Mesolimbic neuropeptide W coordinates stress responses under novel environments.

Neuropeptide B (NPB) and neuropeptide W (NPW) are endogenous neuropeptide ligands for the G protein-coupled receptors NPBWR1 and NPBWR2. Here we report that the majority of NPW neurons in the mesolimbic region possess tyrosine hydroxylase immunoreactivity, indicating that a small subset of dopaminergic neurons coexpress NPW. These NPW-containing neurons densely and exclusively innervate two limbic system nuclei in adult mouse brain: the lateral bed nucleus of the stria terminalis and the lateral part of the central amygdala nucleus (CeAL). In the CeAL of wild-type mice, restraint stress resulted in an inhibition of cellular activity, but this stress-induced inhibition was attenuated in the CeAL neurons of NPW(-/-) mice. Moreover, the response of NPW(-/-) mice to either formalin-induced pain stimuli or a live rat (i.e., a potential predator) was abnormal only when they were placed in a novel environment: The mice failed to show the normal species-specific self-protective and aversive reactions. In contrast, the behavior of NPW(-/-) mice in a habituated environment was indistinguishable from that of wild-type mice. These results indicate that the NPW/NPBWR1 system could play a critical role in the gating of stressful stimuli during exposure to novel environments.

Loss of Goosecoid-like and DiGeorge syndrome critical region 14 in interpeduncular nucleus results in altered regulation of rapid eye movement sleep.

Sleep and wakefulness are regulated primarily by inhibitory interactions between the hypothalamus and brainstem. The expression of the states of rapid eye movement (REM) sleep and non-REM (NREM) sleep also are correlated with the activity of groups of REM-off and REM-on neurons in the dorsal brainstem. However, the contribution of ventral brainstem nuclei to sleep regulation has been little characterized to date. Here we examined sleep and wakefulness in mice deficient in a homeobox transcription factor, Goosecoid-like (Gscl), which is one of the genes deleted in DiGeorge syndrome or 22q11 deletion syndrome. The expression of Gscl is restricted to the interpeduncular nucleus (IP) in the ventral region of the midbrain-hindbrain transition. The IP has reciprocal connections with several cell groups implicated in sleep/wakefulness regulation. Although Gscl(-/-) mice have apparently normal anatomy and connections of the IP, they exhibited a reduced total time spent in REM sleep and fewer REM sleep episodes. In addition, Gscl(-/-) mice showed reduced theta power during REM sleep and increased arousability during REM sleep. Gscl(-/-) mice also lacked the expression of DiGeorge syndrome critical region 14 (Dgcr14) in the IP. These results indicate that the absence of Gscl and Dgcr14 in the IP results in altered regulation of REM sleep.

Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41.

The distal human intestine harbors trillions of microbes that allow us to extract calories from otherwise indigestible dietary polysaccharides. The products of polysaccharide fermentation include short-chain fatty acids that are ligands for Gpr41, a G protein-coupled receptor expressed by a subset of enteroendocrine cells in the gut epithelium. To examine the contribution of Gpr41 to energy balance, we compared Gpr41-/- and Gpr41+/+ mice that were either conventionally-raised with a complete gut microbiota or were reared germ-free and then cocolonized as young adults with two prominent members of the human distal gut microbial community: the saccharolytic bacterium, Bacteroides thetaiotaomicron and the methanogenic archaeon, Methanobrevibacter smithii. Both conventionally-raised and gnotobiotic Gpr41-/- mice colonized with the model fermentative community are significantly leaner and weigh less than their WT (+/+) littermates, despite similar levels of chow consumption. These differences are not evident when germ-free WT and germ-free Gpr41 knockout animals are compared. Functional genomic, biochemical, and physiologic studies of germ-free and cocolonized Gpr41-/- and +/+ littermates disclosed that Gpr41-deficiency is associated with reduced expression of PYY, an enteroendocrine cell-derived hormone that normally inhibits gut motility, increased intestinal transit rate, and reduced harvest of energy (short-chain fatty acids) from the diet. These results reveal that Gpr41 is a regulator of host energy balance through effects that are dependent upon the gut microbiota.

Distinct narcolepsy syndromes in Orexin receptor-2 and Orexin null mice: molecular genetic dissection of Non-REM and REM sleep regulatory processes.

