Characterization of NPY Y2 receptor protein expression in the mouse brain. II. Coexistence with NPY, the Y1 receptor, and other neurotransmitter-related molecules

Neuropeptide Y (NPY) is widely expressed in the brain and its biological effects are mediated through a variety of receptors. We examined, using immunohistochemistry, expression of the Y2 receptor (R) protein in the adult mouse brain and its association with NPY and the Y1R, as well as a range of additional neurotransmitters and signaling-related molecules, which previously have not been defined. Our main focus was on the hippocampal formation (HiFo), amygdaloid complex, and hypothalamus, considering the known functions of NPY and the wide expression of NPY, Y1R, and Y2R in these regions. Y2R-like immunoreactivity (-LI) was distributed in nerve fibers/terminal endings throughout the brain axis, without apparent colocalization with NPY or the Y1R. Occasional coexistence between NPY- and Y1R-LI was found in the HiFo. Following colchicine treatment, Y2R-LI accumulated in cell bodies that coexpressed γ-aminobutyric acid (GABA) in a population of cells in the amygdaloid complex and lateral septal nucleus, but not in the HiFo. Instead, Y2R-positive nerve terminals appeared to surround GABA-immunoreactive (ir) cells in the HiFo and other neuronal populations, e.g., NPY-ir cells in HiFo and tyrosine hydroxylase-ir cells in the hypothalamus. In the HiFo, Y2R-ir mossy fibers coexpressed GABA, glutamic acid decarboxylase 67 and calbindin, and Y2R-LI was found in the same fibers that contained the presynaptic metabotropic glutamate receptor 2, but not together with any of the three vesicular glutamate transporters. Our findings provide further support that Y2R is mostly presynaptic, and that Y2Rs thus have a modulatory role in mediating presynaptic neurotransmitter release.     

大鼠脊髓背角浅层神经元中神经肽Y Y1受体与生长抑素共存

用间接免疫荧光双标记方法,研究大鼠脊髓Ⅱ层神经元中神经肽Ｙ Ｙ１受体与生长抑素共存关系。结果显示在Ⅱ层深部许多神经元含Ｙ１受体－免疫反应性物质,只观察到少量的生长抑素免疫反应阳性神经元胞体,其中个别Ｙ１受体阳性神经元也含生长抑素免疫反应性物质;在局部使用秋水仙素后,大多数Ｙ１受体免疫反应阳性神经元含有生长抑素免疫反应性物质。推论脊髓背角Ⅱ层神经元中的Ｙ１受体可能与生长抑素在脊髓水平的功能有关。     

Effect of neuropeptide Y Y2 receptor deletion on emotional stress‐induced neuronal activation in mice

In different behavioral paradigms including the elevated plus maze (EPM), it was observed previously that deletion of the neuropeptide Y Y2 receptor subtype results in potent suppression of anxiety‐related and stress‐related behaviors. To identify neurobiological correlates underlying this behavioral reactivtiy, expression of c‐Fos, an established early marker of neuronal activation, was examined in Y2 receptor knockout (Y2^−/−) vs. wildtype (WT) mice. Mice were placed on the open arm (OA) or closed arm (CA) of the EPM for 10 min and the effect on regional c‐Fos expression in the brain was investigated. The number of c‐Fos positive neurons was significantly increased in both WT and Y2^−/− lines after OA and CA exposure in 51 of 54 regions quantified. These regions included various cortical, limbic, thalamic, hypothalamic, and hindbrain regions. Genotype influenced c‐Fos responses to arm exposures in 6 of the 51 activated regions: the cingulate cortex, barrel field of the primary somatosensory cortex, nucleus accumbens, dorsal lateral septum, amygdala and lateral periaqueductal gray. These differences in neuronal activity responses to the novel environments were more pronounced after OA than after CA exposure. Mice lacking Y2 receptors exhibited reduced neuronal activation when compared to WT animals in response to the emotional stressors. Reduced neuronal excitability in the identified brain areas relevant to the processing of motivated, explorative as well as anxiety‐related behaviors is suggested to contribute to the reduced anxiety‐related behavior observed in Y2^−/− mice. Synapse 63:236–246, 2009. © 2008 Wiley‐Liss, Inc.     

