W. John Sheward

The VPAC2 Receptor Is Essential for Circadian Function in the Mouse Suprachiasmatic Nuclei

The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are implicated in the photic entrainment of circadian rhythms in the suprachiasmatic nuclei (SCN). We now report that mice carrying a null mutation of the VPAC(2) receptor for VIP and PACAP (Vipr2(-/-)) are incapable of sustaining normal circadian rhythms of rest/activity behavior. These mice also fail to exhibit circadian expression of the core clock genes mPer1, mPer2, and mCry1 and the clock-controlled gene arginine vasopressin (AVP) in the SCN. Moreover, the mutants fail to show acute induction of mPer1 and mPer2 by nocturnal illumination. This study highlights the role of intercellular neuropeptidergic signaling in maintenance of circadian function within the SCN.

The mouse VPAC2 receptor confers suprachiasmatic nuclei cellular rhythmicity and responsiveness to vasoactive intestinal polypeptide in vitro

Expression of coherent and rhythmic circadian (≈ 24 h) variation of behaviour, metabolism and other physiological processes in mammals is governed by a dominant biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). Photic entrainment of the SCN circadian clock is mediated, in part, by vasoactive intestinal polypeptide (VIP) acting through the VPAC2 receptor. Here we used mice lacking the VPAC2 receptor (Vipr2−/−) to examine the contribution of this receptor to the electrophysiological actions of VIP on SCN neurons, and to the generation of SCN electrical firing rate rhythms SCN in vitro. Compared with wild‐type controls, fewer SCN cells from Vipr2−/− mice responded to VIP and the VPAC2 receptor‐selective agonist Ro 25‐1553. By contrast, similar proportions of Vipr2−/− and wild‐type SCN cells responded to gastrin‐releasing peptide, arginine vasopressin or N‐methyl‐d‐aspartate. Moreover, VIP‐evoked responses from control SCN neurons were attenuated by the selective VPAC2 receptor antagonist PG 99‐465. In firing rate rhythm experiments, the midday peak in activity observed in control SCN cells was lost in Vipr2−/− mice. The loss of electrical activity rhythm in Vipr2−/− mice was mimicked in control SCN slices by chronic treatment with PG 99‐465. These results demonstrate that the VPAC2 receptor is necessary for the major part of the electrophysiological actions of VIP on SCN cells in vitro, and is of fundamental importance for the rhythmic and coherent expression of circadian rhythms governed by the SCN clock. These findings suggest a novel role of VPAC2 receptor signalling, and of cell‐to‐cell communication in general, in the maintenance of core clock function in mammals, impacting on the cellular physiology of SCN neurons.

Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a gene associated with pulmonary vascular disease

Heightened expression of the S100 calcium–binding protein, S100A4/Mts1, is observed in pulmonary vascular disease. Loss of serotonin (5-hydroxytryptamine [5-HT]) receptors or of the serotonin transporter (SERT) attenuates pulmonary hypertension in animals, and polymorphisms causing gain of SERT function are linked to clinical pulmonary vascular disease. Because 5-HT induces release of S100&bgr;, we investigated the codependence of 5-HT receptors and SERT in regulating S100A4/Mts1 in human pulmonary artery smooth muscle cells (hPA-SMC). 5-HT elevated S100A4/Mts1 mRNA levels and increased S100A4/Mts1 protein in hPA-SMC lysates and culture media. S100A4/Mts1 in the culture media stimulated proliferation and migration of hPA-SMC in a manner dependent on the receptor for advanced glycation end products. Treatment with SB224289 (selective antagonist of 5-HT1B), fluoxetine (SERT inhibitor), SERT RNA-interference, and iproniazid (monoamine oxidase-A inhibitor), blocked 5-HT–induced S100A4/Mts1. 5-HT signaling mediated phosphorylation (p) of extracellular signal–regulated kinase 1/2 (pERK1/2), but pERK1/2 nuclear translocation depended on SERT, monoamine oxidase activity, and reactive oxygen species. Nuclear translocation of pERK1/2 was required for pGATA-4–mediated transcription of S100A4/Mts1. These data provide evidence for a mechanistic link between the 5-HT pathway and S100A4/Mts1 in pulmonary hypertension and explain how the 5-HT1B receptor and SERT are codependent in regulating S100A4/Mts1.

