Vasoactive Intestinal Polypeptide Signaling Research Articles

Vasopressin Resets the Central Circadian Clock in a Manner Influenced by Sex and Vasoactive Intestinal Polypeptide Signaling

Background/Aims: Circadian rhythms in behavior and physiology are programmed by the suprachiasmatic nucleus (SCN) of the hypothalamus. A subset of SCN neurons produce the neuropeptide arginine vasopressin (AVP), but it remains unclear whether AVP signaling influences the SCN clock directly. Methods: Here, we test that AVP signaling acting through V1A and V1B receptors influences molecular rhythms in SCN neurons. V1 receptor agonists were applied ex vivo to PERIOD2::LUCIFERASE SCN slices, allowing for real-time monitoring of changes in molecular clock function. Results: V1A/B agonists reset the phase of the SCN molecular clock in a time-dependent manner, with larger magnitude responses by the female SCN. Further, we found evidence that both G_αq and G_αs signaling pathways interact with V1A/B-induced SCN resetting, and that this response requires vasoactive intestinal polypeptide (VIP) signaling. Conclusions: Collectively, this work indicates that AVP signaling resets SCN molecular rhythms in conjunction with VIP signaling and in a manner influenced by sex. This highlights the utility of studying clock function in both sexes and suggests that signal integration in central clock circuits regulates emergent properties important for the control of daily rhythms in behavior and physiology.

Absence of Vasoactive Intestinal Peptide Synthesis in Recipients in Allogeneic Bone Marrow Transplant Increases Graft Versus Host Diseases

Introduction: Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide. Activation of donor T cells leading to graft-versus-host disease (GvHD) in allogeneic bone marrow transplant (allo-BMT) occurs via early post transplant interactions between donor T cells and host antigen presenting cells and leads to upregulation of immune co-inhibitory pathways, including expression of VIP in T cells and dendritic cells. We have previously shown VIP signaling favorably regulates the activation status and enhances the graft-versus-leukemia (GvL) activities of donor T cells (Li et al., Cancer Research 2016). The role of the VIP produced by host antigen presenting cells in regulating GvHD in allo-BMT is unknown. We tested the hypothesis that absence of VIP expression by host cells would augment donor T cell activation and increase GvHD.Methods and Results: We used the B10BR->B6 MHC fully mismatched murine model of allogeneic BMT. Recipient wildtype (WT) and VIP knockout B6 (VIP-KO) mice were lethally irradiated with 11Gy and injected i.v. with 5×106 T cell depleted (TCD) bone marrow cells (BM) plus 0, 1 or 3 ×106 splenocytes from WT B10BR donors. Fifteen mice (males and females) were assigned to each splenocytes dosage group. The survival of recipients after BMT was recorded daily and the average clinical GvHD scores were obtained by combining weight-loss, activity, posture, fur texture and skin integrity data following standard published procedures. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had only 33.3% survival at day 75 compared with 73.3% survival among the WT recipients. Chimerism studies on day 10 post-BMT showed equivalent level of donor chimerism: 80% donor in WT recipients vs 72% donor in VIP-KO recipients, indicating increased mortality in the VIP-KO recipients was not due to early graft rejection. The addition of donor splenocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients (Figure 1), with 80% vs 100% day 75 survival among recipients of 1x106 splenocytes (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x106 donor splenocytes, respectively.To determine how the absence of VIP production in KO recipients modulates immune effector mechanisms that contributes to the differential survival rate post allo-BMT, we analyzed the recipients' splenocytes on day 30 post transplant with flow cytometry and staining with CD3, CD4, CD8, CD62L, CD44 and PD1 antibodies. VIP-KO recipients transplanted with 5M TCD BM plus 1M splenocytes had more CD4+ central memory and effector T cells, and CD8+ central memory and effector T cells. VIP-KO recipients transplanted with 5×106 TCD BM alone in addition had significantly more CD4 and CD8 effector T cells (p<0.01). VIP-KO recipients transplanted with 5×106 TCD BM plus 1x106 splenocytes had more PD1 expression on donor CD4+ central memory and effector T cells, and CD8+ central memory effector T cells, consistent with enhanced antigen-mediated activation.To visualize VIP produced by the host cells, we transplanted C57/BL6 recipients expressing a GFP transgene under the control of the VIP promoter (n=10) with 5×106 TCD BM plus 3 ×106 splenocytes from WT B10BR donors, and visualized VIP gene promoter activity as GFP expression in recipient lymphoid tissues and GvHD target organs. Only small number of CD45+ VIP-GFP+ cells were detected from in host liver, spleen and lymph nodes on day 10 post transplant (Figure 2), suggesting residual host leukocytes, particularly dendritic cells, expressed VIP.Conclusion: VIP production by recipient antigen presenting cells is a key pathway in limiting the activation of donor T cells. Physiological expression of VIP by host leukocytes regulate the GvHD activity of donor T cells. Novel methods to enhance VIP production in transplant recipients or the parenteral administration of VIP receptor agonists may limit GvHD, and may be particularly important in allo-BMT where GvL is not necessarily critical for success, such as transplants for hemoglobinopathy. [Display omitted] DisclosuresWaller:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; PRA: Consultancy; AMGEN: Consultancy; National Institutes of Health: Research Funding; Coulter Foundation: Research Funding; Helocyte: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Katz Foundation: Research Funding; Chimerix: Equity Ownership; Cerus: Equity Ownership; Celldex: Consultancy.