Narcolepsy-cataplexy, a neurological disorder associated with the absence of hypothalamic orexin (hypocretin) neuropeptides, consists of two underlying problems: inability to maintain wakefulness and intrusion of rapid eye movement (REM) sleep into wakefulness. Here we document, using behavioral, electrophysiological, and pharmacological criteria, two distinct classes of behavioral arrests exhibited by mice deficient in orexin-mediated signaling. Both OX2R(-/-) and orexin(-/-) mice are similarly affected with behaviorally abnormal attacks of non-REM sleep ("sleep attacks") and show similar degrees of disrupted wakefulness. In contrast, OX2R(-/-) mice are only mildly affected with cataplexy-like attacks of REM sleep, whereas orexin(-/-) mice are severely affected. Absence of OX2Rs eliminates orexin-evoked excitation of histaminergic neurons in the hypothalamus, which gate non-REM sleep onset. While normal regulation of wake/non-REM sleep transitions depends critically upon OX2R activation, the profound dysregulation of REM sleep control unique to the narcolepsy-cataplexy syndrome emerges from loss of signaling through both OX2R-dependent and OX2R-independent pathways.

Distribution of neuropeptide W immunoreactivity and mRNA in adult rat brain.

Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor (GPR) 7, which is highly expressed in several discrete regions of the rodent brain including the central amygdaloid nucleus and bed nucleus of the stria terminalis. Although several reports suggested that NPW is implicated in the regulation of energy homeostasis and nociception, the precise physiological role of NPW has remained unclear. In this study, we examined distribution of NPW messenger RNA and NPW immunoreactivity in the adult rat brain. NPW-immunoreactive (ir) cells were detected in the ventral tegmental area, periaqueductal gray, and Edinger-Westphal nucleus. NPW-ir fibers were observed in several brain regions, including the lateral septum, bed nucleus of the stria terminalis, dorsomedial and posterior hypothalamus, central amygdaloid nucleus, CA1 field of hippocampus, interpeduncular nucleus, inferior colliculus, lateral parabrachial nucleus, facial nucleus, and hypoglossal nucleus. NPW-ir fibers were most abundantly observed in the central amygdaloid nucleus and the bed nucleus of the stria terminalis, which are regions implicated in fear and anxiety. These results suggest that NPW might be involved in the regulation of stress and emotive responses, especially in fear and anxiety-related physiological and behavioral functions.

Orexin neurons function in an efferent pathway of a food-entrainable circadian oscillator in eliciting food-anticipatory activity and wakefulness.

Temporal restriction of feeding can entrain circadian behavioral and physiological rhythms in mammals. Considering the critical functions of the hypothalamic orexin (hypocretin) neuropeptides in promoting wakefulness and locomotor activity, we examined the role of orexin neurons in the adaptation to restricted feeding. In orexin neuron-ablated transgenic mice, the food-entrained rhythmicity of mPer2 expression in the brain and liver, the reversal of the sleep-wake cycle, and the recovery of daily food intake were unaltered compared with wild-type littermates. In contrast, orexin neuron-ablated mice had a severe deficit in displaying the normal food-anticipatory increases in wakefulness and locomotor activity under restricted feeding. Moreover, activity of orexin neurons markedly increased during the food-anticipatory period under restricted feeding in wild-type mice. Orexin neurons thus convey an efferent signal from putative food-entrainable oscillator or oscillators to increase wakefulness and locomotor activity.

Expression of a poly-glutamine-ataxin-3 transgene in orexin neurons induces narcolepsy-cataplexy in the rat.

The sleep disorder narcolepsy has been linked to loss of hypothalamic neurons producing the orexin (hypocretin) neuropeptides. Here, we report the generation of transgenic rats expressing a human ataxin-3 fragment with an elongated polyglutamyl stretch under control of the human prepro-orexin promoter (orexin/ataxin-3 rats). At 17 weeks of age, the transgenic rats exhibited postnatal loss of orexin-positive neurons in the lateral hypothalamus, and orexin-containing projections were essentially undetectable. The loss of orexin production resulted in the expression of a phenotype with fragmented vigilance states, a decreased latency to rapid eye movement (REM) sleep and increased REM sleep time during the dark active phase. Wakefulness time was also reduced during the dark phase, and this effect was concentrated at the photoperiod boundaries. Direct transitions from wakefulness to REM sleep, a defining characteristic of narcolepsy, occurred frequently. Brief episodes of muscle atonia and postural collapse resembling cataplexy were also noted while rats maintained the electroencephalographic characteristics of wakefulness. These findings indicate that the orexin/ataxin-3 transgenic rat could provide a useful model of human narcolepsy.

