Palatability-induced hyperphagia increases hypothalamic Dynorphin peptide and mRNA levels

Opioid involvement in regulating the intake of highly palatable diets was studied by examining the effect of feeding either a cornstarch-based diet (CHO) or a high fat diet containing sucrose (Fat/Sucrose) on hypothalamic opioid levels. Rats received either CHO ad libitum, Fat/Sucrose ad libitum, Fat/Sucrose pair-fed to the caloric intake of CHO, or Fat/Sucrose at 60% of ad libitum Fat/Sucrose intake. Animals receiving Fat/Sucrose ad libitum consumed more calories and gained more weight than animals receiving CHO (P < 0.001). Relative to CHO, ad libitum intake of Fat/Sucrose elevated proDynorphin mRNA levels in the arcuate and Dynorphin A_1–17 levels in the paraventricular nucleus (PVN) (P < 0.05), but did not affect arcuate mRNA levels of proEnkephalin or proOpiomelanocortin (POMC), or PVN levels of Met-Enkephalin or β-Endorphin. Pair-feeding the Fat/Sucrose diet to the level of intake of the CHO diet resulted in levels of proDynorphin and Dynorphin A_1–17 that were similar in the two diet groups. Pair-feeding Fat/Sucrose reduced mRNA levels of proDynorpin, proEnkephalin and POMC, and Dynorphin A_1–17 levels, relative to ad libitum feeding of Fat/Sucrose. Met-Enkephalin and β-Endorphin were not affected by dietary treatment. Feeding Fat/Sucrose at 60% of ad libitum intake resulted in mRNA levels of proDynorphin, proEnkephalin and POMC, and Dynorphin A_1–17 levels that were similar to those observed in CHO group. Hypothalamic Dynorphin A_1–17 and proDynorphin mRNA levels are stimulated by feeding a highly palatable diet rich in fat and sucrose. The increased synthesis may be due in part to a palatability-induced overconsumption of calories. Caloric restriction of the same diet decreases mRNA levels of proDynorphin, proEnkephalin and POMC, as well as levels of Dynorphin A_1–17.     

ARC POMC mRNA and PVN α-MSH are lower in obese relative to lean Zucker rats

Effects of obesity on gene expression for opioid peptides and neuropeptide-Y (NPY) in the arcuate nucleus (ARC), and on opioid peptides and α-melanocyte stimulating hormone (α-MSH) in the paraventricular nucleus (PVN) were examined in obese Zucker rats (18 weeks old). Obese Zucker rats are insulin-resistant, diabetic and hyperleptinemic as indicated by high serum glucose, insulin and leptin levels. ARC proOpiomelanocortin (POMC) mRNA levels were significantly lower in the obese relative to lean Zucker rats and ARC proNeuropeptide Y (proNPY) mRNA levels were higher (P<0.05). There were no differences in proDynorphin and proEnkephalin mRNA levels in the ARC (P>0.05). Obese Zucker rats had lower α-MSH and dynorphin A_1–17 peptide levels in the paraventricular nucleus (PVN) (P<0.05), but did not have lower PVN β-endorphin peptide levels (P>0.05). The decrease in POMC in the ARC and decrease in α-MSH in the PVN seen in the obese Zucker rat in the present study suggest that reduced activity of the melanocortin system in the ARC to PVN pathway may contribute to the related hyperphagia. Reduced activity of the melanocortin system in the ARC to PVN pathway may be due to a disturbance of leptin signaling coupling to POMC.     

Topographical distribution of pro-opiomelanocortin-derived peptides (ACTH/β-END/α-MSH) in the rat median eminence

