Sergueı̈ O. Fetissov

Distribution of NPY receptors in the hypothalamus.

Neuropeptide Y (NPY) neurons abundantly innervate the hypothalamus, where NPY is involved in the regulation and integration of a broad range of homeostatic functions. In order to understand NPY-mediated behavioral, autonomic and neuroendocrine effects, it is important to characterize in detail the distribution of the hypothalamic NPY receptors. In this review, we briefly summarize the origin of NPY and its two related peptides, peptide YY and pancreatic polypeptide in the hypothalamus. Moreover, based on the results obtained with histological techniques such as in situ hybridization, immunohistochemistry and ligand binding, we summarize data on the hypothalamic distribution of the known NPY receptors, the Y1 Y2, Y4 and Y5 receptors as best characterized to date. These NPY receptors are found with individual distribution patterns in many hypothalamic neurons including neuroendocrine motoneurons, magnocellular neurosecretory neurons and numerous neurons connecting the hypothalamus with the limbic and the autonomic nervous systems. The histochemical analyses allow characterization of coexisting molecules and in this way definition of the neurochemistry of NPY circuitries. By showing coexistence of various NPY receptors they provide a morphological basis for in vitro studies showing heterodimerization of NPY receptors. The NPY neurons and their circuitries underlie the integrative role of NPY as a pleiotropic neuropeptide in the regulation of homeostasis.

Pituitary autoantibodies in autoimmune polyendocrine syndrome type 1

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene. High titer autoantibodies (Aabs) toward intracellular enzymes are a hallmark for APS1 and serve as diagnostic markers and predictors for disease manifestations. In this study, we aimed to identify pituitary autoantigens in patients with APS1. A pituitary cDNA expression library was screened with APS1 sera and a tudor domain containing protein 6 (TDRD6) cDNA clone was isolated. Positive immunoreactivity against in vitro translated TDRD6 fragments was shown in 42/86 (49%) APS1 patients but not in patients with other autoimmune diseases or in healthy controls. By using immunohistochemistry, sera from 3/6 APS1 patients with growth hormone (GH) deficiency showed immunostaining of a small number of guinea pig anterior pituitary cells, and 40–50% of these cells were GH-positive. No such immunostaining was seen with sera from healthy controls. The APS1 Aab-positive, GH-negative cells may represent a novel subpopulation of anterior pituitary cells. In addition, 4/6 patient sera showed staining of a fiber-plexus in the pituitary intermediate lobe recognizing enzymes of monoamine and GABA synthesis. Thus, we have identified TDRD6 as a major autoantigen in APS1 patients and shown that several sera from GH-deficient patients stain specific cell populations and nerves in the pituitary gland.

Regulation of feeding and anxiety by α-MSH reactive autoantibodies

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a stress-related neuropeptide involved in the regulation of motivated behavior, appetite and emotion including stimulation of satiety and anxiety. Although autoantibodies (autoAbs) reactive with alpha-MSH have been identified in human subjects and in rats, it remained unknown if these autoAbs are involved in the regulation of feeding and anxiety and if their production is related to stress. Here we show that repeated exposure of rats to anxiolytic mild stress by handling increases the levels and affinity of alpha-MSH reactive IgG autoAbs and that these changes are associated with adaptive feeding and anxiety responses during exposure of rats to a strong stress by food restriction. Importantly, an increase in affinity of alpha-MSH reactive autoAbs was associated with changes of their functional roles from stimulation to inhibition of alpha-MSH-mediated behavioural responses, suggesting that these autoAbs can be a carrier or a neutralizing molecule of alpha-MSH peptide, respectively. Using a model of passive transfer into the brain, we show that alpha-MSH autoAbs affinity purified from blood of rats exposed to repeated mild stress, but not from control rats, are able to increase acutely food intake, suppress anxiety and modify gene expression of hypothalamic neuropeptides in naïve rats. These data provide the first evidence that autoAbs reactive with alpha-MSH are involved in the physiological regulation of feeding and mood, supporting a further role of the immune system in the control of motivated behavior and adaptation to stress.