026 Vasoactive Intestinal Polypeptide Directly Excites Neurons of the Subparaventricular Zone

Abstract Introduction The suprachiasmatic nucleus (SCN) is responsible for generating the circadian rhythmicity in mammals. The ventral region or core of the SCN contains neurons that express the neuropeptide vasoactive intestinal polypeptide (VIP). VIP signaling is central for coherency and synchrony of SCN activity. VIP-expressing neurons in the SCN densely project to the ventral subregions of the subparaventricular zone (vSPZ). We studied the effects of VIP on vSPZ neurons in brain slices of mice with a combined calcium imaging and whole-cell patch-clamp recording techniques. We used calcium imaging to assess the effects of VIP on vSPZ neurons as a population and we acquired patch-clamp recordings to explore the effects of VIP on the electrical properties and the synaptic inputs to vSPZ neurons. Methods We expressed GCamp6 in vSPZ neurons by stereotaxically injecting AAV10-DIO-Ef1a-GCamp6 into the vSPZ of vGAT-IRES-Cre mice. Brain slices were prepared two weeks later and images were captured using a standard GFP filter set. We performed whole-cell recordings of the vSPZ neurons of wild-type mice. We assessed the effects of VIP on the membrane potential and the on excitatory synaptic input in vSPZ neurons. Results Using GCamp6-based in vitro calcium imaging we found that VIP excites 17% of vSPZ neurons and this effect was maintained in the presence of tetrodotoxin (TTX) and synaptic blockers for AMPA/NMDA and GABAA transmissions suggesting a direct effect of VIP on vSPZ neurons. We confirmed this result with patch-clamp recordings. We found that 29% of vSPZ neurons were excited by VIP. VIP produced a membrane depolarization of vSPZ neurons in the presence of antagonists for AMPA/NMDA and GABAA receptors. In addition, we found that in a small percentage of vSPZ neurons VIP increased the frequency of the glutamatergic excitatory postsynaptic currents, suggesting an additional excitatory mechanism. Conclusion Our results demonstrate that exogenous VIP directly excites the vSPZ neurons producing an increase in intracellular calcium and membrane depolarization. In addition, VIP increases glutamatergic afferent inputs to vSPZ neurons indicating an additional synergistic excitation. We conclude that when VIP is released from the SCN VIP fibers it can activate vSPZ neurons. Support (if any) NS091126 and HL149630.

Localization of Vasoactive Intestinal Polypeptide Receptor 1 (VPAC1) in Hypothalamic Neuroendocrine Oxytocin Neurons; A Potential Role in Circadian Prolactin Secretion.