The detailed distribution of adrenocorticotropin (ACTH), beta-endorphin (beta-END) and alpha-melanotropin (alpha-MSH) immunoreactivity was examined in the rat median eminence (ME) and pituitary stalk using light microscopic immunocytochemistry and radioimmunoassay (RIA). Nerve fibers and varicosities immunoreactive for ACTH/beta-END/alpha-MSH had identical distributions in the ME suggesting that they are part of the same arcuate proopiomelanocortin neuronal (POMC) system. The quantitative image analysis of POMC immunoreactive varicosities in the ME indicates no significant differences between the various rostro-caudal segments. In the main (preinfundibular) portion of the ME, a moderate density of immunoreactive elements was located in the lateral part of the internal zone and throughout the postinfundibular ME. Very few scattered varicosities were observed in the neurohemal (external) zone and in the pituitary stalk. By RIA, alpha-MSH is present in a substantially higher concentration than ACTH and beta-END throughout the ME. Knife cuts between the arcuate nucleus and ME indicate that proopiomelanocortin (POMC) fibers enter the ME in its whole rostro-caudal extent. Thus POMC neurons seem to provide innervation of structures in the internal zone but not in the neurohemal/external/zone where the portal capillary system is located. Moreover, the observation that the density of immunoreactive elements is substantially lower in the pituitary stalk than in the ME, suggests that the majority of immunoreactive fibers in the internal zone are not fibers of passage directed towards the neurohypophysis.     

Distribution of the pro-opiomelanocortin derived peptides, adrenocorticotrope hormone, alpha-melanocyte-stimulating hormone and beta-endorphin (ACTH, alpha-MSH, beta-END) in the rat hypothalamus

Rat hypothalamic nuclei were removed and assayed for adrenocorticotropic hormone (ACTH), beta-endorphin (beta-END) and alpha-melanocyte-stimulating hormone (alpha-MSH) content by radioimmunoassay, from the same samples. We also performed immunostaining for these 3 pro-opiomelanocortin (POMC) derived peptides on paraffin embedded serial sections of the hypothalamus. Areas known to project to the external zone of the median eminence receive a dense POMC innervation while those projecting to the posterior pituitary are not innervated. In addition, hypothalamic areas previously suggested to project to medullary autonomic centers are densely innervated. This innervation pattern may provide the morphological basis for the involvement of POMC peptides in neuroendocrine and autonomic functions. The biochemical data raise the possibility that the POMC precursor is processed differently in various brain regions.     

Effects of fluoxetine administration on hypothalamic melanocortin system in obese Zucker rats

The aim of the present work was to study the potential involvement of melanocortin system in the anorectic mechanism of fluoxetine, a selective serotonin reuptake inhibitors, in obese Zucker rats. Male obese Zucker (fa/fa) rats were administered fluoxetine (10 mg/kg; i.p.) daily for two weeks. The control group was given 0.9% NaCl solution. RT-PCR for pro-opiomelanocortin (POMC), Agouti gene related peptide (AgRP) and melanocortin receptor 4 (MC4-R) in the hypothalamus, as well as regional immunostaining for alpha-melanocyte stimulating hormone (alpha-MSH) and MC4-R were carried out. Fluoxetine administration increased POMC expression and reduced MC4-R expression in the hypothalamus, without changes in AgRP mRNA levels. Moreover, an increase in the numbers of alpha-MSH positively immunostained neural cells in the hypothalamic arcuate nucleus (ARC), as well as a significant decrease in the numbers of neural cells positively immunostained for MC4-R in the paraventricular nucleus (PVN), without changes in lateral hypothalamic area (LHA), were observed. These results suggest the involvement of alpha-MSH in central fluoxetine anorectic action.     

Opioid-induced feeding: Localization of sensitive brain sites

These experiments were designed to identify brain sites at which opioids might act to influence ingestive behavior and to determine which opioid receptor types are involved. After food deprivation, rats were given microinjections of naloxone into several brain regions and food intake was measured. Injections into or near the paraventricular (PVN) or ventromedial (VMH) hypothalamic nuclei or the globus pallidus (GP) reduced food intake; injections into the striatum or lateral hypothalamus (LH) were ineffective. A second study examined the ingestive effects of roughly equimolar doses (1.43-1.75 nmol) of dynorphin A (DYN), beta-endorphin (beta-END), and D-Ala2,D-Leu5-enkephalin (DADLE) when injected into 4 different brain regions. Only DYN significantly increased food intake, and this effect was seen only with injections into the PVN and VMH. Beta-END stimulated water intake when injected into the PVN, VMH and GP but not the LH. Further studies indicated that with PVN injections, DYN was effective at a dose as low as 0.47 nmol, and that a higher dose of DADLE (4.39 nmol) did stimulate food intake. These studies support an important role for dynorphin and the kappa opioid receptor in the regulation of feeding and suggest that the opioid regulation of food and water intake can be differentiated both by sites of action and by effective agonists.     