Increased Expression of the 5-HT Transporter Confers a Low- Anxiety Phenotype Linked to Decreased 5-HT Transmission

A commonly occurring polymorphic variant of the human 5-hydroxytryptamine (5-HT) transporter (5-HTT) gene that increases 5-HTT expression has been associated with reduced anxiety levels in human volunteer and patient populations. However, it is not known whether this linkage between genotype and anxiety relates to variation in 5-HTT expression and consequent changes in 5-HT transmission. Here we test this hypothesis by measuring the neurochemical and behavioral characteristics of a mouse genetically engineered to overexpress the 5-HTT. Transgenic mice overexpressing the human 5-HTT (h5-HTT) were produced from a 500 kb yeast artificial chromosome construct. These transgenic mice showed the presence of h5-HTT mRNA in the midbrain raphe nuclei, as well as a twofold to threefold increase in 5-HTT binding sites in the raphe nuclei and a range of forebrain regions. The transgenic mice had reduced regional brain whole-tissue levels of 5-HT and, in microdialysis experiments, decreased brain extracellular 5-HT, which reversed on administration of the 5-HTT inhibitor paroxetine. Compared with wild-type mice, the transgenic mice exhibited a low-anxiety phenotype in plus maze and hyponeophagia tests. Furthermore, in the plus maze test, the low-anxiety phenotype of the transgenic mice was reversed by acute administration of paroxetine, suggesting a direct link between the behavior, 5-HTT overexpression, and low extracellular 5-HT. In toto, these findings demonstrate that associations between increased 5-HTT expression and anxiety can be modeled in mice and may be specifically mediated by decreases in 5-HT transmission.

Entrainment to Feeding but Not to Light: Circadian Phenotype of VPAC2 Receptor-Null Mice

The master clock driving mammalian circadian rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and entrained by daily light/dark cycles. SCN lesions abolish circadian rhythms of behavior and result in a loss of synchronized circadian rhythms of clock gene expression in peripheral organs (e.g., the liver) and of hormone secretion (e.g., corticosterone). We examined rhythms of behavior, hepatic clock gene expression, and corticosterone secretion in VPAC2 receptor-null (Vipr2−/−) mice, which lack a functional SCN clock. Unexpectedly, although Vipr2−/− mice lacked robust circadian rhythms of wheel-running activity and corticosterone secretion, hepatic clock gene expression was strongly rhythmic, but advanced in phase compared with that in wild-type mice. The timing of food availability is thought to be an important entrainment signal for circadian clocks outside the SCN. Vipr2−/− mice consumed food significantly earlier in the 24 h cycle than wild-type mice, consistent with the observed timing of peripheral rhythms of circadian gene expression. When restricted to feeding only during the daytime (RF), mice develop rhythms of activity and of corticosterone secretion in anticipation of feeding time, thought to be driven by a food-entrainable circadian oscillator, located outside the SCN. Under RF, mice of both genotypes developed food-anticipatory rhythms of activity and corticosterone secretion, and hepatic gene expression rhythms also became synchronized to the RF stimulus. Thus, food intake is an effective zeitgeber capable of coordinating circadian rhythms of behavior, peripheral clock gene expression, and hormone secretion, even in the absence of a functional SCN clock.

Endopeptidase EC 3.4.24.15 Presence in the Rat Median Eminence and Hypophysial Portal Blood and its Modulation of the Luteinizing Hormone Surge

The endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is widely distributed in a variety of tissues, including the testes, pituitary and the central nervous system. Among its numerous roles in metabolizing and processing biologically‐active peptides, the enzyme degrades gonadotropin‐releasing hormone (GnRH) by cleaving the central Tyr5‐Gly6 bond. The aim of the present studies was to determine whether EP24.15 can modulate the concentrations of GnRH within the hypothalamo‐hypophysial portal blood and thereby play a physiological role in reproduction. Our data suggest the presence of immunoreactive EP24.15 in the perivascular space of the median eminence and that this enzyme is secreted into portal blood. We have also shown a physiological role for this enzyme in that an inhibition of its activity with a specific inhibitor augmented the steroid‐induced LH increase in ovariectomized rats. The present results suggest that secretory and post‐secretory mechanisms are important in shaping the GnRH signal from the central nervous system; GnRH metabolism by EP24.15 may be one such mechanism.

Circadian control of mouse heart rate and blood pressure by the suprachiasmatic nuclei: behavioral effects are more significant than direct outputs

Background Diurnal variations in the incidence of events such as heart attack and stroke suggest a role for circadian rhythms in the etiology of cardiovascular disease. The aim of this study was to assess the influence of the suprachiasmatic nucleus (SCN) circadian clock on cardiovascular function. Methodology/Principal Findings Heart rate (HR), blood pressure (BP) and locomotor activity (LA) were measured in circadian mutant (Vipr2 −/−) mice and wild type littermates, using implanted radio-telemetry devices. Sleep and wakefulness were studied in similar mice implanted with electroencephalograph (EEG) electrodes. There was less diurnal variation in the frequency and duration of bouts of rest/activity and sleep/wake in Vipr2 −/− mice than in wild type (WT) and short “ultradian” episodes of arousal were more prominent, especially in constant conditions (DD). Activity was an important determinant of circadian variation in BP and HR in animals of both genotypes; altered timing of episodes of activity and rest (as well as sleep and wakefulness) across the day accounted for most of the difference between Vipr2 −/− mice and WT. However, there was also a modest circadian rhythm of resting HR and BP that was independent of LA. Conclusions/Significance If appropriate methods of analysis are used that take into account sleep and locomotor activity level, mice are a good model for understanding the contribution of circadian timing to cardiovascular function. Future studies of the influence of sleep and wakefulness on cardiovascular physiology may help to explain accumulating evidence linking disrupted sleep with cardiovascular disease in man.