Prolactin (PRL) is a versatile hormone and serves a broad variety of physiological functions besides lactation. The release of PRL from lactotrophs in the pituitary has in rodents been shown to be released with a circadian pattern depending on the physiological state of the animal. The circadian release of PRL seems to be complex involving tonic inhibition by dopamine (DA) neurons on lactotrophs and one or even several releasing factors. Because of the circadian releasing pattern of PRL, neurons in the suprachiasmatic nucleus (SCN), “the brain clock,” and especially the neurons expressing neuropeptide vasoactive intestinal polypeptide (VIP), have been suggested to be involved in the circadian regulation of PRL. In the present study, we used fluorescence immunohistochemistry, in situ hybridization histochemistry, confocal microscopy, three-dimensional reconstruction, and highly specific antibodies to visualize the occurrence of VIP receptors 1 and 2 (VPAC1 and VPAC2) in mouse brain hypothalamic sections stained in combination with VIP, oxytocin (OXT), arginine vasopressin (AVP), and DA (tyrosine hydroxylase, TH). We demonstrated that VIP fibers most likely originating from the ventral part of the SCN project to OXT neurons in the magnocellular part of the paraventricular nucleus (PVN). In the PVN, VIP fibers were found in close apposition to OXT neuron exclusively expressing the VPAC1 receptor. Furthermore, we demonstrate that neither OXT neurons nor TH or AVP neurons were expressing the VPAC2 receptor. VPAC1 receptor expression was also found on blood vessels but not in neurons expressing AVP or TH. These findings suggest that VIP signaling from the SCN does not directly target DA neurons involved in PRL secretion. Furthermore, the findings support the notion that VIP from neurons in the SCN could regulate circadian release of OXT in the posterior pituitary or modulate OXT neurons as a releasing factor involved in the circadian regulation of PRL from pituitary lactotrophs.

Reduced VIP Expression Affects Circadian Clock Function in VIP-IRES-CRE Mice (JAX 010908).

Circadian rhythms are programmed by the suprachiasmatic nucleus (SCN), which relies on neuropeptide signaling to maintain daily timekeeping. Vasoactive intestinal polypeptide (VIP) is critical for SCN function, but the precise role of VIP neurons in SCN circuits is not fully established. To interrogate their contribution to SCN circuits, VIP neurons can be manipulated specifically using the DNA-editing enzyme Cre recombinase. Although the Cre transgene is assumed to be inert by itself, we find that VIP expression is reduced in both heterozygous and homozygous adult VIP-IRES-Cre mice (JAX 010908). Compared with wild-type mice, homozygous VIP-Cre mice display faster reentrainment and shorter free-running period but do not become arrhythmic in constant darkness. Consistent with this phenotype, homozygous VIP-Cre mice display intact SCN PER2::LUC rhythms, albeit with altered period and network organization. We present evidence that the ability to sustain molecular rhythms in the VIP-Cre SCN is not due to residual VIP signaling; rather, arginine vasopressin signaling helps to sustain SCN function at both intracellular and intercellular levels in this model. This work establishes that the VIP-IRES-Cre transgene interferes with VIP expression but that loss of VIP can be mitigated by other neuropeptide signals to help sustain SCN function. Our findings have implications for studies employing this transgenic model and provide novel insight into neuropeptide signals that sustain daily timekeeping in the master clock.

Entrainment of Circadian Rhythms Depends on Firing Rates and Neuropeptide Release of VIP SCN Neurons

The mammalian suprachiasmatic nucleus (SCN) functions as a master circadian pacemaker, integrating environmental input to align physiological and behavioral rhythms to local time cues. Approximately 10% of SCN neurons express vasoactive intestinal polypeptide (VIP); however, it is unknown how firing activity of VIP neurons releases VIP to entrain circadian rhythms. To identify physiologically relevant firing patterns, we optically tagged VIP neurons and characterized spontaneous firing over 3days. VIP neurons had circadian rhythms in firing rate and exhibited two classes of instantaneous firing activity. We next tested whether physiologically relevant firing affected circadian rhythms through VIP release. We found that VIP neuron stimulation with high, but not low, frequencies shifted gene expression rhythms invitro through VIP signaling. Invivo, high-frequency VIP neuron activation rapidly entrained circadian locomotor rhythms. Thus, increases in VIP neuronal firing frequency release VIP and entrain molecular and behavioral circadian rhythms. VIDEO ABSTRACT.