α-Melanocyte stimulating hormone discloses a stimulatory effect of β-endorphin on somatostatin release

The influence of alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin (beta-END) on the secretion of somatostatin (SRIF) from the median eminence (ME) was studied using an in vitro incubation system. The MEs from adult male rats were first preincubated at 37 degrees C for 30 min with constant shaking in 0.4 ml of Krebs-Ringer bicarbonate-glucose buffer (pH 7.4) containing bacitracin in an atmosphere of 95% O2/5% CO2. Medium was discarded and replaced by medium containing different doses of alpha-MSH, beta-END, or a fixed dose of alpha-MSH (10(-7) M or 10(-9) M) plus beta-END at various concentrations. By themselves alpha-MSH and beta-END did not alter basal SRIF release, but in the presence of alpha-MSH (10(-7) M) beta-END stimulated somatostatin release. This effect was significant at concentrations of beta-END of 10(-8) M and higher. The permissive effect of alpha-MSH was observed at a concentration as low as 10(-9) M, but in this case the stimulatory effect of beta-END became evident only at higher doses tested (10(-7) M). It is suggested that alpha-MSH and beta-END participate in the modulation of SRIF release. By themselves beta-END and alpha-MSH did not affect basal release of SRIF but in the presence of alpha-MSH, beta-END had a stimulatory effect on SRIF release. The mechanism for this interaction is unknown. The results are consistent with the possibility that beta-END neurons have stimulatory and inhibitory effects on SRIF release and that alpha-MSH, by blocking the inhibitory components, discloses the stimulatory effect of beta-END on SRIF release.     

Enhanced inhibitory feeding response to alpha-melanocyte stimulating hormone in the diet-induced obese rat

A dysregulation in the hypothalamic neuropeptide systems involved in the control of appetite has previously been shown in models of diet-induced obesity. In the present study, male Sprague-Dawley rats were rendered obese by a highly palatable cafeteria-style diet over 20 weeks, while control rats had access to standard laboratory chow. Feeding responses to alpha-melanocyte stimulating hormone (alpha-MSH), an anorectic peptide and neuropeptide Y (NPY), a potent orexigenic agent were investigated in diet-induced obese and control animals. In addition, endogenous hypothalamic peptide levels were determined in these animals. Intracerebroventricular injections of either 4 nmol alpha-MSH or saline vehicle were given 10 min prior to the onset of the dark phase. Diet-induced obese rats had significantly enhanced nocturnal inhibitory feeding responses to alpha-MSH (P<0.05). The orexigenic feeding response induced by 1 nmol NPY was similar for both groups. At sacrifice, both alpha-MSH and NPY peptide content were selectively reduced in the paraventricular nucleus (PVN) of these animals (P<0.05). Although diet-induced obesity had no effect on responses to NPY, the significantly greater inhibition of nocturnal feeding by alpha-MSH and reduction in PVN alpha-MSH peptide level, suggests melanocortinergic signalling may be reduced in obesity which may account for the hyperphagia of these animals when presented with a palatable diet.     

alpha-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression.

