Neuropeptide Y Cells Research Articles

The Effect of Dietary Fat and Sucrose on Cognitive Functioning in Mice Lacking Insulin Signaling in Neuropeptide Y Neurons.

Obesogenic diets can produce hippocampal insulin resistance and impairments to hippocampal-dependent cognition. This study investigated the effect of disrupted insulin signaling in Neuropeptide Y (NPY) neurons on diet-induced deficits in hippocampal-dependent memory. Wild-type mice and mice that had a targeted knockout of insulin receptors on NPY cells (IRlox/lox;NPYCre/+) were given ad libitum access to a high-fat diet (high fat; HF), 10% sucrose solution (high sugar; HS), both high-fat diet and sucrose solution (high fat, high sugar; HFHS), or a normal fat control chow for 12 weeks. Mice were tested in the Morris Water Maze (MWM), a hippocampal-dependent spatial memory task. Glucose homeostasis was assessed via a glucose tolerance test. Independent of genotype, consumption of HF, but not HS, diet increased energy intake, body weight, and plasma leptin, and impaired glucose tolerance. Disrupted insulin signaling in NPY cells and dietary interventions did not significantly affect the ability of mice to learn the location of the platform in the MWM. However, for IRlox/lox control mice, consumption of HF, but not HS, diet resulted in reduced time spent in the target quadrant during the probe trial, suggesting a hippocampal-dependent memory deficit. IRlox/lox;NPYCre/+ mice had poor performance in the probe trial regardless of diet, suggesting a floor effect. This study did not find adverse effects of chronic sucrose intake on metabolic outcomes or hippocampal-dependent memory. These data also suggest that the effects of HF diet on hippocampal-dependent memory may be dependent on insulin signaling in hippocampal NPY cells.

Behavioral and brain morphological analysis of non-inflammatory and inflammatory rat models of preterm brain injury

Investigations using preclinical models of preterm birth have much contributed, together with human neuropathological studies, for advances in our understanding of preterm brain injury. Here, we evaluated whether the neurodevelopmental and behavioral consequences of preterm birth induced by a non-inflammatory model of preterm birth using mifepristone would differ from those after inflammatory prenatal transient hypoxia–ischemia (TSHI) model. Pregnant Wistar rats were either injected with mifepristone, and pups were delivered on embryonic day 21 (ED21 group), or laparotomized on the 18th day of gestation for 60 min of uterine arteries occlusion. Rat pups were tested postnatally for characterization of developmental milestones and, after weaning, they were behaviorally tested for anxiety and for spatial learning and memory. One month later, brains were processed for quantification of doublecortin (DCX)- and neuropeptide Y (NPY)-immunoreactive cells, and cholinergic varicosities in the hippocampus. ED21 rats did not differ from controls with respect to neonatal developmental milestones, anxiety, learning and memory functions, and neurochemical parameters. Conversely, in TSHI rats the development of neonatal reflexes was delayed, the levels of anxiety were reduced, and spatial learning and memory was impaired; in the hippocampus, the total number of DCX and NPY cells was increased, and the density of cholinergic varicosities was reduced. With these results we suggest that a preterm birth, in a non-inflammatory prenatal environment, does not significantly change neonatal development and adult neurologic outcome. On other hand, prenatal hypoxia and ischemia (inflammation) modifies developmental trajectory, learning and memory, neurogenesis, and NPY GABAergic and cholinergic brain systems.

Kisspeptin Stimulates Growth Hormone Release by Utilizing Neuropeptide Y Pathways and Is Dependent on the Presence of Ghrelin in the Ewe.

