Sumit Sarkar

Neuropeptide Y Has a Central Inhibitory Action on the Hypothalamic-Pituitary-Thyroid Axis.

Recent evidence suggests that neuropeptide Y (NPY), originating in neurons in the hypothalamic arcuate nucleus, is an important mediator of the effects of leptin on the central nervous system. As these NPY neurons innervate hypophysiotropic neurons in the hypothalamic paraventricular nucleus (PVN) that produce the tripeptide, TRH, we raised the possibility that NPY may be responsible for resetting of the hypothalamic-pituitary-thyroid (HPT) axis during fasting. To test this hypothesis, the effects of intracerebroventricularly administered NPY on circulating thyroid hormone levels and proTRH messenger RNA in the PVN were studied by RIA and in situ hybridization histochemistry, respectively. NPY administration suppressed circulating levels of thyroid hormone (T(3) and T(4)) and resulted in an inappropriately normal or low TSH. These alterations were associated with a significant suppression of proTRH messenger RNA in the PVN, indicating that NPY infusion had resulted in a state of central hypothyroidism. Similar observations were made in NPY-infused animals pair fed to the vehicle-treated controls. These data are reminiscent of the effect of fasting on the thyroid axis and indicate that NPY may play a major role in the inhibition of HPT axis during fasting.

Intracerebroventricular administration of α-melanocyte stimulating hormone increases phosphorylation of CREB in TRH- and CRH-producing neurons of the hypothalamic paraventricular nucleus

Changes in circulating leptin levels, as determined by nutritional status, are important for the central regulation of neuroendocrine axes. Among these effects, fasting reduces TRH gene expression selectively in the hypothalamic paraventricular nucleus (PVN), which can be reversed by leptin administration. Intracerebroventricular (i.c.v.) infusion of alpha-MSH recapitulates the effects of leptin on hypophysiotropic TRH neurons, completely restoring proTRH mRNA to levels in fed animals despite continuation of the fast, making alpha-MSH a candidate for mediating the central effects of leptin. As alpha-MSH binds to a G-protein coupled receptor that activates cAMP and alpha-MSH stimulates the TRH promoter through the phosphorylation of the transcription factor CREB in vitro, we determined whether i.c.v. injection of alpha-MSH to rats regulates phosphorylation of CREB, specifically in hypophysiotropic TRH neurons of PVN. As alpha-MSH also induces the activation of CRH gene expression in the PVN, we further determined whether i.c.v. injection of alpha-MSH regulates the phosphorylation of CREB in hypophysiotropic CRH neurons. In vehicle-treated animals, only rare neurons contained nuclear phospho-CREB (PCREB) immunoreactivity in the parvocellular PVN. I.c.v. injection of 10 microg alpha-MSH dramatically increased the number of PCREB-immunolabeled cell nuclei in the PVN in fasted groups at 10 min postinjection, particularly in the medial, periventricular, anterior and ventral parvocellular subdivisions, whereas a moderate increase of PCREB immunoreactivity was observed at 30 min and PCREB immunoreactivity was lowest at 1 h postinfusion. Double immunolabeling with proTRH antiserum revealed that following i.c.v. alpha-MSH infusion at 10 min, the majority of TRH neurons contained PCREB in the anterior (71%), medial (83%) and periventricular (63%) parvocellular subdivisions. The percentage of double-labeled TRH neurons declined at 30 min and 1 h post alpha-MSH infusion. Similarly, only 16% of CRH neurons of the medial parvocellular neurons contained PCREB nuclei in vehicle treated animals, whereas 10 min following alpha-MSH infusion the percentage of CRH neurons colocalizing with the PCREB rose to 54%, then fell to 37 and 17% at 30 and 60 min postinfusion, respectively. These data demonstrate that i.c.v. alpha-MSH administration increases the phosphorylation of CREB in several subdivisions of the PVN including TRH and CRH neurons in the medial and periventricular parvocellular subdivisions, suggesting that phosphorylation of CREB may be necessary for alpha-MSH-induced activation of the TRH and CRH genes. The increase in PCREB in the anterior and ventral parvocellular subdivisions of the PVN, regions linked to nonhypophysiotropic functions such as autonomic regulation, would also imply a role for these neurons in anorectic and energy wasting responses of melanocortin signaling.