The hypothalamic arcuate nucleus has an essential role in mediating the homeostatic responses of the thyroid axis to fasting by altering the sensitivity of prothyrotropin-releasing hormone (pro-TRH) gene expression in the paraventricular nucleus (PVN) to feedback regulation by thyroid hormone. Because agouti-related protein (AGRP), a leptin-regulated, arcuate nucleus-derived peptide with alpha-MSH antagonist activity, is contained in axon terminals that terminate on TRH neurons in the PVN, we raised the possibility that alpha-MSH may also participate in the mechanism by which leptin influences pro-TRH gene expression. By double-labeling immunocytochemistry, alpha-MSH-IR axon varicosities were juxtaposed to approximately 70% of pro-TRH neurons in the anterior and periventricular parvocellular subdivisions of the PVN and to 34% of pro-TRH neurons in the medial parvocellular subdivision, establishing synaptic contacts both on the cell soma and dendrites. All pro-TRH neurons receiving contacts by alpha-MSH-containing fibers also were innervated by axons containing AGRP. The intracerebroventricular infusion of 300 ng of alpha-MSH every 6 hr for 3 d prevented fasting-induced suppression of pro-TRH in the PVN but had no effect on AGRP mRNA in the arcuate nucleus. alpha-MSH also increased circulating levels of free thyroxine (T4) 2.5-fold over the levels in fasted controls, but free T4 did not reach the levels in fed controls. These data suggest that alpha-MSH has an important role in the activation of pro-TRH gene expression in hypophysiotropic neurons via either a mono- and/or multisynaptic pathway to the PVN, but factors in addition to alpha-MSH also contribute to the mechanism by which leptin administration restores thyroid hormone levels to normal in fasted animals.     

The effects of beta-endorphin (beta-END) on cardiovascular and behavioral dynamics in conscious rats

Beta-endorphin (beta-END) a product of the proopiomelanocortin (POMC) has been demonstrated to play a role in the regulation of metabolic and autonomic responses. Recent studies have suggested the involvement of the endogenous opioid system in cardiovascular control. Previous studies conducted in our laboratory using anesthetized animals investigated the actions of beta-END and other POMC derived peptides on sympathetic and cardiovascular dynamics. In this study, we determined both the acute and chronic effects of beta-END on cardiovascular and behavioral dynamics in conscious unrestrained rats using radio-telemetry. Animals were instrumented with a radio-telemetry transmitter in the abdominal cavity and the attached catheter inserted into the femoral artery for recording of cardiovascular dynamics and activity. They were subsequently implanted with intracerebroventricular (ICV) cannulas. The acute ICV administration of beta-END significantly increased the mean arterial pressure (MAP) and heart rate (HR) compared to controls. The cardiovascular responses returned toward control levels after 2 h. In contrast, the chronic infusion of beta-END significantly decreased the MAP and HR during both the active and inactive phase. Chronic beta-END administration also decreased physical activity. Food intake was increased initially and later declined and water consumption followed a similar pattern. We conclude that in the conscious unrestrained animal the acute administration of beta-END increases MAP and HR while the chronic infusion of beta-END decreases MAP, HR, physical activity, and stimulate a short-term increase in food and water intake.     

Presence of strong glucocorticoid receptor immunoreactivity within hypothalamic and hypophyseal cells containing pro-opiomelanocortic peptides.

The presence of nuclear glucocorticoid receptor immunoreactivity (GR IR) was studied in the adrenocorticotropin (ACTH), beta-Endorphin (beta-END) and alpha-melanocyte stimulating hormone (alpha-MSH) IR neuronal populations of the rat hypothalamus and hypophysis using double immunolabelling techniques. All the nuclei of the ACTH/beta-END/alpha-MSH IR neurons of the arcuate and periarcuate nuclei were strongly GR IR in the 48 h colchicine treated animal, but very few alpha-MSH-like IR perikarya located in the dorsal and lateral hypothalamus displayed nuclear GR IR. GR IR was present in the ACTH/beta-END corticotrophs and absent in the intermediate lobe of the hypophysis. The data provide morphological evidence for a glucocorticoid action through a nuclear GR in the arcuate ACTH/beta-END/alpha-MSH IR neurons and the ACTH/beta-END corticotrophs, whereas the alpha-MSH-like IR neurons of the lateral hypothalamus and the melanotropes of the intermediate lobe may not be directly affected by glucocorticoids under normal conditions.     