Although kisspeptin is the primary stimulator of gonadotropin-releasing hormone secretion and therefore the hypothalamic-pituitary-gonadal axis, recent findings suggest kisspeptin can also regulate additional neuroendocrine processes including release of growth hormone (GH). Here we show that central delivery of kisspeptin causes a robust rise in plasma GH in fasted but not fed sheep. Kisspeptin-induced GH secretion was similar in animals fasted for 24 hours and those fasted for 72 hours, suggesting that the factors involved in kisspeptin-induced GH secretion are responsive to loss of food availability and not the result of severe negative energy balance. Pretreatment with the neuropeptide Y (NPY) Y1 receptor antagonist, BIBO 3304, blocked the effects of kisspeptin-induced GH release, implicating NPY as an intermediary. Kisspeptin treatment induced c-Fos in NPY and GH-releasing hormone (GHRH) cells of the arcuate nucleus. The same kisspeptin treatment resulted in a reduction in c-Fos in somatostatin (SS) cells in the periventricular nucleus. Finally, blockade of systemic ghrelin release or antagonism of the ghrelin receptor eliminated or reduced the ability of kisspeptin to induce GH release, suggesting the presence of ghrelin is required for kisspeptin-induced GH release in fasted animals. Our findings support the hypothesis that during short-term fasting, systemic ghrelin concentrations and NPY expression in the arcuate nucleus rise. This permits kisspeptin activation of NPY cells. In turn, NPY stimulates GHRH cells and inhibits SS cells, resulting in GH release. We propose a mechanism by which kisspeptin conveys reproductive and hormone status onto the somatotropic axis, resulting in alterations in GH release.

Neuropeptide Y (NPY) promotes inflammation-induced tumorigenesis by enhancing epithelial cell proliferation.

Our work exemplifies a novel role of neuropeptide Y (NPY) in regulating inflammation-induced tumorigenesis via two modalities: first by enhanced proliferation (PI3-K/pAkt), and second by downregulation of microRNA-375 (miR-375)-dependent apoptosis in intestinal epithelial cells. Our data establish the existence of a microRNA-mediated cross talk between enteric neurons producing NPY and intestinal epithelial cells, and the potential of neuropeptide-regulated miRNAs as potential therapeutic molecules for the management of inflammation-associated tumors in the gut.

Neuropeptide Y as a Biomarker and Therapeutic Target for Neuroblastoma

Neuroblastoma (NB) is a pediatric malignant neoplasm of sympathoadrenal origin. Challenges in its management include stratification of this heterogeneous disease and a lack of both adequate treatments for high-risk patients and noninvasive biomarkers of disease progression. Our previous studies have identified neuropeptide Y (NPY), a sympathetic neurotransmitter expressed in NB, as a potential therapeutic target for these tumors by virtue of its Y5 receptor (Y5R)-mediated chemoresistance and Y2 receptor (Y2R)-mediated proliferative and angiogenic activities. The goal of this study was to determine the clinical relevance and utility of these findings. Expression of NPY and its receptors was evaluated in corresponding samples of tumor RNA, tissues, and sera from 87 patients with neuroblastic tumors and in tumor tissues from the TH-MYCN NB mouse model. Elevated serum NPY levels correlated with an adverse clinical presentation, poor survival, metastasis, and relapse, whereas strong Y5R immunoreactivity was a marker of angioinvasive tumor cells. In NB tissues from TH-MYCN mice, high immunoreactivity of both NPY and Y5R marked angioinvasive NB cells. Y2R was uniformly expressed in undifferentiated tumor cells, which supports its previously reported role in NB cell proliferation. Our findings validate NPY as a therapeutic target for advanced NB and implicate the NPY/Y5R axis in disease dissemination. The correlation between elevated systemic NPY and NB progression identifies serum NPY as a novel NB biomarker.

Neuropeptide Y input to the rat basolateral amygdala complex and modulation by conditioned fear.

Within the basolateral amygdaloid complex (BLA), neuropeptide Y (NPY) buffers against protracted anxiety and fear. Although the importance of NPY's actions in the BLA is well documented, little is known about the source(s) of NPY fibers to this region. The current studies identified sources of NPY projections to the BLA by using a combination of anatomical and neurochemical approaches. NPY innervation of the BLA was assessed in rats by examining the degree of NPY coexpression within interneurons or catecholaminergic fibers with somatostatin and tyrosine hydroxylase (TH) or dopamine β-hydroxylase (DβH), respectively. Numerous NPY(+) /somatostatin(+) and NPY(+) /somatostatin(-) fibers were observed, suggesting at least two populations of NPY fibers within the BLA. No colocalization was noted between NPY and TH or DβH immunoreactivities. Additionally, Fluorogold (FG) retrograde tracing with immunohistochemistry was used to identify the precise origin of NPY projections to the BLA. FG(+) /NPY(+) cells were identified within the amygdalostriatal transition area (AStr) and stria terminalis and scattered throughout the bed nucleus of the stria terminalis. The subpopulation of NPY neurons in the AStr also coexpressed somatostatin. Subjecting animals to a conditioned fear paradigm increased NPY gene expression within the AStr, whereas no changes were observed within the BLA or stria terminalis. Overall, these studies identified limbic regions associated with stress circuits providing NPY input to the BLA and demonstrated that a unique NPY projection from the AStr may participate in the regulation of conditioned fear. J. Comp. Neurol. 524:2418-2439, 2016. © 2016 Wiley Periodicals, Inc.

