Thyrotropin-releasing hormone (TRH) inhibits melanin-concentrating hormone neurons: implications for TRH-mediated anorexic and arousal actions

Thyrotropin-releasing hormone (TRH) increases activity and decreases food intake, body weight, and sleep, in part through hypothalamic actions. The mechanism of this action is unknown. Melanin-concentrating hormone (MCH) and hypocretin/orexin neurons in the lateral hypothalamus (LH) together with neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the arcuate nucleus play central roles in energy homeostasis. Here, we provide electrophysiological evidence from GFP-reporter transgenic mouse brain slices that shows TRH modulates the activity of MCH neurons. Using whole-cell current-clamp recording, we unexpectedly found that TRH and its agonist, montrelin, dose-dependently inhibited MCH neurons. Consistent with previous reports, TRH excited hypocretin/orexin neurons. No effect was observed on arcuate nucleus POMC or NPY neurons. The TRH inhibition of MCH neurons was eliminated by bicuculline and tetrodotoxin, suggesting that the effect was mediated indirectly through synaptic mechanisms. TRH increased spontaneous IPSC frequency without affecting amplitude and had no effect on miniature IPSCs or EPSCs. Immunocytochemistry revealed little interaction between TRH axons and MCH neurons, but showed TRH axons terminating on or near GABA neurons. TRH inhibition of MCH neurons was attenuated by Na(+)-Ca(2+) exchanger (NCX) inhibitors, TRPC channel blockers and the phospholipase C inhibitor U-73122. TRH excited LH GABA neurons, and this was also reduced by NCX inhibitors. Finally, TRH attenuated the excitation of MCH neurons by hypocretin. Together, our data suggest that TRH inhibits MCH neurons by increasing synaptic inhibition from local GABA neurons. Inhibition of MCH neurons may contribute to the TRH-mediated reduction in food intake and sleep.     

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.     

Hypocretin/Orexin- and melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: Relationship to the neuropeptide Y and agouti gene-related protein systems

Cells in the lateral hypothalamus and in the arcuate nucleus play prominent roles in the central control of food intake; however, a neurochemical link connecting these potential components of a hypothalamic circuitry regulating energy metabolism remains to be established. In the present study, the topographical relationship between cells expressing mRNAs encoding melanin-concentrating hormone and the newly discovered neuropeptide family hypocretins/orexins was studied in the rat and mouse lateral hypothalamus by using double-labeling in situ hybridization. Cells expressing the two mRNAs formed completely distinct populations, with hypocretin/orexin cells located primarily perifornically and in the magnocellular lateral hypothalamic nucleus; melanin-concentrating hormone cells extended in a wider area both laterally and periventricularly and appeared to partly surround the hypocretin/orexin population. In the arcuate nucleus, cells expressing neuropeptide Y and agouti gene-related protein were studied by routine fluorescence and/or confocal microscopy immunohistochemistry. Double staining demonstrated that a large proportion of the neuropeptide Y-positive cell bodies in this nucleus also contained agouti gene-related protein-like immunoreactivity. Moreover, these two peptides also coexisted in nerve terminals surrounding and in close relationship to perikarya and processes of both hypocretin/orexin- and melanin-concentrating hormone-immunoreactive cells in the lateral hypothalamus, whereby the former appeared to receive a more dense innervation. These results thus provide evidence for an arcuate-lateral hypothalamic neuropeptide Y/agouti gene-related protein pathway. Furthermore, the results implicate hypocretin/orexin and melanin-concentrating hormone-expressing cells as downstream targets in neuropeptide Y-induced feeding. J. Comp. Neurol. 402:460–474, 1998. © 1998 Wiley-Liss, Inc.     

CART peptides in the central control of feeding and interactions with neuropeptide Y

While CART peptides have been implicated as novel, putative peptide neurotransmitters/cotransmitters, behavioral effects of these peptides have not yet been demonstrated. In this study, we show the first behavioral effect of CART peptides. Icv administration of CART peptide fragments inhibits feeding in rats. Moreover, injection of an antibody to CART peptide 82–103 stimulates feeding, suggesting that endogenous CART peptides exert an inhibitory tone on feeding. Injection of CART peptide 82–103 five min before NPY reduces the increase in feeding caused by injection of NPY alone. Also, in light microscopic immunohistochemical studies, NPY-positive varicosities were observed around CART peptide-positive cell bodies in the paraventricular nucleus of the hypothalamus. These data suggest functional interactions between CART peptides and NPY. These results indicate that CART peptides play a role in the control of food intake by the brain. Synapse 29:293–298, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Melanin concentrating hormone (MCH): a novel neural pathway for regulation of GnRH neurons

The link between the state of energy balance and reproductive function is well known. Thus, signals denoting negative energy balance and the accompanying hyperphagic drive are likely to be factors in the suppression of gonadotropin releasing hormone (GnRH) activity. We have previously found that appetite-regulating systems, such as neuropeptide Y (NPY) in the arcuate nucleus (ARH) and orexin in the lateral hypothalamic area (LHA), send fiber projections that come in close apposition with GnRH neurons. Furthermore, the appropriate receptors, NPY Y5 and OR-1, respectively, are coexpressed on GnRH neurons, providing neuroanatomical evidence for a direct link between the NPY and orexin systems and GnRH neurons. Therefore, these orexigenic neuropeptide systems are potential candidates that convey information about energy balance to GnRH neurons. The current studies focused on melanin concentrating hormone (MCH), another orexigenic neuropeptide system located in the LHA that is sensitive to energy balance. The results showed that MCH fiber projections came in close apposition with approximately 85-90% of GnRH cell bodies throughout the preoptic area and anterior hypothalamic area in the rat. In addition, the MCH receptor (MCHR1) was coexpressed on about 50-55% of GnRH neurons. These findings present evidence for a possible direct neuroanatomical pathway by which MCH may play a role in the regulation of GnRH neuronal function. Thus, MCH is another potential signal that may serve to integrate energy balance and reproductive function.     