Characterization of neuropeptide Y Y2 and Y5 receptor expression in the mouse hypothalamus

Neuropeptide Y (NPY) neurons abundantly innervate the hypothalamus, where NPY is involved in the regulation of a broad range of homeostatic functions. In the present work we studied NPY Y2 and Y5 receptor (R) gene expression in the mouse hypothalamus by using immunohistochemical detection of beta-galactosidase (beta-gal), a gene reporter molecule for Y2R and Y5R in Y2R-knockout (KO) and Y5R-KO mice, respectively. With this approach, cells normally expressing Y2R or Y5R are immunopositive for beta-gal. In the hypothalamus of the Y2R-KO mouse, beta-gal immunoreactivity (-ir) was found in numerous neurons of the medial preoptic nucleus as well as in the lateral anterior, periventricular, dorsomedial, tuberal, perifornical, and arcuate nuclei. Most of the dopaminergic neurons in the A13 dorsal hypothalamic group were beta-gal positive, whereas other hypothalamic dopaminergic neurons rarely displayed beta-gal-ir. In the arcuate nucleus, most of the beta-gal-positive neurons expressed NPY, but colocalizations with beta-endorphin were also found; in the tuberal and perifornical nuclei, many beta-gal-positive neurons contained nitric oxide synthase. beta-Gal-ir was also found in other forebrain regions of the Y2R-KO mouse, including the amygdala, thalamic nuclei, hippocampal CA3 area, and cortex. In the hypothalamus of the Y5R-KO mouse, beta-gal-positive neurons were found mainly in the arcuate nucleus and contained beta-endorphin. The present data show that Y2R and Y5R are expressed in distinct groups of hypothalamic neurons. High levels of Y2R expression in the preoptic nuclei suggest an involvement of Y2R in the regulation of reproductive behavior, whereas Y2R expression in the arcuate, dorsomedial, and perifornical nuclei may be relevant to feeding and body weight control. The finding that A13 dopaminergic neurons express Y2R suggests a new mechanism putatively involved in the central control of feeding, in which NPY can modulate dopamine secretion. The distribution of Y5R expression supports earlier evidence for involvement of this receptor in control of feeding and body weight via NPY's action on proopiomelanocortin-expressing neurons. J. Comp. Neurol. 470:256-265, 2004.     

Distribution of neuropeptide Y Y1 receptors in rodent peripheral tissues

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking alpha-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R.     

Spatiotemporal distribution of vasoactive intestinal polypeptide receptor 2 in mouse suprachiasmatic nucleus

Vasoactive intestinal polypeptide (VIP) signaling is critical for circadian rhythms. For example, the expression of VIP and its main receptor, VPAC2R, is necessary for maintaining synchronous daily rhythms among neurons in the suprachiasmatic nucleus (SCN), a master circadian pacemaker in animals. Where and when VPAC2R protein is expressed in the SCN and other brain areas has not been examined. Using immunohistochemistry, we characterized a new antibody and found that VPAC2R was highly enriched in the SCN and detectable at low levels in many brain areas. Within the SCN, VPAC2R was circadian, peaking in the subjective morning, and abundantly expressed from the rostral to caudal margins with more in the dorsomedial than ventrolateral area. VPAC2R was found in nearly all SCN cells including neurons expressing either VIP or vasopressin (AVP). SCN neurons mainly expressed VPAC2R in their somata and dendrites, not axons. Finally, constant light increased VIP and AVP expression, but not VPAC2R. We conclude that the circadian clock, not the ambient light level, regulates VPAC2R protein localization. These results are consistent with VPAC2R playing a role in VIP signaling at all times of day, broadly throughout the brain and in all SCN cells.     