Neuroglobin, a novel member of the globin family, is expressed in focal regions of the brain.

Hemoproteins are widely distributed among unicellular eukaryotes, plants, and animals. In addition to myoglobin and hemoglobin, a third hemoprotein, neuroglobin, has recently been isolated from vertebrate brain. Although the functional role of this novel member of the globin family remains unclear, neuroglobin contains a heme-binding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. In this study we utilized in situ hybridization (ISH) and RT-PCR analyses to examine the expression of neuroglobin in the normoxic and hypoxic murine brain. In the normoxic adult mouse, neuroglobin expression was observed in focal regions of the brain, including the lateral tegmental nuclei, the preoptic nucleus, amygdala, locus coeruleus, and nucleus of the solitary tract. Using ISH and RT-PCR techniques, no significant changes in neuroglobin expression in the adult murine brain was observed in response to chronic 10% oxygen. These results support the hypothesis that neuroglobin is a hemoprotein that is expressed in the brain and may have diverse functional roles.

Low blood pressure in endothelial cell-specific endothelin 1 knockout mice.

Endothelin (ET) 1 is a potent vasoconstrictor peptide produced by vascular endothelial cells and implicated in various pathophysiologic states involving abnormal vascular tone. Homozygous ET-1 null mice have craniofacial and cardiac malformations that lead to neonatal death. To study the role of ET-1 in adult vascular physiology, we generated a mouse strain (ET-1(flox/flox);Tie2-Cre mice) in which ET-1 is disrupted specifically in endothelial cells. ET-1 peptide levels in plasma, heart, lung, kidney, and brain homogenates were reduced by 65% to 80% in these mice. mRNA levels for ET receptors were unaltered except that the ET(A) receptor mRNA was upregulated in the heart. ET-1(flox/flox);Tie2-Cre mice had mean blood pressures 10 to 12 mm Hg lower than genetic controls. In contrast, the blood pressure of mice systemically heterozygous for the ET-1 null allele (ET-1(dlox/+) mice) was unchanged compared with wild-type littermates. Despite the lower basal blood pressure, acute pharmacological responses to angiotensin II, captopril, phenylephrine, bradykinin, N(G)-nitro-L-arginine methyl ester, and exogenous ET-1 were normal in ET-1(flox/flox);Tie2-Cre mice. These results support an essential role of endothelial-derived ET-1 in the maintenance of basal vascular tone and blood pressure. Normal pharmacological responses of ET-1(flox/flox);Tie2-Cre mice suggest that the renin-angiotensin system, the adrenergic system, and NO are not significantly altered by endothelial ET-1. Taken in conjunction with other lines of genetically altered mice, our results provide evidence for a paracrine vasoregulatory pathway mediated by endothelial cell-derived ET-1 acting on the vascular smooth muscle ET(A) receptor.

Enhanced orexin receptor-2 signaling prevents diet-induced obesity and improves leptin sensitivity.

The hypothalamic orexin neuropeptide acutely promotes appetite, yet orexin deficiency in humans and mice is associated with obesity. Prolonged effects of increased orexin signaling upon energy homeostasis have not been fully characterized. Here, we examine the metabolic effects of orexin gain of function utilizing genetic and pharmacologic techniques in mice. Transgenic orexin overexpression confers resistance to high-fat diet-induced obesity and insulin insensitivity by promoting energy expenditure and reducing consumption. Genetic studies indicate that orexin receptor-2 (OX2R), rather than OX1R signaling, predominantly mediates this phenotype. Likewise, prolonged central administration of an OX2R-selective peptide agonist inhibits diet-induced obesity. While orexin overexpression enhances the anorectic-catabolic effects of central leptin administration, obese leptin-deficient mice are completely resistant to the metabolic effects of orexin overexpression or OX2R agonist infusion. We conclude that enhanced orexin-OX2R signaling confers resistance to diet-induced features of the metabolic syndrome through negative energy homeostasis and improved leptin sensitivity.