GABA-ergic innervation of thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

To determine whether GABA-ergic axons are anatomically situated to directly influence TRH neurons in the PVN, double-labeling light- and electronmicroscopic immunocytochemistry was performed using antisera against glutamic acid decarboxylase (GAD) and prothyrotropin-releasing hormone (proTRH). In the anterior, periventricular and medial parvocellular subdivisions of the PVN, GAD-immunoreactive (IR) axon varicosities were closely apposed to all proTRH containing cell bodies and proximal dendrites. Ultrastucturally, GAD-IR nerve terminals established symmetric type synapses with both perikarya and dendrites of proTRH-IR neurons, indicating the inhibitory nature of the contacts. Since a subpopulation of neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus co-synthesize GABA, and NPY-containing neurons of arcuate nucleus origin densely innervate TRH neurons in the PVN, we performed triple labeling immunocytochemistry to elucidate the origin of the GAD-IR innervation of hypophysiotropic TRH neurons. While axons co-containing GAD and NPY were observed throughout the PVN, only approximately 10% of GAD-IR terminals in contact with TRH neurons were found to contain NPY-immunoreactivity. We conclude that GABA-ergic neurons are in position to act directly on hypophysiotropic TRH neurons and while this innervation arises partly from neurons in the arcuate nucleus that co-synthesize NPY, the majority of the GABA-ergic input arises from other neuronal groups.     

Projections of RFamide-related Peptide-3 Neurones in the Ovine Hypothalamus, with Special Reference to Regions Regulating Energy Balance and Reproduction

RFamide-related peptide-3 (RFRP-3) is a neuropeptide produced in cells of the paraventricular nucleus and dorsomedial nucleus of the ovine hypothalamus. In the present study, we show that these cells project to cells in regions of the hypothalamus involved in energy balance and reproduction. A retrograde tracer (FluoroGold) was injected into either the arcuate nucleus, the lateral hypothalamic area or the ventromedial nucleus. The distribution and number of retrogradely-labelled RFRP-3 neurones was determined. RFRP-3 neurones projected to the lateral hypothalamic area and, to a lesser degree, to the ventromedial nucleus and the arcuate nucleus. Double-label immunohistochemistry was employed to identify cells receiving putative RFRP-3 input to cells in these target regions. RFRP-3 cells were seen to project to neuropeptide Y and pro-opiomelanocortin neurones in the arcuate nucleus, orexin and melanin-concentrating hormone neurones in the lateral hypothalamic area, as well as orexin cells in the dorsomedial nucleus and corticotrophin-releasing hormone and oxytocin cells in the paraventricular nucleus. Neurones expressing gonadotrophin-releasing hormone in the preoptic area were also seen to receive input from RFRP-3 projections. We conclude that RFRP-3 neurones project to hypothalamic regions and cells involved in regulation of energy balance and reproduction in the ovine brain.     

Hypothalamic neuropeptide Y, its gene expression and receptor activity: relation to circulating corticosterone in adrenalectomized rats

Previous evidence has suggested a possible relationship between the adrenal steroid, corticosterone (CORT) and neuropeptide Y (NPY) in the brain. To provide a more systematic analysis of this interaction, the present study employed a variety of techniques, including in sity hybridization to measure NPY gene expression, radioimmunoassay to examine peptide levels and radioligand [¹²⁵I]peptide YY (PYY) binding for analysis of peptide receptors. The results show that adrenalectomy (ADX), which caused a decline in CORT to levels < 0.3 μg%, has generally little impact on the hypothalamic NPY projection system under normal, basal conditions. This includes peptide gene expression or content in the area of its cell bodies (arcuate nucleus, ARC), in addition to peptide binding at its receptor sites. While it also includes peptide content at most hypothalamic terminal sites, there are three notable exceptions, namely, the medial paraventricular (PVN) and dorsomedial nuclei and medial preoptic area, where NPY nerve terminals and glucocorticoid receptors are particularly dense and the decline in CORT through ADX markedly reduces NPY content. In contrast, evidence obtained from CORT replacement in ADX rats shows that this steroid has profound impact on all components of the hypothalamic NPY system. This peptide-steroid interaction is apparent at the level of the cell body (ARC), as well as at the nerve terminal or receptor site (PVN and ARC), where CORT levels > 10 μg% strongly potentiate NPY gene expression, peptide content and radioligand binding. These and other findings suggest that this CORT-NPY interaction in the hypothalamus occurs physiologically under conditions, e.g., at the onset of the active feeding cycle, when circulating CORT normally rises.