Morphofunctional interaction of CART peptide and dopaminergic brain neurons

In Wistar rats, after 6 h of sleep deprivation and subsequent 2 h postdeprivation sleep, we found significant changes in optical density of CART peptide in neurons of nucleus accumbens and hypothalamic nucleus arcuatus as well as in processes coming into substantia nigra from nucleus accumbens. The obtained data revealed unidirectional changes of optical density of CART and tyrosine hydroxylase in the studied structures: a decrease after sleep deprivation (p < 0.05) and, on the contrary, an increase after postdeprivation sleep (p < 0.05). Confocal laser microscopy showed morphological connections of CART and dopaminergic neurons and possible colocalization of these both substances in the same neuron at the postdeprivation sleep. In experiments in vitro, after 1 h of incubation of surviving brain sections from the substantia nigra area in the medium with CART peptide there was revealed a rise of optical density of tyrosine hydroxylase in the substantia nigra pars compacta by 55% (p < 0.05). The obtained data indicate an activating effect of CART peptide on brain dopaminergic neurons and its role as a modulator of their functional activity.     

Tyrosine hydroxylase and tryptophan hydroxylase activities and its cofactor biopterin level in brain regions of the rolling mouse: The influence of thyrotropin releasing hormone

Biopterin, the cofactor for tyrosine hydroxylase and tryptophan hydroxylase, was decreased in caudate nucleus, hypothalamus and cerebellum of the rolling mouse. Though there were not significant differences of tyrosine hydroxylase and tryptophan hydroxylase activities between the rolling and normal control mouse in the hypothalamus, the rolling showed significant increase of biopterin concentration and tyrosine hydroxylase activity after administration of thyrotropin releasing hormone (TRH). These results suggest that ataxic gait of the rolling mouse may be partly due to some abnormalities of catecholaminergic neurons, especially noradrenergic neurons, and that TRH may improve the abnormalities of catecholaminergic neurons. The changes of biopterin concentration by TRH administration indicate that biopterin may be a regulatory factor in catecholamine biosynthesis.     

Effect of apoptotic proteins on function of rat vasopressin-and dopaminergic hypothalamic neurons

To study character of effect of apoptotic signal proteins on activities of neurosecretory cells and neurons of rat hypothalamus, pharmacological inhibitors of proapoptotic protein p53 Pifithrin-α and antiapoptotic protein Bcl-2 HA14-1 were injected into hypothalamus. Activation of vasopressinergic neurosecretory cells at administration of the blocker Bcl-2 HA14-1 was shown: there were observed an increase of vasopressin mRNA in neurons of hypothalamic supraoptical and paraventricular nuclei, a decrease of the immunoreactive vasopressin content in posterior pituitary, and reduction of diuresis. Inactivation of p53 inhibited release of vasopressin from hypothalamus cell bodies, which is indicated by an elevated content of immunoreactive vasopressin in neurosecretory cell bodies with its unchanged synthesis, a decrease of the neurohormone content in the posterior pituitary, and an increase of diuresis rate. Activation of vasopressinergic neurons of the suprachiasmatic nucleus was also shown. Administration of the blocker of Bcl-2 has been revealed to decrease functional activity both of dopaminergic neurons (zona incerta) and of dopaminergic neurosecretory cells (arcuate nucleus), in which a decrease of the tyrosine hydroxylase content was observed. The p53 inactivation also led to a decrease of activity of dopaminergic neurosecretory cells of arcuate nucleus, whereas activity of the neurons of zona incerta did not change. Thus, it has been shown that a change of the apoptotic protein content in vasopressinergic and dopaminergic neurons and neurosecretory cells leads to a change of their functional activity, the character and possibly mechanisms of effects of apoptotic proteins on activities of vasopressin-and dopaminergic cells being different.     

The effect of dopaminergic nigrostriatal system on sleep deprivation in rats

The analysis of the electrophysiological features of sleep-wakefulness cycle in Wistar rats for 9h after a 6h sleep deprivation was carried out. The delay of sleep rebound (since 2.5-3 h after deprivation) was found in the form of moderate increasing of slow-wave sleep and fast-wave sleep phases. According to these sleep-wakefulness cycle changes, a quantitative immunohistochemical study of tyrosine hydroxylase: a key enzyme of dopamine synthesis--and D1 and D2 receptors in nigro-striatal projections has been performed. After sleep, an elevation of D1 receptors immunoreactivity in caudate nucleus and reduction of tyrosine hydroxylase immunoreactivity in compact part of substancia nigra was found. After postdeprivation sleep, a decrease of D1 receptors immunoreactivity and increase of D2 receptors immunoreactivity in caudate nucleus together some increase of tyrosine hydroxilase immunoreactivity in substancia nigra compacta has been observed. These data can testify about active role of dopaminergic nigrostriatal system which provide at the same time with another neurotransmitters of the central nervous system the telencephalo-diencephalic interaction in sleep-wakefulness-sleep cycle.     