Transgenic approach reveals expression of the VPAC2 receptor in phenotypically defined neurons in the mouse suprachiasmatic nucleus and in its efferent target sites.

Circadian rhythms in mammals depend on the properties of cells in the suprachiasmatic nucleus (SCN). The retino‐recipient core of the mouse SCN is characterized by vasoactive intestinal peptide (VIP) neurons. Expression within the SCN of VPAC2, a VIP receptor, is required for circadian rhythmicity. Using transgenic mice with β‐galactosidase as a marker for VPAC2, we have phenotyped VPAC2‐expressing cells within the SCN and investigated expression of the VPAC2 marker at sites previously shown to receive VIP‐containing SCN efferents. In situ hybridization and immunohistochemistry demonstrated identical distributions for VPAC2 mRNA and β‐galactosidase and coexpression of the two signals in the SCN. Double‐label confocal immunofluorescence identified β‐galactosidase in 32% of the VIP and 31% of the calretinin neurons in the SCN core. Of the arginine‐vasopressin neurons that characterize the SCN shell, 45% expressed β‐galactosidase. In contrast, this marker was not apparent in astrocytes within the SCN core or shell. Cell bodies containing β‐galactosidase were detected at sites reportedly receiving VIP‐containing SCN efferents, including the subparaventricular zone and lateral septum and the anteroventral periventricular, preoptic suprachiasmatic, medial preoptic and paraventricular hypothalamic nuclei. The detection of a marker for VPAC2 expression in the SCN in almost one‐third of the VIP and calretinin core neurons and nearly half of the arginine‐vasopressin shell neurons and also in cell bodies at sites receiving VIP‐immunoreactive projections from the SCN indicates that VPAC2 may contribute to autoregulation and/or coupling within the SCN core and to the control of the SCN shell and sites distal to this nucleus.

Atrial Natriuretic Factor is Released into Hypophysial Portal Blood: Direct Evidence that Atrial Natriuretic Factor may be a Neurohormone Involved in Hypothalamic Pituitary Control.

Atrial natriuretic factor (ANF) is produced in atriomyocytes (1, 2), released as a 28 amino‐acid peptide into the systemic circulation and probably plays an important role in fluid and electrolyte balance (3, 4). The facts that immunoreactive ANF (ir‐ANF) is also present in nerve terminals in the external layer of the median eminence (ME) (5), ir‐ANF can be released from the hypothalamus in vitro by potassium depolarization (6) and specific ANF binding sites are present at a high concentration in rat pituitary tissue (7) suggest that ANF may be involved in hypothalamic‐pituitary regulation. We report here that ir‐ANF concentrations in hypophysial portal blood are about two to four times greater than in peripheral plasma from hypophysectomized as well as pituitary‐intact adult female rats. These results show for the first time that ir‐ANF is secreted from the hypothalamus into the hypophysial portal circulation at a concentration (≈10−9 M) consistent with a role for ANF as a hypothalamic‐pituitary regulator or modulator.

The neurotransmitter VIP expands the pool of symmetrically dividing postnatal dentate gyrus precursors via VPAC2 receptors or directs them toward a neuronal fate via VPAC1 receptors

The controlled production of neurons in the postnatal dentate gyrus and thoughout life is important for hippocampal learning and memory. The mechanisms underlying the necessary coupling of neuronal activity to neural stem/progenitor cell (NSPC) function remain poorly understood. Within the dentate subgranular stem cell niche, local interneurons appear to play an important part in this excitation‐neurogenesis coupling via GABAergic transmission, which promotes neuronal differentiation and integration. Here we show that vasoactive intestinal polypeptide, a neuropeptide coreleased with GABA under specific firing conditions, is uniquely trophic for proliferating postnatal nestin‐positive dentate NSPCs, mediated via the VPAC2 receptor. We also show that VPAC2 receptor activation shifts the fate of symmetrically dividing NSPCs toward a nestin‐only phenotype, independent of the trophic effect. In contrast, selective VPAC1 receptor activation shifts NSPC fate toward granule cell neurogenesis without any trophism. We confirm a trophic role for VPAC2 receptors in vivo, showing reduced progeny survival and dentate neurogenesis in adult Vipr2−/− mice. We also show a specific reduction in type 2 nestin‐positive precursors in vivo, consistent with a role for VPAC2 in maintaining this cell population. This work provides the first evidence of differential fate modulation of neurogenesis by neurotransmitter receptor subtypes and extends the fate‐determining effects of neurotransmitters to maintaining the nestin‐positive pool of NSPCs. This differential receptor effect may support the independent pharmacological manipulation of precursor pool expansion and neurogenic instruction for therapeutic application in the treatment of cognitive deficits associated with a decline in neurogenesis. STEM CELLS 2009;27:2539–2551