Hypothalamic, feeding/arousal-related peptidergic projections to the paraventricular thalamic nucleus in the rat

Based on the importance of paraventricular thalamic nucleus (PVT) as a relay station of energy balance, arousal, and food reward, we aimed in the present study to determine projection patterns of neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and orexin (ORX)-ergic fibers to the PVT. First, the distribution of peptidergic axon terminals within the PVT was examined. NPY and CART terminals were confined within the boundary of the thalamic nucleus, exhibiting almost identical distribution. MCH terminals were rarely observed. In contrast, ORX terminals were as extensive as NPY/CART terminals, but spread into the peri-PVT region. Second, neuronal origin of feeding/arousal-related peptides projecting to the PVT was investigated. NPY neurons were observed in the medial subdivision of the arcuate nucleus (Arc), whereas CART cells were in the lateral Arc as well as other hypothalamic regions including the paraventricular hypothalamic nucleus, lateral hypothalamus (LH), dorsal hypothalamic area, and zona incerta. Both NPY- and CART-fiber projections to the PVT were bilateral; ipsilateral proportion was 54.0%±3.6% (n=6) for NPY and 57.1%±2.5% (n=6) for CART. The total number of CART neurons projecting to the PVT exceeded that of NPY cells; the ratio of labeled CART neurons to NPY cells was 2.4±0.2 (n=6). In contrast, ORX-ergic fiber projection to the PVT exhibited a slight ipsilateral dominance (62.7%±1.6%, n=6), with majority of labeled cells located in the LH medial to the fornix (72.2%±2.3%, n=6). Third, based on heavy projection from the PVT to the nucleus accumbens shell (NAcSh), the convergence of NPY and CART terminals on a single PVT neuron was identified; the proportion of labeled PVT neurons that received converging NPY/CART terminals compared with the total PVT neurons projecting to the NAcSh was 2.7%±0.6% (n=3). Finally, PVT cells receiving NPY, CART, or ORX terminals provided divergent axon collaterals to NAcSh and medial prefrontal cortex. The present observations provided the anatomical evidence that the PVT might play an essential role in the integration of antagonistically-acting, feeding/arousal-related peptidergic inputs on their way to the cortical reward circuit.

Identification and localization of gastrointestinal hormones in the skin of the bullfrog Rana catesbeiana during periods of activity and hibernation

Amphibian skin and its secretions contain a wide variety of biogenic amines and biologically active peptides, some of which are either identical or highly homologous to gastrointestinal hormones (GHs) of higher vertebrates. This study investigated the distribution density and immunoreactive (IR) intensity of 5-hydroxytryptamine (5-HT), gastrin (GAS), somatostatin (SS), pancreatic polypeptide (PP), neuropeptide Y (NPY) and glucagon (GLU) IR cells in the skin of the bullfrog Rana catesbeiana during periods of activity and hibernation. The results indicated that the six types of GHs were all present in the bullfrog skin and were most predominant in the epidermis and mucous glands. In dorsal skin, the density of the GHs-IR cells in mucous glands was higher than that in epidermis except for GAS-IR cells. In ventral skin, the density of 5-HT, PP and NPY-IR cells in mucous glands was also higher than that in the epidermis. During hibernation, the density of the six types of GHs-IR cells and the IR intensity of GAS, SS, NPY and GLU-IR cells in the epidermis of dorsal skin increased significantly. The IR intensity of SS, PP and NPY-IR cells in granular glands of ventral skin also increased significantly during hibernation. These results suggested that multiple types of GHs-IR cells present in the skin of R. catesbeiana, may play important roles in the regulation of the physiological functions of skin. Also, adaptive changes in the density and IR intensity of GHs-IR cells occurred during hibernation.