Alterations of arcuate nucleus neuropeptidergic development in contactin-deficient mice : comparison with anorexia and food-deprived mice

A mutation in the Contactin‐1 gene results in an ataxic and anorectic phenotype that is apparent by postnatal day 10 and lethal by postnatal day 19 [ Berglund et al. (1999)Neuron 24, 739–750]. The resemblance of this phenotype with the anorexia (anx/anx) mouse mutation prompted us to investigate the hypothalamic neurochemistry of Contactin knock‐out (KO) mice. Contactin was expressed in the hypothalamic neuropil of wild‐type (WT) but not Contactin KO mice. In the KO condition, neuropeptide Y (NPY) and agouti‐related protein (AgRP) immunoreactivity (IR) accumulated in the somata of arcuate nucleus neurons, whereas IR for these neuropeptides as well as for α‐melanocyte‐stimulating hormone (α‐MSH) decreased in the corresponding axon projections. These changes in the pattern of neuropeptide expression in the Contactin‐deficient hypothalamus were similar but more pronounced than those found in anx/anx mice. Increased levels of NPY and AgRP and decreased concentrations of pro‐opiomelanocortin mRNA in arcuate neurons accompanied these changes. In relating these alterations a 24‐h food deprivation period, we observed in 3‐week‐old WT mice an elevation of NPY‐ and AgRP‐IR in the perikarya of arcuate neurons without notable reduction of NPY‐ or AgRP‐IR in nerve fibers, suggesting that the decrease of arcuate projections can be associated with postnatal anorectic phenotype. Our data implicate Contactin in the postnatal development of the NPY/AgRP and α‐MSH arcuate neurons and suggest that similar to anx/anx mutant mice, compromised orexigenic signaling via NPY/AgRP neurons may contribute to reduced food intake by the Contactin‐mutant animals.

Expression of hypothalamic neuropeptides after acute TCDD treatment and distribution of Ah receptor repressor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant originating from industrial waste. At sublethal concentrations it induces anorexia and weight loss as part of the so-called wasting syndrome. To gain insight into its possible underlying mechanisms, mRNA expression of some key hypothalamic neuropeptides involved in the regulation of body weight was studied using in situ hybridization histochemistry in adult male Sprague-Dawley rats 6 days after single oral administration of TCDD (15 microg/kg) and in age-paired control rats. In TCDD-treated rats which displayed a decrease in body weight gain vs. controls, arcuate nucleus expression of neuropeptide Y (NPY), proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) mRNA was increased. In the lateral hypothalamic area, melanin-concentrating hormone (MCH) mRNA expression was also increased, while levels of CART and orexin/hypocretin mRNA were not significantly changed. Since TCDD is known to bind to the aryl hydrocarbon receptor (AhR), the distribution of the AhR repressor (AhRR), which is co-expressed with AhR in the same cells, was studied by immunohistochemistry in the mouse hypothalamus using mouse AhRR specific antiserum. AhRR immunoreactivity was present in the nuclei of neurons found in all main hypothalamic groups including NPY, CART, MCH and orexin/hypocretin neurons. Xenobiotic response elements were found in these neuropeptide genes with the exception of MCH. Thus changes in expression of orexigenic and anorexigenic neuropeptides after TCDD treatment may help to explain the occurrence of the TCDD-induced weight loss, which may be either directly or indirectly related to the effects of TCDD on neuropeptide expression.

Gastric electrical stimulation increases ghrelin production and inhibits catecholaminergic brainstem neurons in rats

Gastric electrical stimulation (GES) is a new therapeutic option for functional dyspepsia and gastroparesis. In addition to ameliorating nausea and vomiting, GES results in improved appetite which is not always associated with accelerated gastric emptying. To explore the central and peripheral factors underlying GES‐associated improvement of appetite we developed a GES model in anaesthetized Wistar rats. During laparotomy, two electrodes were implanted into the stomach and high‐frequency low‐energy GES (14 Hz, 5 mA) was applied. The effects of 1 h GES were compared with sham stimulation. After GES, c‐Fos expression was increased in the mucosal and submucosal layers of the stimulated area (174%). In the stomach, GES increased ghrelin mRNA (178%) and doubled the number of ghrelin‐positive cells, resulting in elevated plasma levels of ghrelin (2.3 ± 0.2 vs. 1.6 ± 0.2 ng/mL). In the arcuate nucleus of the hypothalamus, GES increased c‐Fos (277%) and agouti‐related protein (AgRP) mRNA expression (135%). GES reduced the number of c‐Fos‐positive cells throughout the nucleus of the solitary tract (between 93 and 75% from rostral to caudal levels) including catecholaminergic neurons (81% at caudal level). Gastric emptying, plasma glucose and heart rate variability were not affected by GES. This study shows that GES may improve appetite via stimulation of main orexigenic pathways, including ghrelin production in the stomach and AgRP in the hypothalamus, as well as by reducing the activity of catecholaminergic brainstem neurons.