The dorsomedial hypothalamic nucleus as a putative food-entrainable circadian pacemaker.

Temporal restriction of feeding can phase-shift behavioral and physiological circadian rhythms in mammals. These changes in biological rhythms are postulated to be brought about by a food-entrainable oscillator (FEO) that is independent of the suprachiasmatic nucleus. However, the neural substrates of FEO have remained elusive. Here, we carried out an unbiased search for mouse brain region(s) that exhibit a rhythmic expression of the Period genes in a feeding-entrainable manner. We found that the compact part of the dorsomedial hypothalamic nucleus (DMH) demonstrates a robust oscillation of mPer expression only under restricted feeding. The oscillation persisted for at least 2 days even when mice were given no food during the expected feeding period after the establishment of food-entrained behavioral rhythms. Moreover, refeeding after fasting rapidly induced a transient mPer expression in the same area of DMH. Taken in conjunction with recent findings (i) that behavioral expression of food-entrainable circadian rhythms is blocked by cell-specific lesions of DMH in rats and (ii) that DMH neurons directly project to orexin neurons in the lateral hypothalamus, which are essential for proper expression of food-entrained behavioral rhythms, the present study suggests that DMH plays a key role as a central FEO in the feeding-mediated regulation of circadian behaviors.

A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice.

Here, we report the isolation and characterization of an endogenous peptide ligand of GPR103 from rat brains. The purified peptide was found to be the 43-residue RF-amide peptide QRFP. We also describe two mouse homologues of human GPR103, termed mouse GPR103A and GPR103B. QRFP binds and activates the human GPR103, as well as mouse GPR103A and GPR103B, with nanomolar affinities in transfected cells. Systematic in situ hybridization analysis in mouse brains showed that QRFP is expressed exclusively in the periventricular and lateral hypothalamus, whereas the two receptor mRNAs are distinctly localized in various brain areas without an overlap to each other. When administered centrally in mice, QRFP induced feeding behavior, accompanied by increased general locomotor activity and metabolic rate. QRFP-induced food intake was abolished by preadministration of BIBP3226, a specific antagonist for the Y1 neuropeptide Y receptor. Hypothalamic prepro-QRFP mRNA expression was up-regulated upon fasting and in genetically obese ob/ob and db/db mice. Central QRFP administration also evoked highly sustained elevation of blood pressure and heart rate. Our findings suggest that QRFP and GPR103A/B may regulate diverse neuroendocrine and behavioral functions and implicate this neuropeptide system in metabolic syndrome.

Neuropeptide B-deficient mice demonstrate hyperalgesia in response to inflammatory pain.

Neuropeptide B (NPB) and neuropeptide W (NPW) have been recently identified as ligands for the G protein-coupled receptor (GPR) 7 and GPR8. The precise in vivo role of this neuropeptide-receptor pathway has not been fully demonstrated. In this paper, we report that NPB-deficient mice manifest a mild adult-onset obesity, similar to that reported in GPR7-null mice. NPB-deficient mice also exhibit hyperalgesia in response to inflammatory pain. Hyperalgesia was not observed in response to chemical pain, thermal pain, or electrical stimulation. NPB-deficient mice demonstrated intact behavioral responses to pain, and learning from the negative reinforcement of electrical stimulation was unaltered. Baseline anxiety was also unchanged as measured in both the elevated plus maze and time spent immobile in a novel environment. These data support the idea that NPB is a factor in the modulation of responses to inflammatory pain and body weight homeostasis.

Extrarenal ETB plays a significant role in controlling cardiovascular responses to high dietary sodium in rats.