Ontogeny of Circadian Rhythms and Synchrony in the Suprachiasmatic Nucleus.

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus coordinates daily rhythms including sleep-wake, hormone release, and gene expression. The cells of the SCN must synchronize to each other to drive these circadian rhythms in the rest of the body. The ontogeny of circadian cycling and intercellular coupling in the SCN remains poorly understood. Recent in vitro studies have recorded circadian rhythms from the whole embryonic SCN. Here, we tracked the onset and precision of rhythms in PERIOD2 (PER2), a clock protein, within the SCN isolated from embryonic and postnatal mice of undetermined sex. We found that a few SCN cells developed circadian periodicity in PER2 by 14.5 d after mating (E14.5) with no evidence for daily cycling on E13.5. On E15.5, the fraction of competent oscillators increased dramatically corresponding with stabilization of their circadian periods. The cells of the SCN harvested at E15.5 expressed sustained, synchronous daily rhythms. By postnatal day 2 (P2), SCN oscillators displayed the daily, dorsal-ventral phase wave in clock gene expression typical of the adult SCN. Strikingly, vasoactive intestinal polypeptide (VIP), a neuropeptide critical for synchrony in the adult SCN, and its receptor, VPAC2R, reached detectable levels after birth and after the onset of circadian synchrony. Antagonists of GABA or VIP signaling or action potentials did not disrupt circadian synchrony in the E15.5 SCN. We conclude that endogenous daily rhythms in the fetal SCN begin with few noisy oscillators on E14.5, followed by widespread oscillations that rapidly synchronize on E15.5 by an unknown mechanism.SIGNIFICANCE STATEMENT We recorded the onset of PER2 circadian oscillations during embryonic development in the mouse SCN. When isolated at E13.5, the anlagen of the SCN expresses high, arrhythmic PER2. In contrast, a few cells show noisy circadian rhythms in the isolated E14.5 SCN and most show reliable, self-sustained, synchronized rhythms in the E15.5 SCN. Strikingly, this synchrony at E15.5 appears before expression of VIP or its receptor and persists in the presence of blockers of VIP, GABA or neuronal firing. Finally, the dorsal-ventral phase wave of PER2 typical of the adult SCN appears ∼P2, indicating that multiple signals may mediate circadian synchrony during the ontogeny of the SCN.

Presence of Vasoactive Intestinal Polypeptide Signaling in Plasmacytoid Dendritic Cells Improves Survival in a Murine Allo-BMT Model