Involvement of serotonin 2C receptor RNA editing in accumbal neuropeptide Y expression and behavioural despair

Serotonin 2C receptors (5‐HT_2CRs) are widely expressed in the central nervous system, and are associated with various neurological disorders. 5‐HT_2CR mRNA undergoes adenosine‐to‐inosine RNA editing at five sites within its coding sequence, resulting in expression of 24 different isoforms. Several edited isoforms show reduced activity, suggesting that RNA editing modulates serotonergic systems in the brain with causative relevance to neuropsychiatric disorders. Transgenic mice solely expressing the non‐edited 5‐HT_2CR INI‐isoform (INI) or the fully edited VGV‐isoform exhibit various phenotypes including metabolic abnormalities, aggressive behaviour, anxiety‐like behaviour, and depression‐like behaviour. Here, we examined the behavioural phenotype and molecular changes of INI mice on a C57BL/6J background. INI mice showed an enhanced behavioural despair in the forced swimming test, elevated sensitivity to the tricyclic antidepressant desipramine, and significantly decreased serotonin in the nucleus accumbens (NAc), amygdala, and striatum. They also showed reduced expression of neuropeptide Y (NPY) mRNA in the NAc. In addition, by stereotactic injection of adeno‐associated virus encoding NPY into the NAc, we demonstrated that accumbal NPY overexpression relieved behavioural despair. Our results suggest that accumbal NPY expression may be regulated by 5‐HT_2CR RNA editing, and its impairment may be linked to mood disorders.     

Distribution of neuropeptide S receptor mRNA and neurochemical characteristics of neuropeptide S-expressing neurons in the rat brain

Neuropeptide S (NPS) and its receptor (NPSR) constitute a novel neuropeptide system that is involved in regulating arousal and anxiety. The NPS precursor mRNA is highly expressed in a previously undescribed group of neurons located between the locus coeruleus (LC) and Barrington's nucleus. We report here that the majority of NPS-expressing neurons in the LC area and the principal sensory trigeminal nucleus are glutamatergic neurons, whereas many NPS-positive neurons in the lateral parabrachial nucleus coexpress corticotropin-releasing factor (CRF). In addition, we describe a comprehensive map of NPSR mRNA expression in the rat brain. High levels of expression are found in areas involved in olfactory processing, including the anterior olfactory nucleus, the endopiriform nucleus, and the piriform cortex. NPSR mRNA is expressed in several regions mediating anxiety responses, including the amygdaloid complex and the paraventricular hypothalamic nucleus. NPSR mRNA is also found in multiple key regions of sleep neurocircuitries, such as the thalamus, the hypothalamus, and the preoptic region. In addition, NPSR mRNA is strongly expressed in major output and input regions of hippocampus, including the parahippocampal regions, the lateral entorhinal cortex, and the retrosplenial agranular cortex. Multiple hypothalamic nuclei, including the dorsomedial and the ventromedial hypothalamic nucleus and the posterior arcuate nucleus, express high levels of NPSR mRNA, indicating that NPS may regulate energy homeostasis. These data suggest that the NPS system may play a key role in modulating a variety of physiological functions, especially arousal, anxiety, learning and memory, and energy balance.     

Distribution of neuropeptide Y in the lower brainstem: an immunohistochemical analysis

The distribution of neuropeptide Y (NPY)-like immunoreactive (NPYI) structures in the rat lower brainstem was examined by means of indirect immunofluorescence or peroxidase-antiperoxidase methods. In addition to the well known immunoreactive NPYI containing cell groups, the present study demonstrated a much wider distribution of immunoreactive NPYI cells in the lower brainstem, i.e. substantia nigra pars lateralis, interpeduncular nucleus, inferior colliculus, nucleus cuneiformis, dorsal tegmental nucleus of Gudden, nucleus laterodorsalis tegmenti, nucleus vestibularis medialis, nucleus vestibularis inferioris, in the fasciculus longitudinalis medialis and nucleus parvocellularis compacta. We also demonstrated an extensive network of NPYI fibers in various areas of the lower brainstem including the auditory system, viscerosensory system, visceromotor system, raphe nuclei, reticular formation, parabrachial area, locus coeruleus and interpeduncular nucleus, etc. The possible importance of NPY is briefly discussed.     