Glucagon like peptide-1 (7-36) amide (GLP-1) nerve terminals densely innervate corticotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Glucagon like peptide-1 (7-36) amide (GLP-1), a potent regulator of glucose homeostasis, is also produced in the central nervous system and has been implicated in the control of hypothalamic-pituitary function and food intake. GLP-1 immunoreactive (IR) fibers and terminals are widely distributed in the septum, hypothalamus, thalamus and brainstem, likely originating from GLP-1-IR neuronal cell bodies from the nucleus of the solitary tract of the medulla oblongata. Central administration of GLP-1 increases plasma corticosterone levels and elicits c-fos expression in corticotropin releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN). To identify the endogenous neurocircuitry that may underlie this response, the present study determined whether there is an innervation of PVN CRH neurons by GLP-1-containing nerve terminals. GLP-1-IR fibers and nerve terminals were found in the parvocellular parts of the PVN, with highest concentrations in the anterior and medial parvocellular subdivisions. The magnocellular divisions of the PVN also showed moderate numbers of GLP-1-IR nerve fibers. Double immunolabelling revealed numerous GLP-1-IR nerve fibers in close apposition to approximately 65% of detectable CRH neurons in the medial parvocellular subdivision of the rat PVN. At the ultrastructural level, GLP-1-IR terminals were observed to establish synapses on both perikarya and dendrites of CRH neurons. These findings support the hypothesis that the GLP-1-induced activation of CRH neurons and the associated pituitary-adrenocortical activation may be accomplished by GLP-1s direct action on hypophysiotropic CRH neurons. Since central CRH is also thought to be an anorexigenic factor and GLP-1 neurons contain leptin receptors, activation of CRH neurons in the PVN by GLP-1 may contribute to the complex neuroendocrine and metabolic actions by the adipostatic hormone, leptin.

Central administration of cocaine- and amphetamine-regulated transcript increases phosphorylation of cAMP response element binding protein in corticotropin-releasing hormone-producing neurons but not in prothyrotropin-releasing hormone-producing neurons in the hypothalamic paraventricular nucleus

Cocaine- and amphetamine-regulated transcript (CART) has an important action on hypophysiotropic thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) neurons to regulate the hypothalamic-pituitary-thyroid and adrenal axis, respectively. To elucidate the mechanisms by which CART mediates its effect on TRH and CRH neurons, we determined whether the exogenous administration of CART into the cerebrospinal fluid (CSF) phosphorylates the transcription factor, cyclic adenosine 5-monophosphate response element binding protein (CREB), in the nucleus of TRH and CRH neurons. CART dramatically increased the percentage of phosphoCREB (PCREB) immunolabeled cell nuclei in the hypothalamic paraventricular nucleus (PVN) in fasted as well as fed rats at 10-min postinjection, particularly in the medial parvocellular subdivision of the PVN. Double immunolabelling with CRH antiserum revealed that CART increased the number of CRH neurons containing PCREB from 10.5+/-1.2 % to 87+/-1.2% (P<0.001) in fasting animals and from 3.7+/-0.8% to 74+/-5.3% (P<0.001) in fed animals. In contrast, no significant change was observed in the percentage of proTRH neurons colocalizing with PCREB either in the fasted (11.7+/-1.85%) or fed animals (4.2+/-2.2%) as compared to their respective vehicle controls (2.5+/-1.4% and 4.6+/-1%). Ultrastructural analysis revealed that CART establishes axosomatic and axodendritic contacts with CRH neurons in the PVN. These data demonstrate a selective effect of CART to phosphorylate CREB in CRH, but not TRH neurons in the PVN. Since CART is capable of increasing the gene expression of both CRH and TRH in hypophysiotropic neurons, and CART-containing axon terminals establish synaptic relationships with hypophysiotropic CRH and TRH neurons, we propose that CART may signal to the nucleus by more than one pathway.

GABAergic agents prevent alpha-melanocyte stimulating hormone induced anxiety and anorexia in rats

Alpha-melanocyte stimulating hormone (alpha-MSH) is a hypothalamic peptide believed to play a tonic inhibitory role in feeding and energy homeostasis. Systemic administration of alpha-MSH is known to produce anorexia and anxiety. Since synaptic contacts between gamma-aminobutyric acid (GABA)ergic terminals and alpha-MSH neurons in the hypothalamus have been reported, the present work was undertaken to refine our knowledge on the role of GABAergic systems in anxiety and anorexia induced by intracerebroventricular (icv) administration of alpha-MSH in rats. The anxiety was assessed by elevated plus maze, and spontaneous food consumption was monitored during dark cycle. Prior administration of diazepam and muscimol that promote the function of GABA(A) receptors reversed the anxiogenic response and decreased food intake elicited by alpha-MSH. In contrast, bicuculline, the GABA(A) receptor antagonist, not only enhanced the effects of alpha-MSH but also prevented the influence of GABAergic drugs on alpha-MSH-induced anorexia and anxiety. These findings suggest that alpha-MSH-induced anxiety and anorexia are due to its negative influence on GABAergic system.