Leptin sensitive neurons in the hypothalamus.

A major paradigm in the field of obesity research is the existence of an adipose tissue-brain endocrine axis for the regulation of body weight. Leptin, the peptide mediator of this axis, is secreted by adipose cells. It lowers food intake and body weight by acting in the hypothalamus, a region expressing an abundance of leptin receptors and a variety of neuropeptides that influence food intake and energy balance. Among the most promising candidates for leptin-sensitive cells in the hypothalamus are arcuate nucleus neurons that co-express the anabolic neuropeptides, neuropeptide Y (NPY) and agouti-related peptide (AGRP), and those that express proopiomelanocortin (POMC), the precursor of the catabolic peptide, alphaMSH. These cell types contain mRNA encoding leptin receptors and show changes in neuropeptide gene expression in response to changes in food intake and circulating leptin levels. Decreased leptin signaling in the arcuate nucleus is hypothesized to increase the expression of NPY and AGRP. Levels of leptin receptor mRNA and leptin binding are increased in the arcuate nucleus during fasting, principally in NPY/AGRP neurons. These findings suggest that changes in leptin receptor expression in the arcuate nucleus are inversely associated with changes in leptin signaling, and that the arcuate nucleus is an important target of leptin action in the brain.     

Involvement of stomach ghrelin and hypothalamic neuropeptides in tumor necrosis factor-alpha-induced hypophagia in mice

This study aimed to clarify the interaction of tumor necrosis factor-alpha (TNF-alpha), an anorexigenic cytokine, with ghrelin, an orexigenic peptide secreted by the stomach lining, and hypothalamic neuropeptides in the regulation of food intake in mice. The peripheral administration of TNF-alpha dose-dependently decreased the 24-h cumulative food intake compared with the administration of saline. Reduced food intake was observed at 6 h and 24 h. The same TNF-alpha treatment significantly decreased the plasma level of ghrelin at 6 h and 24 h after treatment compared with the control levels. These changes were accompanied by a significant reduction in the expression of ghrelin mRNA in the stomach at 24 h after treatment. TNF-alpha treatment also resulted in a significant increase in expression of pro-opiomelanocortin (POMC) mRNA and a significant decrease in expression of agouti-related protein (AGRP) mRNA in the hypothalamus at 6 h after treatment. Finally, the pre-administration of ghrelin, reversed the TNF-alpha-induced hypophagia in mice at 6 and 24 h. Taken together, these findings suggest that hypothalamic POMC and AGRP and stomach ghrelin may be involved in TNF-alpha-induced hypophagia in mice.     

Tyrosine hydroxylase-immunoreactive neurons in the hypothalamus of the crested newt. An electron microscopic study

The fine structure and specialized neuronal, vascular and ventricular relations of tyrosine hydroxylase (TH)-immunopositive neurons and processes were examined in the hypothalamus of the crested newt. TH-immunoreactive neurons form a well developed system. Its possible role in the hypothalamic neuroendocrine mechanisms is discussed.     

Role of the AMP-activated protein kinase (AMPK) signaling pathway in the orexigenic effects of endogenous ghrelin

Ghrelin, released from the stomach, stimulates food intake through activation of the ghrelin receptor (GHS-R) located on neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons in the hypothalamus. A role for the energy sensor AMP-activated protein kinase (AMPK) and its downstream effector uncoupling protein 2 (UCP2) in the stimulatory effect of exogenous ghrelin on NPY/AgRP expression and food intake has been suggested. This study aimed to investigate whether a rise in endogenous ghrelin levels is able to influence hypothalamic AMPK activity, pACC, UCP2 and NPY/AgRP expression through activation of GHS-R. An increase in endogenous ghrelin levels was established by fasting (24h) or by induction of streptozotocin(STZ)-diabetes (15 days) in GHS-R(+/+) and GHS-R(-/-) mice. GHS-R(+/+) mice showed a significant increase in AgRP and NPY mRNA expression after fasting, which was not observed in GHS-R(-/-) mice. Fasting did not affect AMPK activity nor ACC phosphorylation in both genotypes and increased UCP2 mRNA expression. The hyperghrelinemia associated with STZ-induced diabetes was accompanied by an increased NPY and AgRP expression in GHS-R(+/+) but not in GHS-R(-/-) mice. AMPK activity and UCP2 expression in GHS-R(+/+) mice after induction of diabetes were decreased to a similar extent in both genotypes. Exogenous ghrelin administration tended to decrease hypothalamic AMPK activity. In conclusion, an increase in endogenous ghrelin levels triggered by fasting or STZ-induced diabetes stimulates the expression of AgRP and NPY via interaction with the GHS-R. The changes in AMPK activity, pACC and UCP2 occur independently from GHS-R suggesting that they do not play a major role in the orexigenic effect of endogenous ghrelin.     