Paradoxical Effect of Gonadotrophin‐Inhibiting Hormone to Negatively Regulate Neuropeptide Y Neurones in Mouse Arcuate Nucleus

Regulation of reproduction and energy homeostasis are linked, although our understanding of the central neural mechanisms subserving this connection is incomplete. Gonadotrophin-inhibiting hormone (GnIH) is a neuropeptide that negatively regulates reproduction and stimulates food intake. Neuropeptide Y (NPY) and products of the pro-opiomelanocortin (POMC) precursor (β-endorphin melanocortins) are appetite regulating peptides produced in the neurones of the arcuate nucleus; these peptides also regulate reproduction. In the present study, we determined the effects of GnIH on NPY and POMC neurones. Using brain slices from mice with transgenes for fluorescent tags in the two types of neurone and patch clamp electrophysiology, a predominant inhibitory effect of GnIH was observed. GnIH (100 nM) inhibited the firing rate in POMC cells, confirming the results of previous studies and consistent with the stimulatory effect of GnIH on food intake. Paradoxically (i.e. because both GnIH and NPY stimulate food intake), GnIH also had a predominantly inhibitory effect on action potential activity in NPY cells. GnIH also inhibited the secretion of NPY and α-melanocyte-stimulating hormone secretion in incubated hypothalamic blocks. GnIH (100 ng) injected into the cerebral ventricles of mice did not increase the number of NPY cells that were positively immunostained for c-Fos. Finally, dual label immunocytochemistry showed that 20% of NPY neurones had close contacts from GnIH fibres/varicosities. In conclusion, we confirm a negative effect of GnIH on POMC cells and demonstrate a paradoxical reduction of electrophysiological and functional activity in NPY cells.

Functional differences between neurochemically defined populations of inhibitory interneurons in the rat spinal dorsal horn

In order to understand how nociceptive information is processed in the spinal dorsal horn we need to unravel the complex synaptic circuits involving interneurons, which constitute the vast majority of the neurons in laminae I–III. The main limitation has been the difficulty in defining functional populations among these cells. We have recently identified 4 non-overlapping classes of inhibitory interneuron, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) and parvalbumin, in the rat spinal cord. In this study we demonstrate that these form distinct functional populations that differ in terms of sst2A receptor expression and in their responses to painful stimulation. The sst2A receptor was expressed by nearly all of the nNOS- and galanin-containing inhibitory interneurons but by few of those with NPY and none of the parvalbumin cells. Many galanin- and NPY-containing cells exhibited phosphorylated extracellular signal-regulated kinases (pERK) after mechanical, thermal or chemical noxious stimuli, but very few nNOS-containing cells expressed pERK after any of these stimuli. However, many nNOS-positive inhibitory interneurons up-regulated Fos after noxious thermal stimulation or injection of formalin, but not after capsaicin injection. Parvalbumin cells did not express either activity-dependent marker following any of these stimuli. These results suggest that interneurons belonging to the NPY, nNOS and galanin populations are involved in attenuating pain, and for NPY and nNOS cells this is likely to result from direct inhibition of nociceptive projection neurons. They also suggest that the nociceptive inputs to the nNOS cells differ from those to the galanin and NPY populations.

Retrograde study of CART- or NPY-neuronal projection from the hypothalamic arcuate nucleus to the dorsal raphe and/or the locus coeruleus in the rat

The present study was designed to reveal cocaine- and amphetamine-regulated transcript (CART)- or neuropeptide Y (NPY)-immunoreactive neuronal projections from the hypothalamic arcuate nucleus (Arc) to the dorsal raphe (DR) and/or the locus coeruleus (LC) in the rat. Our results demonstrated that CART or NPY axon terminals formed close appositions to the neuronal profiles in the DR and the LC. Thus, arcuate sections were immunostained for the CART or NPY after the injections of green RetroBeads™ into the DR and red tracer into the LC (or vice versa). First, retrogradely-labeled CART cells were mainly observed in the lateral Arc without colchicine. Of the total population of arcuate CART neurons, DR- and LC-projecting cells were 5.7%±0.9% and 6.6%±0.7%, respectively. In addition, a subset (3.3%±0.7%) of CART neurons provided divergent axon collaterals to the DR and the LC. Second, retrogradely-labeled NPY cells were observed in lateral or ventral borders of the medial Arc only after colchicine injection. Of the entire NPY cell population, DR- and LC-projecting neurons were 1.5%±0.3% and 1.3%±0.3%, respectively. Only a scanty proportion (0.1%±0.0%) sent axon collaterals to the DR and the LC. These observations suggested that arcuate CART or NPY system might have a potential influence on the brainstem monoaminergic nuclei, modulating their roles in feeding, nociception, emotional behaviors, arousal, and stress responses. Furthermore, a portion of arcuate CART neurons (along with only a few NPY cells) sending divergent axon collaterals to the DR/LC might have a simultaneous (and possibly more efficient) way to exert their specific influences on the monoaminergic nuclei.

