2) in CX female mice for three neuropeptides involved in feeding behaviour: two anorexigenic peptides, (i) the pro-opiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone and (ii) corticotrophin-releasing hormone (CRH), and the orexigenic peptide, (iii) neuropeptide Y (NPY). POMC-expressing neurones were mostly laterally located in the arcuate nucleus. POMC mRNA expression was decreased following CX and a single injection of E2 induced an increase in mRNA levels at 12- and 24-h time intervals. In the parvocellular area of the paraventricular nucleus, CRH mRNA levels were similarly decreased after CX and completely restored to normal levels at 12 and 24 h following E2 injection. On the other hand, the levels of NPY mRNA expressed in neurones located in the inner zone of the arcuate nucleus were increased by CX and decreased to the levels observed in intact animals by E2 injection (3-24 h). The present data suggest that oestrogen might exert an anorexigenic action by stimulating POMC and CRH mRNA expression and decreasing NPY mRNA expression in the hypothalamus.     

Preproneuropeptide Y Messenger Ribonucleic Acid in the Hypothalamic Arcuate Nucleus of the Rat is Increased by Food Deprivation or Dehydration

The distribution of messenger ribonucleic acid (mRNA) encoding preproneuropeptide Y (prepro-NPY) in the hypothalamus of rats subjected to food deprivation or dehydration has been investigated by quantitative in situ hybridization. Levels of prepro-NPY mRNA in the arcuate nucleus (ARC) were selectively increased by both treatments. The very high concentration of prepro-NPY mRNA seen following 96 h of food deprivation had returned towards control levels after 24 h of refeeding. Levels of preprogalanin (prepro-GAL) mRNA throughout the hypothalamus were essentially unaffected by both regimes. These results demonstrate that hypothalamic NPY gene expression is regulated by peripheral metabolic status (and osmolality), and confirm the key physiological role of NPY in controlling ingestive behaviour.     

Acute oral dexamethasone administration reduces levels of orphan GPCR glucocorticoid-induced receptor (GIR) mRNA in rodent brain: potential role in HPA-axis function

Glucocorticoid-induced receptor (GIR) is an orphan G-protein-coupled receptor (GPCR) with predominant expression in brain and thymus. More specifically, high levels of GIR expression have been described in brain regions of mouse, rat and human including limbic forebrain and hypothalamic regions, suggesting a role for GIR in memory, cognition, stress, reward or the control of emotion. Previous in vitro studies performed in murine thymocytes demonstrated an induction of GIR following dexamethasone treatment, suggesting the potential in vivo regulation of GIR by glucocorticoids. Glucocorticoids have been implicated in a number of disorders. In this study we employed in situ hybridisation with semi-quantitative image analysis to assess the level of GIR expression in mouse brain following acute dexamethasone administration. GIR was highly expressed in the nucleus accumbens, striatum, olfactory tubercle and nuclei of the hypothalamus. Three hours following acute dexamethasone treatment (0.05 mg/kg, p.o.), levels of GIR mRNA were found to be significantly decreased in striatum (25%, P<0.05), nucleus accumbens (19%, P<0.05), olfactory tubercle (19%, P<0.05) and CA2 sub-region of the hippocampus (30%, P<0.05) compared with vehicle. Significant decreases in GIR expression were also observed in hypothalamic nuclei including the dorsomedial (48%, P<0.05) and ventrolateral (58%, P<0.05) part of the ventromedial hypothalamic nuclei, dorsomedial hypothalamic nuclei (39%, P<0.01) and arcuate nucleus (54%, P<0.05), compared with vehicle. These data demonstrate the in vivo regulation of GIR in response to glucocorticoids and suggest a potential role for GIR in mediating the response to altered levels of glucocorticoids in disease states.     

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.