Introduction:Pre-clinical murine experiments and clinical data from allogeneic bone marrow transplantation (allo-BMT) have shown that increased numbers of plasmacytoid dendritic cells (pDC) in the bone marrow graft results in better clinical outcomes with less severe graft-versus-host disease (GvHD) and improved survival. The mechanism by which donor pDC modulate GvHD is unknown. Knowing that vasoactive intestinal polypeptide (VIP) is an immunosuppressive peptide , we reasoned that VIP signaling might play a role in regulating T-cell activation and expansion, and the VIP pathway may be a potential therapeutic target for regulating GvHD in allo-BMT. We have tested the hypothesis that VIP synthesis by donor pDCs can modulate T cell alloreactivity.Methods:To explore the mechanisms by which pDC and VIP signaling regulate T cell activation in murine allo-BMT, we prepared B6-background donor cell grafts and transplanted them into lethally irradiated B10.BR recipients. In experiment 1, recipients were transplanted with grafts containing the combination of 5 x 103 VIP-GFP hematopoietic stem cells (HSC) and 3 x 106 VIP-wild type (VIP-WT) or VIP-knock out (VIP-KO) splenocytes. At day 7, splenocytes were isolated for flow cytometric analysis looking for GFP signal, which represents VIP-promotor activity. Experiment 2 used combinations of 5 x 103 VIP-WT HSC, 1 x 106 luciferase+ T cells, and 50 x 103 VIP-WT or VIP-KO pDC from B6 as donor grafts. Recipients were monitored for survival and GvHD based on fur texture, posture, activity, skin integrity and weight loss. T cell expansion was measured by bioluminescent imaging (BLI). Serum cytokines from bleeds at day 3 and day 8 post-transplant were analyzed using a Luminex 38 plex panel. Some recipients were euthanized on day 3 for intracellular cytokine analysis of splenic T cells.Results:In experiment 1, 7 days post-transplant, analysis of splenocytes from all mice showed increased activity of the VIP gene promoter in donor pDC that were derived from HSC, compared to other cell types. The VIP promoter signal was also stronger in donor HSC-derived pDCs, if originally transplanted with VIP-KO splenocytes. In experiment 2 over 70% of mice receiving HSC+T+VIP-WT pDC in the BM graft survived to day 100 post-transplant, while those getting VIP-KO pDC instead only had 30% survival (Fig 1A). All surviving recipients were fully engrafted by day 30. BLI revealed greater total T-cell proliferation (measured as radiance) in recipients of VIP-KO pDC (Fig 1B). Furthermore, recipients of VIP-KO pDC had more severe acute GvHD, with increased weight loss and GvHD clinical scores (Fig 1C, 1D). Some recipients were euthanized and their serum were collected for cytokine analysis on day 8 post-transplant, which showed up-regulation of pro-inflammatory or chemotactic cytokines MCP1, IL-1, IL-12, IL-17 in T cells co-transplanted with VIP-KO pDC compared to WT pDC.Conclusion:The present findings show that: 1) VIP is produced by donor pDC early after allo-BMT; 2) absence of VIP production by donor pDC leads to increased T-cell expansion in a murine allo-BMT model. Thus the pDC-T cell VIP signaling pathway is a critical element in controlling donor T cell alloreactivity after allo-BMT. Future studies will include VIP qPCR to confirm VIP production in donor pDC post-transplant, and determine the mechanism by which VIP production by pDC regulates T cell activity and modulates GvHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of vasoactive intestinal polypeptide signaling augments T cell activation by plasmacytoid dendritic cells: implications for cellular immunotherapy

Abstract Introduction Murine models of allogeneic hematopoietic stem cell transplant (allo-HSCT) have shown that adding donor plasmacytoid dendritic cell (pDC) to allogeneic HSC and T cell grafts can: 1) augment the graft-versus-leukemia (GvL) activity of T cells by inducing donor Th1 polarization; and 2) limit the subsequent development of graft-versus-host disease (GvHD) through interferon gamma-licensed IDO production by pDC. Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide, which may regulate activation of allo-specific T cells, making it a potential target for regulating GvHD and GvL in allo-HSCT. Methods and results We used a model system of indirect presentation by pDC of a I-Ed derived allo-peptide (EaP 52–68) to transgenic T cells expressing the TEa TCR. The effect of blocking VIP signaling from pDC was assessed with VIP-knockout (KO) pDC or by adding a VIP antagonist, VIPhyb. Peptide-primed pDCs from VIP-KO mice increased expression of activation markers (CD69 and CD71) and stimulated TEa T cell proliferation compared with pDCs from WT mice. Similar results were observed in T cells cultured with VIPhyb treated pDCs. T cells proliferated more when cultured with BM VIP-KO pDCs than splenic VIP-KO pDCs. Conclusions These data indicate a novel role for VIP signaling in T cell activation and proliferation in response to indirect antigen presentation. These data support published data on the role of donor pDC in modulating the GvHD activity of T cells and indicate important functional differences in pDC activity according to their source. Ongoing experiments support the immune regulatory effect of VIP on GvHD and GvL in murine allo-HSCT.