Neuropeptide Y cells represent a distinct glucose-sensing population in the lateral hypothalamus.

The maintenance of appropriate glucose levels is necessary for survival. Within the brain, specialized neurons detect glucose fluctuations and alter their electrical activity. These glucose-sensing cells include hypothalamic arcuate nucleus neurons expressing neuropeptide Y (NPY) and lateral hypothalamic area (LHA) neurons expressing orexin/hypocretins (ORX) or melanin-concentrating hormone (MCH). Within the LHA, a population of NPY-expressing cells exists; however, their ability to monitor energy status is unknown. We investigated whether NPY neurons located in the LHA, a classic hunger center, detect and respond to fluctuations in glucose availability and compared these responses with those of known LHA glucose sensors expressing ORX or MCH. Using mice expressing green fluorescent protein under the control of NPY regulatory elements, we identified LHA NPY cells and explored their anatomical distribution, neurochemical and electrical properties, in vivo responses to fasting and insulin-induced hypoglycemia, and in situ electrical responses to extracellular glucose. We report that NPY, ORX, and MCH are expressed in nonoverlapping populations within the LHA. Subpopulations of LHA NPY neurons were activated in vivo by both a 6-h fast and insulin-induced hypoglycemia. Likewise, increased extracellular glucose suppressed the electrical activity of approximately 70% of LHA NPY neurons in situ, eliciting hyperpolarization and activating background K+ currents. Furthermore, we report that the glucose sensitivity of LHA NPY neurons is significantly different from neighboring ORX and MCH neurons. These data suggest that NPY-expressing cells in the LHA are a novel population of glucose-sensing neurons that represent a new player in the brain circuitry integrating information about glucose homeostasis.

Seizure-induced changes in neuropeptide Y-containing cortical neurons: Potential role for seizure threshold and epileptogenesis

Seizure activity induces transient changes in the levels of neuropeptide Y (NPY) and somatostatin (SS) in various brain regions, but it remains unclear whether this effect can persist for long periods and whether it is relevant to epileptogenesis. We report that brief seizures evoked by electroshock produced an increase in the number of NPY neurons in the dentate hilus and retrosplenial cortex, an effect that lasted 10 weeks. The number of hilar SS neurons remained unchanged. However, the pentylenetetrazole seizure threshold was somewhat decreased in electroshock-treated rats. Despite this, no spontaneous seizures were detected in this group. In contrast, status epilepticus (pilocarpine model) produced loss of the hilar NPY and SS cells. Moreover, all rats with status epilepticus showed spontaneous behavioral seizures and their seizure threshold was markedly decreased. These findings support the notion that sustained NPY overexpression induced by brief seizures can be important in preventing epileptogenesis.

Kisspeptin Cells in the Ewe Brain Respond to Leptin and Communicate with Neuropeptide Y and Proopiomelanocortin Cells

Kisspeptin stimulates reproduction, and kisspeptin cells in the arcuate nucleus (ARC) express Ob-Rb in the mouse. Herein we report studies in ewes to determine whether kisspeptin cells express Ob-Rb and respond to leptin and whether reciprocal connections exist between kisspeptin cells and proopiomelanocortin (POMC) or neuropeptide Y (NPY) cells to modulate reproduction and metabolic function. Kiss1 mRNA was measured by in situ hybridization in ovariectomized ewes that were normal body weight, lean, or lean with leptin treatment by intracerebroventricular (icv) infusion (4 microg/h, 3 d). Kiss1 expression in the ARC and the preoptic area was lower in hypogonadotropic lean animals than animals of normal weight, and icv infusion of leptin partially restored Kiss1 expression in lean animals. Single-cell laser capture microdissection coupled with real-time PCR showed that Kiss1 cells in the preoptic area and ARC express Ob-Rb. Double-label fluorescent immunohistochemistry showed that reciprocal connections exist between kisspeptin cells and NPY and POMC cells. Accordingly, we treated ovariectomized ewes with kisspeptin (5 microg/h, icv) or vehicle for 20 h and examined POMC and NPY gene expression by in situ hybridization. Kisspeptin treatment reduced POMC and increased NPY gene expression. Thus, kisspeptin neurons respond to leptin and expression of Kiss1 mRNA is affected by leptin status. Kisspeptin cells communicate with NPY and POMC cells, altering expression of the relevant genes in the target cells; reciprocal connections also exist. This network between the three cell types could coordinate brain control of reproduction and metabolic homeostatic systems.