Chronic repeated restraint stress increases prolactin-releasing peptide/tyrosine-hydroxylase ratio with gender-related differences in the rat brain

In this study, we investigated the effect of chronic repeated restraint (RR) on prolactin-releasing peptide (PrRP) expression. In the brainstem, where PrRP colocalize with norepinephrine in neurons of the A1 and A2 catecholaminergic cell groups, the expression of tyrosine hydroxylase (TH) has also been examined. In the brainstem, but not in the hypothalamus, the basal PrRP expression in female rats was higher than that in the males that was abolished by ovariectomy. RR evoked an elevation of PrRP expression in all areas investigated, with smaller reaction in the brainstems of females. There was no gender-related difference in the RR-evoked TH expression. Elevation of PrRP was relatively higher than elevation of TH, causing a shift in PrRP/TH ratio in the brainstem after RR. Estrogen α receptors were found in the PrRP neurons of the A1 and A2 cell groups, but not in the hypothalamus. Bilateral lesions of the hypothalamic paraventricular nucleus did not prevent RR-evoked changes. Elevated PrRP production parallel with increased PrRP/TH ratio in A1/A2 neurons indicate that: (i) there is a clear difference in the regulation of TH and PrRP expression after RR, and (ii) among other factors this may also contribute to the changed sensitivity of the hypothalamo-pituitary–adrenal axis during chronic stress.     

Extrapyramidal and neuroendocrine effects of AM404, an inhibitor of the carrier-mediated transport of anandamide.

A selective inhibitor of the carrier-mediated transport of endogenous cannabinoids, N-(4-hydroxyphenyl)-arachidonylethanolamide (AM404), has been recently synthesized and proposed as a useful tool for studying the physiological effects of endogenous cannabinoids and as a potential therapeutic agent in a variety of diseases. In the present study, we have examined the effects of this compound in two important brain processes in which a role for anandamide and other endogenous cannabinoids has been claimed: neuroendocrine regulation and extrapyramidal motor activity. A single and well-characterized dose of AM404, which presumably resulted in a significant elevation of the levels of endogenous cannabinoids, produced a marked decrease in plasma prolactin (PRL) levels, with no changes in luteinizing hormone (LH) levels. This decrease in PRL levels was accompanied by an increase in the activity of tyrosine hydroxylase (TH) in the medial basal hypothalamus. Both decreased PRL secretion and increased hypothalamic TH activity have been reported to occur after the administration of anandamide. Administration of AM404 also produced a marked motor inhibition in the open-field test, as also reported for anandamide, with a decrease in ambulatory and exploratory activities and an increase in the time spent in inactivity. This was accompanied by a decrease in the activity of TH in the substantia nigra, an effect also previously observed for anandamide.     

Developmental changes in embryonic hypothalamic neurons during prenatal fat exposure

Maternal consumption of a fat-rich diet during pregnancy, which causes later overeating and weight gain in offspring, has been shown to stimulate neurogenesis and increase hypothalamic expression of orexigenic neuropeptides in these postnatal offspring. The studies here, using an in vitro model that mimics in vivo characteristics after prenatal high-fat diet (HFD) exposure, investigate whether these same peptide changes occur in embryos and if they are specific to neurons. Isolated hypothalamic neurons were compared with whole hypothalamus from embryonic day 19 (E19) embryos that were prenatally exposed to HFD and were both found to show similar increases in mRNA expression of enkephalin (ENK) and neuropeptide Y (NPY) compared with that of chow-exposed embryos, with no change in melanin-concentrating hormone, orexin, or galanin. Further examination using immunofluorescence cytochemistry revealed an increase in the number of cells expressing ENK and NPY. By plotting the fluorescence intensity of each cell as a probability density function, three different populations of neurons with low, medium, or high levels of ENK or NPY were found in both HFD and chow groups. The prenatal HFD shifted the density of neurons from the population containing low peptide levels to the population containing high peptide levels. This study indicates that neuronal culture is a useful in vitro system for studying diet effects on neuronal development and shows that prenatal HFD exposure alters the population of hypothalamic neurons containing ENK and NPY in the embryo. These changes may contribute to the increase in HFD intake and body weight observed in offspring.     