Neuropeptide Y targets in the hypothalamus: Nitric oxide synthesizing neurones express Y1 receptor

Neuropeptide Y (NPY)‐expressing neurones in the arcuate nucleus densely innervate many hypothalamic nuclei. To determine the neurochemical phenotype of target neurones for NPY, we studied the immunohistochemical localization of the NPY Y1 receptor (Y1R) in discrete subpopulations of neurones in the rat hypothalamus. Among several tested populations, including hypocretin/orexin‐, melanin‐concentrating hormone (MCH)‐ and nitric oxide synthase (NOS)‐positive neurones, only the latter were found to coexpress the Y1R. Numerous Y1R/NOS‐positive neurones were found as a densely packaged group of cells located ventrolateral to the ventromedial nucleus, forming a band ascending towards the fornix. Lower numbers of Y1R/NOS‐positive neurones were found in the perifornical area and in the peri‐ and paraventricular nuclei. Expression of the Y1R gene was found in the same locations in the mouse by colocalizing β‐galactosidase, a Y1R gene reporter, with NOS in a Y1R knockout mouse. To explore possible downstream targets of NO in the rat hypothalamus, the NO‐regulated molecule cGMP was analysed immunohistochemically after incubation of brain slices with sodium nitroprusside, an NO donor. We observed several cGMP‐positive cell bodies in the arcuate nucleus, cGMP‐positive blood vessels and a cGMP‐positive network of thin fibres, some of which colocalized with choline acetyltransferase.

Intestinal inflammation influences α-MSH reactive autoantibodies: relevance to food intake and body weight.

Autoantibodies reacting with alpha-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, are involved in regulation of feeding. In this work we studied if intestinal inflammation (mucositis) may influence α-MSH autoantibodies production relevant to food intake and body weight. Mucositis and anorexia were produced in Sprague-Dawley rats by methotrexate (MTX, 2.5mg/kg/day, for three days, subcutaneously). Plasma levels of total IgG and of α-MSH autoantibodies were measured during and after MTX-induced mucositis and were compared with pair-fed and ad libitum-fed controls. Effects of intraperitoneal injections of rabbit anti-α-MSH IgG (3 or 10 μg/day/rat) on MTX-induced anorexia and on plasma α-MSH peptide concentration were separately studied. Here we show that in MTX rats, intestinal mucositis and anorexia were accompanied by decreased plasma levels of both total IgG and of α-MSH autoantibodies while refeeding was characterized by their elevated levels. In spite of similar food intake in MTX and pair-fed rats, recovery of body weight was delayed by at least 1 week in the MTX group. During refeeding and body weight deficit in MTX rats, α-MSH IgG autoantibody levels correlated negatively with food to water intake ratios. Injections of anti-α-MSH IgG induced a dose-dependent attenuation of food intake and body weight regain in MTX-treated rats accompanied by increased concentrations of α-MSH peptide which correlated positively with plasma levels of α-MSH autoantibodies. These data show that intestinal inflammation, independently from food restriction, affects general humoral immune response which may influence food intake and body weight control via modulation of α-MSH plasma concentration by α-MSH reactive autoantibodies.

Expression of microsomal glutathione S-transferase type 3 mRNA in the rat nervous system

Microsomal glutathione S-transferase type 3 (MGST3) is a recently identified member of a large superfamily of enzymes involved in biotransformation of xenobiotics and biosynthesis of eicosanoids, including prostaglandins and leukotrienes. Using in situ hybridization histochemistry and reverse transcription polymerase chain reaction, we characterized the expression of MGST3 mRNA in the rat nervous system based on the cloned rat MGST3 gene, under normal conditions and after systemic administration of lipopolysaccharide (LPS). The MGST3 mRNA seemed to be confined to neurons. The broad distribution in the brain was characterized by a strong signal in the hippocampal formation and in the nuclei of the cranial nerves. A moderate signal was found in the cortex, thalamus, amygdala and substantia nigra and a weak signal in the hypothalamus. Motoneurons in the spinal cord and sensory neurons in dorsal root ganglia displayed strong MGST3 mRNA signal. No significant changes in the level of expression of MGST3 mRNA in the brain were found 1, 3 or 6 h after LPS administration. The pattern of distribution of MGST3 mRNA in the rat nervous system and the lack of response to LPS do not support a role for MGST3 in the biosynthesis of proinflammatory eicosanoids but rather suggest other functions, perhaps in metabolic detoxication and neuroprotection.