Neuromedin B and Gastrin-Releasing Peptide Excite Arcuate Nucleus Neuropeptide Y Neurons in a Novel Transgenic Mouse Expressing StrongRenillaGreen Fluorescent Protein in NPY Neurons

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright Renilla green fluorescent protein (GFP) in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single-cell reverse transcription-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole-cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin-releasing peptide and neuromedin B, which are found in axons in the mediobasal hypothalamus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni(2+), inclusion of BAPTA in the pipette, KB-R7943, and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around -20 mV. Together, these data suggest two mechanisms, activation of nonselective cation channels and the sodium/calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis.

Reproduction phase‐related variations in neuropeptide Y immunoreactivity in the olfactory system, forebrain, and pituitary of the female catfish, Clarias batrachus (Linn.)

The aim of this study was to determine whether neuropeptide Y (NPY) immunoreactivity in the cells and fibers in the forebrain and pituitary of Clarias batrachus is linked to the annual reproductive cycle. A steady rise in luteinizing hormone (LH) immunoreactivity was seen in the pituitary through preparatory (February-April) and prespawning (May-June) phases; it was greatly reduced during spawning (July-August; P < 0.001) and partially replenished during postspawning (September-November; P < 0.01) through resting (December-January) phases. Although NPY immunoreactivity in olfactory receptor neurons and olfactory nerve layer in olfactory bulb was gradually augmented during resting through prespawning phases (P < 0.001), attaining a peak in spawning phase (P < 0.001), a dramatic decline was encountered during postspawning phase (P < 0.001). A similar pattern was also observed in NPY-containing fibers of the medial olfactory tract (MOT) and pituitary. However, a different pattern of NPY immunoreactivity was observed in the neurons of nucleus entopeduncularis (NE) and nucleus preopticus periventricularis (NPP). Whereas these neurons and fibers in the forebrain showed significant augmentation during the resting through prespawning phases (P < 0.001), the immunoreactivity dramatically declined during spawning (P < 0.001) and was partially replenished in the postspawning phase. Testosterone injection of juveniles significantly augmented (P < 0.001) NPY immunoreactivity in NE neurons. We suggest that NPY cells of NE and NPP, and related fiber systems, might be involved in processing of sex steroid-borne information and regulation of the gonadotropin-releasing hormone-LH axis.

GABA excitation in mouse hilar neuropeptide Y neurons

Neuropeptide Y-containing interneurons in the dentate hilar area play an important role in inhibiting the activity of hippocampal circuitry. Hilar cells are often among the first lost in hippocampal epilepsy. As many types of neurons are found in the hilus, we used a new transgenic mouse expressing green fluorescent protein (GFP) in a subset of neurons that colocalized neuropeptide Y (NPY), somatostatin (SST), and GABA for whole-cell, perforated, and cell-attached recording in 240 neurons. As these neurons have not previously been identifiable in live slices, they have not been the focus of physiological analysis. Hilar NPY neurons showed modest spike frequency adaptation, a large 15.6 +/- 1.0 mV afterhyperpolarization, a mean input resistance of 335 +/- 26 M Omega, and were capable of fast-firing. Muscimol-mediated excitatory actions were found in a nominally Ca(2+)-free/high-Mg(2+) bath solution using cell-attached recording. GABA(A) receptor antagonists inhibited half the recorded neurons and blocked burst firing. Gramicidin perforated-patch recording revealed a GABA reversal potential positive to both the resting membrane potential and spike threshold. Together, these data suggest GABA is excitatory to many NPY cells. NPY and SST consistently hyperpolarized and reduced spike frequency in these neurons. No hyperpolarization of NPY on membrane potential was detected in the presence of tetrodotoxin, AP5, CNQX and bicuculline, supporting an indirect effect. Under similar conditions, SST hyperpolarized the cells, suggesting a direct postsynaptic action. Depolarizing actions of GABA and GABA-dependent burst-firing may synchronize a rapid release of GABA, NPY, and SST, leading to pre- and postsynaptic inhibition of excitatory hippocampal circuits.