Changes in Pro-Opiomelanocortin and Pre-proenkephalin mRNA Levels in the Ovine Brain during Pregnancy, Parturition and Lactation and in Response to Oestrogen and Progesterone

In the female sheep opioids act centrally to influence both oxytocin release and maternal behaviour. We have used in situ hybridization histochemistry to investigate the changes in mRNA expression of the two opioid precursor genes, proopiomelanocortin (POMC) and pre-proenkephalin (PPE), in discrete hypothalamic nuclei as a function of pregnancy, parturition and lactation and following treatment with oestrogen and progesterone. Quantitative in situ hybridization histochemistry demonstrated that POMC mRNA expression in the arcuate nucleus (ARC) decreased at parturition and increased during lactation compared to late pregnant and ovariectomized animals. Oestradiol and progesterone treatments increased POMC mRNA expression compared to ovariectomized controls. Pre-proenkephalin mRNA expression was quantified in three discrete hypothalamic nuclei, the ventromedial nucleus (VMN), the paraventricular nucleus (PVN) and the suprachiasmatic nucleus (SCN). In the VMN, PPE mRNA expression increased during lactation compared to late pregnancy and parturition. Expression levels during late pregnancy and parturition were decreased compared to ovariectomized animals. Oestradiol increased, and progesterone decreased, PPE mRNA levels compared to ovariectomized controls. Combined progesterone followed by oestrogen treatment produced significant increases in PPE mRNA expression. In the PVN, PPE expression increased at parturition compared to late pregnant, lactating and ovariectomized animals. Expression levels in late pregnant animals were decreased compared to lactating or ovariectomized ones. However, sex steroid treatment produced no changes in PPE expression in the PVN. No changes were observed in PPE mRNA expression in the SCN in response to any of the experimental conditions. This data shows that both POMC and PPE mRNA levels are altered in the sheep brain during pregnancy, parturition and lactation and in response to sex steroids, although the direction of the changes is not always the same and in the case of PPE only the VMN and PVN are affected. Levels of gene expression found following exogenous steroid treatment do not precisely mirror those found during pregnancy, parturition and lactation and this suggests that factors other than changing sex steroids levels are involved. In the context of maternal behaviour it is interesting to note that PPE mRNA expression increases at parturition in the PVN when oxytocin mRNA expression levels are also increased.     

Enhanced binocular interaction in the visual cortex of normal kittens subjected to intracortical norepinephrine perfusion

Immunoreactive fibers and varicosities in the hypothalamic paraventricular nucleus (PVN) were examined by light- and electronmicroscopy, following treatment of brain slices with specific antibodies to adrenocorticotropin (ACTH), beta-endorphin (beta-End) and alpha-melanotropin (alpha-MSH) peptides. In an attempt to provide a more precise, quantitative definition of the densities of immunoreactive elements, sections were analyzed by computer based image-analysis techniques. Fibers and varicosities immunostained with the 3 different antibodies displayed an identical distribution pattern throughout the nucleus suggesting that they are parts of the same, arcuate pro-opiomelanocortin (POMC) neuron system. Although immunoreactive varicosities were found all over the PVN, it was possible to identify a characteristic, density distribution pattern. At the ultrastructural level, immunoreactive presynaptic nerve terminals were observed forming symmetrical synaptic contacts with unlabeled dendrites. The majority of immunoreactive elements were found in the dorsal parvo- and caudal magnocellular subdivisions which give rise to long projections to the lower brainstem. Moderate density of POMC neural elements was observed in the anterior and medial (ventral portion) parvocellular subdivisions which project to the external zone of the median eminence. Only a few, widely scattered immunostained varicosities are found in the medial and lateral magnocellular subdivisions which project to the neurohypophysis. A combined lesion and immunocytochemical approach has shown that the bulk of the afferent neuronal input from arcuate POMC cells enters the PVN from a ventral direction.     