The anti-epileptic actions of neuropeptide Y in the hippocampus are mediated by Y and not Y receptors

Neuropeptide Y (NPY) potently inhibits glutamate release and seizure activity in rodent hippocampus in vitro and in vivo, but the nature of the receptor(s) mediating this action is controversial. In hippocampal slices from rats and several wild-type mice, a Y2-preferring agonist mimicked, and the Y2-specific antagonist BIIE0246 blocked, the NPY-mediated inhibition both of glutamatergic transmission and of epileptiform discharges in two different slice models of temporal lobe epilepsy, stimulus train-induced bursting (STIB) and 0-Mg2+ bursting. Whereas Y5 receptor-preferring agonists had small but significant effects in vitro, they were blocked by BIIE0246, and a Y5 receptor-specific antagonist did not affect responses to any agonist tested in any preparation. In slices from mice, NPY was without effect on evoked potentials or in either of the two slice seizure models. In vivo, intrahippocampal injections of Y2- or Y5-preferring agonists inhibited seizures caused by intrahippocampal kainate, but again the Y5 agonist effects were insensitive to a Y5 antagonist. Neither Y2- nor Y5-preferring agonists affected kainate seizures in mice. A Y5-specific antagonist did not displace the binding of two different NPY ligands in WT or mice, whereas all NPY binding was eliminated in the mouse. Thus, we show that Y2 receptors alone mediate all the anti-excitatory actions of NPY seen in the hippocampus, whereas our findings do not support a role for Y5 receptors either in vitro or in vivo. The results suggest that agonists targeting the Y2 receptor may be useful anticonvulsants.     

Distribution of neuropeptide Y in the forebrain and diencephalon: an immunohistochemical analysis

The distribution of neuropeptide Y (NPY)-like immunoreactive (NPYI) structures in the rat forebrain and upper brainstem was examined by means of indirect immunofluorescence and peroxidase-antiperoxidase methods. The present study has demonstrated a much wider and more abundant distribution of NPYI structures in the forebrain and diencephalon than earlier studies in which antisera against avian pancreatic polypeptide or bovine pancreatic polypeptide were used, suggesting that NPY is involved in a variety of brain functions.     

Combined gene overexpression of neuropeptide Y and its receptor Y5 in the hippocampus suppresses seizures

We recently demonstrated that recombinant adeno-associated viral vector-induced hippocampal overexpression of neuropeptide Y receptor, Y2, exerts a seizure-suppressant effect in kindling and kainate-induced models of epilepsy in rats. Interestingly, additional overexpression of neuropeptide Y in the hippocampus strengthened the seizure-suppressant effect of transgene Y2 receptors. Here we show for the first time that another neuropeptide Y receptor, Y5, can also be overexpressed in the hippocampus. However, unlike Y2 receptor overexpression, transgene Y5 receptors in the hippocampus had no effect on kainate-induced motor seizures in rats. However, combined overexpression of Y5 receptors and neuropeptide Y exerted prominent suppression of seizures. This seizure-suppressant effect of combination gene therapy with Y5 receptors and neuropeptide Y was significantly stronger as compared to neuropeptide Y overexpression alone. These results suggest that overexpression of Y5 receptors in combination with neuropeptide Y could be an alternative approach for more effective suppression of hippocampal seizures.     