Peptide YY3-36Inhibits Both Anorexigenic Proopiomelanocortin and Orexigenic Neuropeptide Y Neurons: Implications for Hypothalamic Regulation of Energy Homeostasis

Peptide YY(3-36) (PYY(3-36)) is released by endocrine cells of the gut and may serve as an important long-distance neuropeptide signal relating energy balance information to the brain to depress food intake. The postulated mechanism is the activation of anorexigenic proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. In striking contrast, using voltage and current-clamp recording, we found that PYY(3-36) consistently, dose dependently, and reversibly inhibited POMC cells by reducing action potentials, hyperpolarizing the membrane potential, decreasing input resistance and inward calcium currents, increasing G-protein-gated inwardly rectifying K+ channel currents, and presynaptically inhibiting release of excitatory glutamate. Importantly, we found PYY(3-36) had similar inhibitory effects on identified orexigenic neuropeptide Y (NPY) neurons. In both cell types, these effects were blocked by BIIE0246, a Y2 receptor antagonist. Together, these data argue that anorexigenic actions of PYY(3-36) are mediated more likely by inhibition of NPY neurons. Dual PYY(3-36) inhibition of both NPY and POMC cells may temporarily reduce the contribution of arcuate cells to feeding circuits, enhancing the role of other CNS loci.

Orexin fibers form appositions with Fos expressing neuropeptide-Y cells in the grass rat intergeniculate leaflet

Neuropeptide-Y (NPY) cells in the intergeniculate leaflet (IGL) are known to modulate effects of arousal on the mammalian circadian system. However, the route through which this information reaches the IGL has not been established. Here, we provide evidence that the orexins (hypocretins) are uniquely positioned as a potential source of activity state feedback to the IGL in the grass rat, Arvicanthis niloticus. Specifically, many NPY cells in the grass rat IGL exhibit orexin-A (OXA) fiber appositions. Furthermore, NPY cells contacted by OXA fibers are significantly more likely to express Fos during nocturnal wheel running than are NPY cells without such contacts ( P < 0.001).

Changes in Expression of the Genes for the Leptin Receptor and the Growth Hormone‐Releasing Peptide/Ghrelin Receptor in the Hypothalamic Arcuate Nucleus with Long‐Term Manipulation of Adiposity by Dietary Means

Changes in leptin and ghrelin levels occur with alterations in adiposity, but signalling may be affected by levels of the relevant receptors. We measured expression of the leptin receptor (Ob-Rb) and the ghrelin/growth hormone releasing peptide receptor (GHS-R) in the arcuate nucleus of sheep held at either high or low levels of adiposity. Plasma growth hormone (GH) levels were lower in Fat animals and higher in Lean animals. Plasma insulin and leptin levels were higher in Fat animals and lower in Lean animals. Frozen hypothalamic sections of arcuate nucleus were extracted and mRNA levels measured for mRNA for Ob-Rb and GHS-R. Gene expression for both Ob-Rb and GHS-R was higher in Lean animals than in Fat animals, with no difference in expression between Fat and Normal animals. A second group of animals (n = 4 per group) was used for double-labelling immunohistochemistry to determine whether the increase in Ob-Rb gene expression was translated into Ob-Rb protein and to ascertain whether this effect is localised to the cells of the arcuate nucleus that produce either neuropeptide Y (NPY) and/or pro-opiomelanocortin-derived peptides. Lean animals displayed a 255% increase in immunoreactive NPY cells (P < 0.005), a 167% increase in cells with Ob-Rb (P < 0.037) protein and a 344% increase in cells that were staining for both NPY and Ob-Rb (P < 0.02). There was no difference between the Normal and Lean animals in the number of cells that were detected with an adrenocorticotrophic hormone (ACTH) antibody or the number of ACTH-immunoreactive cells that also stained for Ob-Rb. Finally, we measured plasma ghrelin levels in Normal, Fat and Lean ewes (n = 4/group); levels were higher (P < 0.05) in Fat animals than in Lean animals. We conclude that lowering body weight leads to increased expression of Ob-Rb, ghrelin/GHS-R expression and proportion of NPY cells that express Ob-Rb in the arcuate nucleus. This may be an adaptive mechanism to increase responsivity to both leptin and ghrelin.