Leptin-induced increase in sympathetic nervous and cardiovascular tone is mediated by proopiomelanocortin (POMC) products

The mechanism underlying the leptin-induced increased sympathetic nerve activity and cardiovascular tone was investigated in normal rats. The melanocortin (MC) peptides and other fragments derived from proopiomelancortin (POMC) have a diverse array of biological activities and have been implicated in mediating the feeding behavioral responses to leptin. In this study we evaluated the possible involvement of two major products of POMC, alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin, in mediating the effects of leptin on sympathetic activity and mean arterial pressure (MAP) in normal rats. Intraventricular (i.c.v.) cannulas were implanted in normal rats and allowed to recover. On the day of the study the animals were anesthetized with urethane alpha-chloralose and instrumented for the recording of MAP, lumbar sympathetic nerve activity (LSNA), and heart rate (HR). To determine the correlation between the leptin response and the POMC products, alpha-MSH and beta-endorphins were also injected into the lateral ventricle. alpha-MSH acted to increase MAP and LSNA while beta-endorphin decreased these parameters. Leptin administration by i.c.v. cannula increased the MAP and LSNA in normal rats. The i.c.v. administration of agouti protein, an alpha-MSH receptor antagonist, prior to leptin infusion blocked this response. Likewise, pretreatment with naloxone a beta-endorphin receptor antagonist also blocked the response to leptin. From these studies we conclude that the acute increased LSNA and MAP in response to i.c.v. leptin may be mediated by increased POMC and its subsequent production of breakdown product alpha-MSH and/or beta-endorphin and it is the subsequent action of alpha-MSH that increases MAP and LSNA by activation of the MC4 receptor. The naloxone antagonism of the leptin response is likely due to the blockade of presynaptic opioid inhibition of the MC4 receptor-mediated pressor response.     

Alpha-melanophore-stimulating hormone in the brain,cranial placode derivatives,and retina of Xenopus laevis during development in relation to background adaptation

The amphibian Xenopus laevis can adapt the color of its skin to the light intensity of the background. A key peptide in this adaptation process is alpha-melanophore-stimulating hormone (alpha-MSH), which is derived from proopiomelanocortin (POMC) and released by the endocrine melanotrope cells in the pituitary pars intermedia. In this study, the presence of alpha-MSH in the brain, cranial placode derivatives, and retina of developing Xenopus laevis was investigated using immunocytochemistry, to test the hypothesis that POMC peptide-producing neurons and endocrine cells have a common embryonic origin and a common function, i.e., controlling each other's activities and/or being involved in the process of physiological adaptation. The presence of alpha-MSH-positive cells in the suprachiasmatic nucleus, ventral hypothalamic nucleus, epiphysis, and endocrine melanotrope and corticotrope cells, which are all involved in regulation of adaptation processes, has been detected from stage 37/38 onward. This is consistent with the presumed common origin of these cells, the anterior neural ridge (ANR) of the neural-plate-stage embryo. The olfactory epithelium and the otic and epibranchial ganglia also contain alpha-MSH, indicating that these placodal derivatives originate from a common placodal domain continuous with the ANR. Furthermore, we demonstrate the presence of alpha-MSH in a subpopulation of retinal ganglion cells (RGCs), which is possibly also derived from the ANR. Immunoreactivity for alpha-MSH in RGCs that are located in the dorsal part of the retina is dependent on the background light intensity, suggesting that these cells are involved in the regulation of background adaptation. Taken together, the results support the hypothesis that POMC peptide-producing cells have a common embryonic origin and are involved in adaptation processes.     

Reactive oxygen species in paraventricular nucleus modulates cardiac sympathetic afferent reflex in rats

Our previous studies showed that angiotensin II (Ang II) in the paraventricular nucleus (PVN) potentiated the cardiac sympathetic afferent reflex (CSAR) in rats. This study investigated whether the reactive oxygen species (ROS) in the PVN modulated the CSAR and contributed to the effect of Ang II on the CSAR in rats. Under alpha-chloralose and urethane anesthesia, renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate were recorded in sinoaortic-denervated and cervical-vagotomized rats. The CSAR was evaluated by the RSNA response to epicardial application of bradykinin (0.04 and 0.4 mug). Compared with microinjection of saline into the PVN, superoxide anion scavenger, either tempol (20 nmol) or tiron (10 nmol), significantly decreased the CSAR (P < 0.05). Conversely, superoxide dismutase (SOD) inhibitor diethyldithio-carbamic acid (DETC, 10 nmol) potentiated the CSAR (P < 0.05). Microinjection of Ang II (0.3 nmol) into the PVN resulted in an enhanced CSAR (P < 0.05). The effect of Ang II on the CSAR was completely inhibited by pretreatment with either tempol or tiron (P < 0.05) but was not affected by DETC. On the other hand, either tempol or tiron decreased the RSNA (P < 0.05), but DETC increased the RSNA (P < 0.05). Ang II increased the RSNA (P < 0.05) and MAP (P < 0.05). The effect of Ang II on the RSNA and MAP was abolished by pretreatment with either tempol or tiron but was not affected by DETC. These results indicated that the ROS in the PVN modulated the CSAR and contributed to the effect of Ang II in the PVN on the CSAR.     