T Cell Activation By Plasmacytoid Dendritic Cells Is Augmented By Inhibition of Vasoactive Intestinal Polypeptide Signaling: Implications for Cellular Immunotherapy

Introduction:Data from clinical allogeneic bone marrow transplant (allo-BMT) and pre-clinical murine models of allo-BMT have shown that donor plasmacytoid dendritic cells (pDC) have important roles in regulating graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activities of donor T cells. Using murine models of allo-BMT we have previously shown that 1) donor pDCs induce Th1 polarization of donor T cells and augment the GvL activity of T cells; and 2) the addition of pDC to grafts composed of purified T cells and HSC limited the subsequent development of GvHD. VIP is an immunosuppressive neuropeptide that regulates adaptive immune responses. We reasoned that VIP signaling may regulate activation of allo-specific T-cells, and the VIP pathway may be target for regulating GvHD and GvL in allo-BMT.Methods:To explore the mechanisms by which pDC and VIP signaling regulate T cell activation we used: 1) transgenic mice expressing GFP under the control of the VIP promoter to measure VIP expression in vivo in allo-BMT recipients; 2) one-way mixed lymphocyte reaction (MLR) to measure the proliferative response of transgenic luciferase positive T cells in response to allo-antigens via bioluminescence imaging (BLI); and 3) a model system of indirect presentation of allo-peptides derived from a H2-Ab MHC class II molecule by pDC to transgenic T cells expressing the TEa TCR. The effect of blocking vasoactive intestinal polypeptide (VIP) signaling during activation of allo-reactive T cells was assessed by using VIP-KO cells and by the addition of VIP peptide or a peptide antagonist of VIP (VIPhyb) to the one-way MLR. T cell proliferation and activation was measured by flow cytometry.Results:Analysis of VIP expression in donor pDC in murine models of allo-BMT showed >100-fold induction of VIP promoter activity in donor pDC and donor T cells during the first two weeks post-transplant, indicating that VIP expression in donor pDC may regulate T cell activation.Addition of endogenous native VIP suppressed T cell proliferation in one-way MLR but was reversed by addition of a 10x concentration of the VIP antagonist peptide (Figure 1B). Furthermore, adding 3 uM of the VIP antagonist to the MLR cultured significantly enhanced T cell proliferation. TEa peptide-primed T cells cultures with peptide-primed pDC from VIP knock-out mice had increased proliferation and expressed more of the activation markers CD69 and CD71 compared with T cells cultured with VIP-WT pDCs. Comparing pDC purified from marrow versus spleen, we found significantly more proliferation in T cells cultured with bone marrow VIP-KO pDCs than splenic VIP-KO pDCs, indicating that the less mature marrow pDC have greater antigen presenting ability than the more mature splenic pDC.Conclusion:These data suggest that 1) VIP is produced by donor pDC early after allo-BMT; 2) VIP inhibits T cell allo-proliferation in one-way MLR's; and 3) blocking VIP signaling using donor cells that cannot produce VIP or through the use of pharmacological inhibitors of VIP can augment activation and proliferation of T cells in response to indirect antigen presentation.The present findings support studies of VIP antagonist in allo-BMT to augment the GvL activity of T cells through indirect antigen presentation. Future studies include using the BLI analysis of MLR to determine the effect of novel drugs on T cell proliferation.**Jingxia Li and Reema Panjwani were equal contributors to this abstract. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Regulation of Appetite, Body Composition, and Metabolic Hormones by Vasoactive Intestinal Polypeptide (VIP).

Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide that belongs to the secretin-glucagon superfamily of peptides and has 68 % homology with PACAP. VIP is abundantly expressed in the central and peripheral nervous system and in the gastrointestinal tract, where it exercises several physiological functions. Previously, it has been reported that VIP regulates feeding behavior centrally in different species of vertebrates such as goldfishes, chicken and rodents. Additional studies are necessary to analyze the role of endogenous VIP on the regulation of appetite/satiety, feeding behavior, metabolic hormones, body mass composition and energy balance. The aim of the study was to elucidate the physiological pathways by which VIP regulates appetite/satiety, feeding behavior, metabolic hormones, and body mass composition. VIP deficient (VIP -/-) and age-matched wild-type (WT) littermates were weekly monitored from 5 to 22 weeks of age using a whole body composition EchoMRI analyzer. Food intake and feeding behavior were analyzed using the BioDAQ automated monitoring system. Plasma levels of metabolic hormones including active-ghrelin, GLP-1, leptin, PYY, pancreatic polypeptide (PP), adiponectin, and insulin were measured in fasting as well as in postprandial conditions. The genetic lack of VIP led to a significant reduction of body weight and fat mass and to an increase of lean mass as the mice aged. Additionally, VIP-/- mice had a disrupted pattern of circadian feeding behavior resulting in an abolished regular nocturnal/diurnal feeding. These changes were associated with an altered secretion of adiponectin, GLP-1, leptin, PYY and insulin in VIP-/- mice. Our data demonstrates that endogenous VIP is involved in the control of appetite/satiety, feeding behavior, body mass composition and in the secretion of six different key regulatory metabolic hormones. VIP plays a key role in the regulation of body phenotype by significantly enhancing body weight and fat mass accumulation. Therefore, VIP signaling is critical for the modulation of appetite/satiety and body mass phenotype and is a potential target for future treatment of obesity.

Mo1789 Sulforaphane Inhibits Expression of the Central Iron Regulator Hepcidin STAT3-Independently in an Inflammatory Cell Model

Glucagon-like peptide-2 (GLP-2) has potent anti-inflammatory effects in the intestinal mucosa. Vasoactive intestinal polypeptide (VIP) neurons in the enteric neuronal plexus have been shown to be important in these actions using pharmacologic blockers; but little is known about other specific subsets of neurotransmitters, such as nNOS. Aim: To investigate the role of VIP and nNOS signaling, individually and together, in mediating the antiinflammatory effects of GLP-2. Methods: With animal committee use approval, mice with selective knockouts of VIP-/-, nNOS-/-, and combined VIP-/-/ nNOS-/were used, with wild type (WT) controls; all animals were treated with trinitrobenzene sulfonic acid (TNBS) to induce colitis. Animals received (1-33) GLP-2 (50 μg/kg s.c. bid) or saline for 5 days. Mice were sacrificed and colonic tissue was taken for inflammation indices (myeloperoxidase [MPO] and histological damage scores), immunohistochemistry (BrdU:proliferation marker; Caspase-3: apoptosis marker), and mucosal cytokine levels (TNFα, IL1β, IFNγ, IL10). Results: GLP-2 treatment resulted in significant anti-inflammatory effects in VIP-/-and nNOS-/animals, but had no effects in the VIP-//nNOS-/treated animals. The response included improved weight and mucosal inflammation indices with reduced levels of proinflammatory cytokines (TNFα, IL1β, IFNγ), and reduced proliferation and apoptosis rates. In VIP-/nNOS-/animals treated with GLP-2 these cytokines and cell kinetic rates remained unchanged. (See table, n=8 per group, data: Mean±SEM, *p<0.05 by ANOVA) Conclusions: These findings suggest that the anti-inflammatory effects of GLP-2 can be mediated by either VIP or nNOS pathways, however when both pathways are abolished concomitantly, the anti-inflammatory effects of GLP-2 are lost. Further study is required to identify the enteric neuronal circuitry which mediates these potent anti-inflammatory effects

Mo1788 Vasoactive Intestinal Polypeptide and Neuronal Nitric Oxide Synthase Act via Parallel Pathways As Mediators of GLP-2 Anti-Inflammatory Activity