Bacterial lipopolysaccharide (LPS)-induced type 2 iodothyronine deiodinase (D2) activation in the mediobasal hypothalamus (MBH) is independent of the lps-induced fall in serum thyroid hormone levels

By administration of bacterial lipopolysaccharide (LPS) to intact and T4-replaced thyroidectomized rats, we demonstrate that in contrast to the cortex and anterior pituitary, there is a persistent increase in type 2 iodothyronine deiodinase (D2) activity in the mediobasal hypothalamus (MBH). We propose that endotoxin-induced D2 activation in the MBH is independent of circulating levels of thyroid hormone and that this mechanism may contribute to central hypothyroidism associated with infection.

Galanin- but not galanin-like peptide-containing axon terminals innervate hypophysiotropic TRH-synthesizing neurons in the hypothalamic paraventricular nucleus

Galanin and galanin-like peptide (GALP) are both orexigenic peptides involved in the regulation of food intake and energy metabolism. To determine whether these peptides may directly influence the hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons, double-labeling immunocytochemistry was performed at light and electron microscopic levels using antisera against proTRH, galanin and GALP. Galanin-IR axons densely innervated all of the major parvocellular subdivisions of the PVN where proTRH neurons were identified. The periventricular and anterior parvocellular subdivisions exhibited a prominent network of galaninergic nerve fibers, while the density of fibers was less intense in the medial parvocellular subdivision. Galanin-immunoreactive (IR) axon varicosities were juxtaposed to the majority of TRH-synthesizing neurons in the anterior, medial and periventricular subdivisions of the PVN. Ultrastucturally, galanin-IR nerve terminals established symmetric type synapses with the perikarya of proTRH-IR neurons, suggesting an inhibitory nature of these contacts. In contrast, GALP immunoreactive fibers and nerve terminals concentrated primarily in the anterior parvocellular subdivision of the PVN and were found in association with only few proTRH-IR neurons in the periventricular and medial parvocellular subdivisions. In conclusion, the dense innervation of TRH neurons in all subdivisions of the PVN by galanin-IR axons indicates that galanin may be involved in the central regulation of the hypothalamic-pituitary-thyroid axis. In contrast, the relative paucity of GALP-containing axons in juxtapsoition to TRH neurons in the medial and periventricular parvocellular subdivisions of the PVN, the origin of hypophysiotropic TRH neurons, makes it unlikely that GALP similarly exerts direct regulatory effects on hypophysiotropic TRH neurons.

Relative contribution of brainstem afferents to the cocaine- and amphetamine-regulated transcript (CART) innervation of thyrotropin-releasing hormone synthesizing neurons in the hypothalamic paraventricular nucleus (PVN)

To determine the relative contribution of the brainstem to the CART innervation of the TRH neurons in the PVN, the major ascending brainstem axonal pathways to the PVN were unilaterally transected in the hypothalamus. After 2 weeks survival time, hypothalamic sections were prepared for immunocytochemistry. PNMT-IR axon density decreased 76.0 +/- 3.8% on the side of the knifecut compared to the contralateral side, demonstrating satisfactory disconnection of the ascending brainstem pathways. In contrast, the density of CART-IR axons in the PVN on the lesioned side was reduced by only 26.9 +/- 2.7%. Disconnection of brainstem pathways reduced the total number of TRH neurons contacted by CART from 99.4 +/- 0.9% on the intact side to 74.3 +/- 9.4% on the lesioned side, as well as the number of CART varicosities on the surface of TRH neurons from 6.0 +/- 0.9 to 2.3 +/- 0.4 CART-IR varicosities/cell. These data indicate that CART-IR neurons residing in the brainstem give rise to only approximately one third of the CART input to the PVN as a whole, but serve as a major source of the CART-IR innervation of hypophysiotropic TRH neurons.