Adenosine A2A receptor gene disruption provokes marked changes in melanocortin content and pro-opiomelanocortin gene expression

A2A receptor knockout (A2AR-/-) mice are more anxious and aggressive, and exhibit reduced exploratory activity than their wild-type littermates (A2AR+/+). Because alpha-melanocyte-stimulating hormone (alpha-MSH) influences anxiety, aggressiveness and motor activity, we investigated the effect of A2AR gene disruption on alpha-MSH content in discrete brain regions and pro-opiomelanocortin (POMC) expression in the hypothalamus and pituitary. No modification in alpha-MSH content was observed in the hypothalamus and medulla oblongata where POMC-expressing perikarya are located. In the arcuate nucleus of the hypothalamus, POMC mRNA levels were not affected by A2AR disruption. Conversely, in A2AR-/- mice, a significant increase in alpha-MSH content was observed in the amygdala and cerebral cortex, two regions that are innervated by POMC terminals. In the pars intermedia of the pituitary, A2AR disruption provoked a significant reduction of POMC mRNA expression associated with a decrease in alpha-MSH content. By contrast, in the anterior lobe of the pituitary, a substantial increase in POMC mRNA and adrenocorticotropin hormone concentrations was observed, and plasma corticosterone concentration was significantly higher in A2AR-/- mice, revealing hyperactivity of their pituitary-adrenocortical axis. Together, these results suggest that adenosine, acting through A2A receptors, may modulate the release of alpha-MSH in the cerebral cortex and amygdala. The data also indicate that A2A receptors are involved in the control of POMC gene expression and biosynthesis of POMC-derived peptides in pituitary melanotrophs and corticotrophs.     

Neuroanatomical evidence for participation of the hypothalamic dorsomedial nucleus (DMN) in regulation of the hypothalamic paraventricular nucleus (PVN) by alpha-melanocyte stimulating hormone

To test the hypothesis that neurons in the hypothalamic paraventricular nucleus (PVN) may be under both direct and indirect regulation by alpha melanocyte-stimulating hormone (alpha-MSH)-synthesizing neurons of the arcuate nucleus, we determined whether the retrogradely transported marker substance, cholera toxin beta-subunit (CtB), when injected into the PVN, labels distinct populations of neurons in the hypothalamic dorsomedial nucleus (DMN) that are innervated by axon terminals containing alpha-MSH. Following iontophoresis of CtB into the PVN, retrogradely labeled neurons were identified in the DMN primarily on the same side as the injection, although a few neurons were also identified in the opposite side of the DMN. The greatest percentage of retrogradely labeled DMN neurons were located in the medial portion of the ventral subdivision of the DMN (DMNv), accounting for approximately 64.8 +/- 1.1% of all CtB-labeled cells in the DMN. The second largest population, comprising 25.9 +/- 1.6% of the total number of CtB cells in the DMN, was diffusely distributed in the dorsal subdivision of the DMN (DMNd). Only 9.4 +/- 0.3% of the CtB-labeled cells were located in the compact zone of the DMN (DMNc). In double-labeling immunofluorescent preparations, 61.1 +/- 1.0% of the CtB cells in the DMNv, 38.6 +/- 0.9% of the CtB cells in the DMNd, and 13.1 +/- 1.3% of the CtB cells in the DMNc were contacted by axon terminals containing alpha-MSH. These data establish that neurons in discrete regions in the DMN may be influenced by the melanocortin signaling system and thereby, could serve as important relay sites to the PVN.