Effect of apoptosis proteins on function of vasopressin- and dopaminergic hypothalamic neurons

To study character of effect of apoptosis signal proteins on activities of neurosecretory cells and neurons of rat hypothalamus, pharmacologic inhibitors of proapoptotic protein p53 Pifithrin-alpha and antiapoptotic protein Bcl-2 HA14-1 were injected into the hypothalamus. Activation of vasopressinergic neurosecretory cells at administration of the blocker Bcl-2 HA14-1 was shown: there were observed an increase of vasopressin mRNA in neurons of hypothalamus supraoptical and paraventricular nuclei, a decrease of the immunoreactive vasopressin content in posterior pituitary, and reduction of diuresis. Inactivation of p53 inhibited release of vasopressin from hypothalamus cell bodies, which is indicated by an elevated content of immunoreactive vasopressin in neurosecretory cell bodies with its unchanged synthesis, a decrease of the neurohormone content in the posterior pituitary, and an increase of diuresis rate. Activation of vasopressinergic neurons of the suprachiasmatic nucleus was also shown. Administration of the blocker Bcl-2 has been revealed to decrease functional activity both of dopaminergic neurons (Zona Incerta) and of dopaminergic neurosecretory cells (arcuate nucleus), in which a decrease of the tyrosine hydroxylase content was observed. The p53 inactivation also led to a decrease of activity of dopaminergic neurosecretory cells of arcuate nucleus, whereas activity of the proteins Zone Incerta did not change. Thus, it has been shown that a change of the apoptotic protein content in vasopressinergic and dopaminergic neurons and neurosecretory cells leads to a change of their functional activity, the character and possibly mechanisms of effects of apoptotic proteins on activities of vasopressin- and dopaminergic cells being different.     

Deoxyglucose-autoradiography of the hypothalamus after food deprivation in the mouse

[14C] deoxyglucose-autoradiography of mouse brain after food deprivation showed no specific changes in the ratio of autoradiographic optical density of the lateral nucleus of the hypothalamus (LH) versus ventromedial nucleus of the hypothalamus (VMH). The results show that even if there were differences in functional activity between these hypothalamic nuclei during conditions of hunger and satiety, they are not reflected as autoradiographically detectable in relative deoxyglucose uptake.     

Critical Role of Arcuate Y4 Receptors and the Melanocortin System in Pancreatic Polypeptide-Induced Reduction in Food Intake in Mice

Background

Pancreatic polypeptide (PP) is a potent anti-obesity agent known to inhibit food intake in the absence of nausea, but the mechanism behind this process is unknown.

Methodology/Principal Findings

Here we demonstrate that in response to i.p. injection of PP in wild type but not in Y4 receptor knockout mice, immunostaining for the neuronal activation marker c-Fos is induced specifically in neurons of the nucleus tractus solitarius and the area postrema in the brainstem, notably in cells also showing immunostaining for tyrosine hydroxylase. Importantly, strong c-Fos activation is also detected in the arcuate nucleus of the hypothalamus (ARC), particularly in neurons that co-express alpha melanocyte stimulating hormone (α-MSH), the anorexigenic product of the proopiomelanocortin (POMC) gene. Interestingly, other hypothalamic regions such as the paraventricular nucleus, the ventromedial nucleus and the lateral hypothalamic area also show c-Fos induction after PP injection. In addition to c-Fos activation, PP injection up-regulates POMC mRNA expression in the ARC as detected by in situ hybridization. These effects are a direct consequence of local Y4 signaling, since hypothalamus-specific conditional Y4 receptor knockout abolishes PP-induced ARC c-Fos activation and blocks the PP-induced increase in POMC mRNA expression. Additionally, the hypophagic effect of i.p. PP seen in wild type mice is completely absent in melanocortin 4 receptor knockout mice.

Conclusions/Significance

Taken together, these findings show that PP reduces food intake predominantly via stimulation of the anorexigenic α-MSH signaling pathway, and that this effect is mediated by direct action on local Y4 receptors within the ARC, highlighting a potential novel avenue for the treatment of obesity.