Direct inhibition of arcuate kisspeptin neurones by neuropeptide Y in the male and female mouse

Adverse energy states exert a potent suppressive influence on the reproductive axis by inhibiting the pulsatile release of gonadotrophin‐releasing hormone and luteinising hormone. One potential mechanism underlying this involves the metabolic‐sensing pro‐opiomelanocortin and agouti‐related peptide/neuropeptide Y (AgRP/NPY) neuronal populations directly controlling the activity of the arcuate nucleus kisspeptin neurones comprising the gonadotrophin‐releasing hormone pulse generator. Using acute brain slice electrophysiology and calcium imaging approaches in Kiss1‐GFP and Kiss1‐GCaMP6 mice, we investigated whether NPY and α‐melanocyte‐stimulating hormone provide a direct modulatory influence on the activity of arcuate kisspeptin neurones in the adult mouse. NPY was found to exert a potent suppressive influence upon the neurokinin B‐evoked firing of approximately one‐half of arcuate kisspeptin neurones in both sexes. This effect was blocked partially by the NPY1R antagonist BIBO 3304, whereas the NPY5R antagonist L152,804 was ineffective. NPY also suppressed the neurokinin B‐evoked increase in intracellular calcium levels in the presence of tetrodotoxin and amino acid receptor antagonists, indicating that the inhibitory effects of NPY are direct on kisspeptin neurones. By contrast, no effects of α‐melanocyte‐stimulating hormone were found on the excitability of arcuate kisspeptin neurones. These studies provide further evidence supporting the hypothesis that AgRP/NPY neurones link energy status and luteinising hormone pulsatility by demonstrating that NPY has a direct suppressive influence upon the activity of a subpopulation of arcuate kisspeptin neurones.     

Expression of the melanin-concentrating hormone (MCH) receptor mRNA in the rat brain

The melanin-concentrating hormone (MCH) system is thought to be an important regulator of food intake. Recently the orphan G protein-coupled receptor SLC-1 was identified as the MCH receptor (MCHR). Preliminary analyses of MCHR mRNA distribution have supported a role for the MCH system in nutritional homeostasis. We report here a complete anatomical distribution of the MCHR mRNA. We have found high levels of expression of MCHR mRNA in most anatomical areas implicated in control of olfaction, with the exception of the main olfactory bulb. Dense labeling was also detected in the hippocampal formation, subiculum, and basolateral amygdala, all of which are important in learning and memory, and in the shell of the nucleus accumbens, a substrate for motivated behavior and feeding. Within the hypothalamus, MCHR mRNA was moderately expressed in the ventromedial nucleus, arcuate nucleus, and zona incerta, all of which serve key roles in the neuronal circuitry of feeding. In the brainstem, strong expression was observed in the locus coeruleus, which is implicated in arousal, as well as in nuclei that contribute to orofacial function and mastication, including the facial, hypoglossal, motor trigeminal, and dorsal motor vagus nuclei. In most regions there was a good correspondence between MCHR mRNA distribution and that of MCH-immunoreactive fibers. Taken together, these data suggest that MCH may act at various levels of the brain to integrate various aspects of feeding behavior. However, the extensive MCHR distribution throughout the brain suggests that this receptor may play a role in other functions, most notably reinforcement, arousal, sensorimotor integration, and autonomic control.     

Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain of the crested newt

The newt brain represents a simplified model for the increasingly complex vertebrate neuronal organization. The localization of neuropeptide Y-like (NPY-like) containing neurons in the brain of Triturus cristatus was studied by means of indirect immunofluorescence, peroxidase-antiperoxidase, and avidin-biotin techniques using a highly specific antiserum. NPY-like positive cell bodies were observed in several areas, most notably in the telencephalon (primordium hippocampi and amygdaloid complex), the preoptic and suprachiasmatic areas, the hypothalamus, the dorsal thalamus, the tegmentum, and the rhombencephalon (laterolateral grey column and raphe area). Nerve fibres were particularly abundant in the pallium, striatum, septum, amygdaloid, preoptic neuropils, and pars intercalaris diencephali. Bundles of NPY-immunoreactive fibres also were visualized in the dorsal thalamus and in the posterior hypothalamus. The pars intermedia lacked any NPY-like positive fibres. Neuronal processes also were found in the tectum mesencephali and in the body of the cerebellum. A prominent NPY-like fibre network was observed in the octavolateralis. Concentrations of NPY measured by means of a specific radioimmunoassay were threefold higher in the hypothalamus (15.2 +/- 1.3 ng/mg proteins) than in the rhombencephalon (4.9 +/- 0.3) and the mesencephalon (4.3 +/- 0.2). The concentration found in the telencephalon was 2.1 +/- 0.3 ng/mg proteins. Sephadex G-50 gel chromatography of whole brain extracts indicated the presence of high molecular weight forms of NPY-like material in addition to the authentic peptide. Both amphibian and mammalian NPY peptides had an apparent molecular weight of 4,000 daltons, as evidenced by immunoblotting analysis. High-performance liquid chromatography demonstrated, however, that the newt peptide was slightly less hydrophobic than porcine NPY. The present findings indicate that NPY-immunoreactive neurons are widely distributed in the brain of urodeles. Our data indicate that the NPY molecule has been relatively well preserved during evolution.     

Y1 receptor of neuropeptide Y as a glial marker in proliferative vitreoretinopathy and diseased human retina

The Y1 receptor of neuropeptide Y (NPY) has been demonstrated in glial cells of astrocytic lineage in vitro. We have studied the immunohistochemical expression of Y1 receptors in the glia of the diseased human retina, in tissue samples obtained after surgery for proliferative vitreoretinopathy. In this condition, glia and other cell types migrate and form epi- or subretinal membranes. Both diseased retinas (n = 8) and PVR membranes (n = 43) contained numerous Y1-immunoreactive cells. In the diseased retina, the Y1 antiserum labeled cells with the morphological radial pattern characteristic of Müller cells, whereas in the membranes, label appeared in a large population of elongate cells, measuring up to 250 microm. In both retina and membranes, double labeling demonstrated that the vast majority of Y1-immunoreactive cells were also labeled by a glial fibrillary acidic protein (GFAP) antibody, indicating their glial origin. Retinal regions devoid of GFAP immunoreactivity also lacked the Y1 label. None of these markers was detected in Müller cells of normal retina. Y1 immunoreactivity did not co-localize with smooth muscle actin immunoreactivity, a marker of myofibroblasts. Expression of Y1 receptors would characterize reactive and proliferating glial cells of the diseased retina and could perhaps be involved in the proliferation of injured glial cells causing regrowth of PVR membranes and the consequent secondary retinal detachments.     

Inhibitory effect of neuropeptide Y on morphine withdrawal is accompanied by reduced c-fos expression in specific brain regions

Neuropeptide Y (NPY) was previously shown in our laboratory to attenuate behavioral signs of morphine withdrawal. To further characterize the anti-withdrawal effect of NPY, the present study attempted to identify specific brain regions where NPY inhibits neuronal activity during withdrawal. Morphine dependence was induced in male Wistar rats by two daily subcutaneous injections of morphine at increasing doses, and the withdrawal syndrome was precipitated acutely by intraperitoneal administration of naloxone. Rats were pre-treated with an intracerebroventricular (icv) injection of NPY (12 nmol) or vehicle 30 min before the naloxone challenge. Withdrawal behavior was quantified using a point scoring technique based on motor- and non-motor-related signs. Brain areas involved in the attenuation of morphine withdrawal were delineated by radioactive in situ hybridization for the immediate early gene c-fos, which is a marker for neuronal activity. The present study confirmed the inhibitory effect of NPY on withdrawal behavior. Inhibition of behavioral signs of naloxone-precipitated morphine withdrawal was accompanied by significantly reduced c-fos expression in the locus coeruleus, lateral septal nucleus, ventral part of the periaqueductal grey, cingulate and frontal cortices, and septohippocampal nucleus. Our data suggest that neo- and allo-cortical areas as well as specific brainstem nuclei are involved in the anti-withdrawal effects of NPY.     