Endothelin-B receptor (ET(B))-deficient rats have low-renin, salt-sensitive hypertension. We hypothesized this was caused by an absence of renal ET(B) signaling and performed a series of experiments to examine the effect of dietary sodium (Na) on endothelin-1 (ET1) expression and renal function in wild-type (WT) and ET(B)-deficient rats. We found that ET(B) deficiency, but not dietary Na, increases circulating and tissue (kidney and aorta) ET1 levels. Quantitative reverse-transcription polymerase chain reaction reveals that aortic and renal ET1 and endothelin-A receptor (ET(A)) mRNA, however, are similarly increased by dietary Na in ET(B)-WT and ET(B)-deficient rats. We then determined the effect of chronic ET(A) blockade on blood pressure (direct conscious measurements), urinary protein excretion, and creatinine clearance (Crcl). On a Na-deficient diet, ET(B)-deficient rats have mild proteinuria and impaired Crcl. On a high-Na diet, severe hypertension and renal dysfunction develop in ET(B)-deficient rats. Chronic ET(A) blockade prevents hypertension and renal injury. To determine the role of the renal versus the extrarenal endothelin system, we performed renal cross-transplantation. We found that ET(B) deficiency in the body is associated with renal injury and an impaired ability to excrete an Na load. We also found that ET(B) deficiency in the body affects blood pressure response to dietary Na. Expression of ET1 and ET(A) are regulated by dietary Na. ET(B) receptors outside of the kidney, likely by functioning as a clearance receptor for ET1, limit salt-sensitivity in rats.

Cell-autonomous and nonautonomous actions of endothelin-A receptor signaling in craniofacial and cardiovascular development.

Craniofacial and cardiac development relies on the proper patterning of the neural crest-derived ectomesenchyme of the pharyngeal arches, from which many craniofacial and great vessel structures arise. One of the intercellular signaling molecules that is involved in this process, endothelin-1 (ET-1), is expressed in the arch epithelium and influences arch development by binding to its cognate receptor, the endothelin A (ET(A)) receptor, found on ectomesenchymal cells. We have previously shown that absence of ET(A) signaling in ET(A)(-/-) mouse embryos disrupts neural crest cell development, resulting in craniofacial and cardiovascular defects similar in many aspects to those in mouse models of DiGeorge syndrome. These changes may reflect a cell-autonomous requirement for ET(A) signaling during crest cell development because the ET(A) receptor is an intracellular signaling molecule. However, it is also possible that some of the observed defects in ET(A)(-/-) embryos could arise from the absence of downstream signaling that act in a non-cell-autonomous manner. To address this question, we performed chimera analysis using ET(A)(-/-) embryonic stem cells. We observe that, in almost all early ET(A)(-/-) --> (+/+) chimeric embryos, ET(A)(-/-) cells are excluded from the caudoventral aspects of the pharyngeal arches, suggesting a cell-autonomous role for ET(A) signaling in crest cell migration and/or colonization. Interestingly, in the few embryos in which mutant cells do reach the ventral arch, structures derived from this area are either composed solely of wild type cells or are missing, suggesting a second cell-autonomous role for ET(A) signaling in postmigratory crest cell differentiation. In the cardiac outflow tract and great vessels, ET(A)(-/-) cells are excluded from the walls of the developing pharyngeal arch arteries, indicating that ET(A) signaling also acts cell-autonomously during cardiac neural crest cell development.

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8.

GPR7 and GPR8 are orphan G protein-coupled receptors that are highly similar to each other. These receptors are expressed predominantly in brain, suggesting roles in central nervous system function. We have purified an endogenous peptide ligand for GPR7 from bovine hypothalamus extracts. This peptide, termed neuropeptide B (NPB), has a C-6-brominated tryptophan residue at the N terminus. It binds and activates human GPR7 or GPR8 with median effective concentrations (EC(50)) of 0.23 nM and 15.8 nM, respectively. In situ hybridization shows distinct localizations of the prepro-NPB mRNA in mouse brain, i.e., in paraventricular hypothalamic nucleus, hippocampus, and several nuclei in midbrain and brainstem. Intracerebroventricular (i.c.v.) injection of NPB in mice induces hyperphagia during the first 2 h, followed by hypophagia. Intracerebroventricular injection of NPB produces analgesia to s.c. formalin injection in rats. Through EST database searches, we identified a putative paralogous peptide. This peptide, termed neuropeptide W (NPW), also has an N-terminal tryptophan residue. Synthetic human NPW binds and activates human GPR7 or GPR8 with EC(50) values of 0.56 nM and 0.51 nM, respectively. The expression of NPW mRNA in mouse brain is confined to specific nuclei in midbrain and brainstem. These findings suggest diverse physiological functions of NPB and NPW in the central nervous system, acting as endogenous ligands on GPR7 andor GPR8.