Reduced expression of the KATP channel subunit, Kir6.2, is associated with decreased expression of neuropeptide Y and agouti-related protein in the hypothalami of Zucker diabetic fatty rats

The link between obesity and diabetes is not fully understood but there is evidence to suggest that hypothalamic signalling pathways may be involved. The hypothalamic neuropeptides, pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and agouti-related protein (AGRP) are central to the regulation of food intake and have been implicated in glucose homeostasis. Therefore, the expression of these genes was quantified in hypothalami from diabetic Zucker fatty (ZDF) rats and nondiabetic Zucker fatty (ZF) rats at 6, 8, 10 and 14 weeks of age. Although both strains are obese, only ZDF rats develop pancreatic degeneration and diabetes over this time period. In both ZF and ZDF rats, POMC gene expression was decreased in obese versus lean rats at all ages. By contrast, although there was the expected increase in both NPY and AGRP expression in obese 14-week-old ZF rats, the expression of NPY and AGRP was decreased in 6-week-old obese ZDF rats with hyperinsulinaemia and in 14-week-old rats with the additional hyperglycaemia. Therefore, candidate genes involved in glucose, and insulin signalling pathways were examined in obese ZDF rats over this age range. We found that expression of the ATP-sensitive potassium (K(ATP)) channel component, Kir6.2, was decreased in obese ZDF rats and was lower compared to ZF rats in each age group tested. Furthermore, immunofluorescence analysis showed that Kir6.2 protein expression was reduced in the dorsomedial and ventromedial hypothalamic nuclei of 6-week-old prediabetic ZDF rats compared to ZF rats. The Kir6.2 immunofluorescence colocalised with NPY throughout the hypothalamus. The differences in Kir6.2 expression in ZF and ZDF rats mimic those of NPY and AGRP, which could infer that the changes occur in the same neurones. Overall, these data suggest that chronic changes in hypothalamic Kir6.2 expression may be associated with the development of hyperinsulinaemia and hyperglycaemia in ZDF rats.     

Characterization of the neuropeptide Y5 receptor in the human hypothalamus: a lack of correlation between Y5 mRNA levels and binding sites

Neuropeptide Y (NPY) is a 36-amino-acid peptide that appears to play a central role in the control of feeding behavior. Recently, a cDNA encoding a novel NPY receptor subtype (Y5) was cloned from the rat and human hypothalamus, and shown to have a pharmacology consistent with NPY-induced feeding. We have subsequently cloned this cDNA from human hypothalamus and stably expressed it in CHO cells. Consistent with earlier reports, hY5 has a high affinity for NPY, [Leu³¹,Pro³⁴]NPY, and NPY(3–36), but low affinity for larger C-terminal deletions of NPY and BIBP3226. High levels of hY5 mRNA were found in the human testis, brain, spleen and pancreas, with lower levels in several other tissues. In the human brain, hY5 mRNA levels were typically higher than hY2, but lower in comparison to hY1 receptor mRNA. To quantify the relative amounts of hY1, hY2 and hY5 mRNA in the human hypothalamus, we employed competitive RT-PCR. Interestingly, the relative amount of hY5 mRNA was substantially higher than either hY1 or hY2. However, pharmacological characterization of NPY binding sites in human hypothalamus membranes revealed predominantly the hY2 subtype. These data establish that while hY5 mRNA levels are very high in the human hypothalamus, conventional radioligand binding techniques do not detect hY5-like binding site. Whether hY5-like binding sites exist in the other human tissues that express hY5 mRNA (and what function hY5 has in those tissues) awaits future investigation.