Glucagon-like peptide-2 (GLP-2) has potent anti-inflammatory effects in the intestinal mucosa. Vasoactive intestinal polypeptide (VIP) neurons in the enteric neuronal plexus have been shown to be important in these actions using pharmacologic blockers; but little is known about other specific subsets of neurotransmitters, such as nNOS. Aim: To investigate the role of VIP and nNOS signaling, individually and together, in mediating the antiinflammatory effects of GLP-2. Methods: With animal committee use approval, mice with selective knockouts of VIP-/-, nNOS-/-, and combined VIP-/-/ nNOS-/were used, with wild type (WT) controls; all animals were treated with trinitrobenzene sulfonic acid (TNBS) to induce colitis. Animals received (1-33) GLP-2 (50 μg/kg s.c. bid) or saline for 5 days. Mice were sacrificed and colonic tissue was taken for inflammation indices (myeloperoxidase [MPO] and histological damage scores), immunohistochemistry (BrdU:proliferation marker; Caspase-3: apoptosis marker), and mucosal cytokine levels (TNFα, IL1β, IFNγ, IL10). Results: GLP-2 treatment resulted in significant anti-inflammatory effects in VIP-/-and nNOS-/animals, but had no effects in the VIP-//nNOS-/treated animals. The response included improved weight and mucosal inflammation indices with reduced levels of proinflammatory cytokines (TNFα, IL1β, IFNγ), and reduced proliferation and apoptosis rates. In VIP-/nNOS-/animals treated with GLP-2 these cytokines and cell kinetic rates remained unchanged. (See table, n=8 per group, data: Mean±SEM, *p<0.05 by ANOVA) Conclusions: These findings suggest that the anti-inflammatory effects of GLP-2 can be mediated by either VIP or nNOS pathways, however when both pathways are abolished concomitantly, the anti-inflammatory effects of GLP-2 are lost. Further study is required to identify the enteric neuronal circuitry which mediates these potent anti-inflammatory effects

Vasoactive intestinal polypeptide mediates circadian rhythms in mammalian olfactory bulb and olfaction.

Accumulating evidence suggests that the olfactory bulbs (OBs) function as an independent circadian system regulating daily rhythms in olfactory performance. However, the cells and signals in the olfactory system that generate and coordinate these circadian rhythms are unknown. Using real-time imaging of gene expression, we found that the isolated olfactory epithelium and OB, but not the piriform cortex, express similar, sustained circadian rhythms in PERIOD2 (PER2). In vivo, PER2 expression in the OB of mice is circadian, approximately doubling with a peak around subjective dusk. Furthermore, mice exhibit circadian rhythms in odor detection performance with a peak at approximately subjective dusk. We also found that circadian rhythms in gene expression and odor detection performance require vasoactive intestinal polypeptide (VIP) or its receptor VPAC2R. VIP is expressed, in a circadian manner, in interneurons in the external plexiform and periglomerular layers, whereas VPAC2R is expressed in mitral and external tufted cells in the OB. Together, these results indicate that VIP signaling modulates the output from the OB to maintain circadian rhythms in the mammalian olfactory system.

VPAC receptor signaling modulates grouping behavior and social responses to contextual novelty in a gregarious finch: A role for a putative prefrontal cortex homologue

In both mammals and birds, vasoactive intestinal polypeptide (VIP) neurons and fibers are present in virtually every brain area that is important for social behavior. VIP influences aggression in birds, social recognition in rodents, and prolactin secretion in both taxa, but other possible functions in social modulation remain little explored. VIP effects are mediated by VPAC receptors, which bind both VIP and pituitary adenylate cyclase activating peptide. Within the lateral septum and medial bed nucleus of the stria terminalis, VPAC receptors are found at higher densities in gregarious finch species relative to territorial species, suggesting that VPAC receptor activation promotes social contact and/or preference for larger groups. Here we here test this hypothesis in zebra finches (Taeniopygia guttata), and also examine the relevance of VPAC receptors to anxiety-like processes. Intraventricular infusions of the VPAC receptor antagonist, neurotensin6–11 mouseVIP7–28, strongly reduce social contact when animals are tested in a novel environment, and exert sex-specific effects on grouping behavior. Specifically, VPAC receptor antagonism reduces gregariousness in females but increases gregariousness in males. Interestingly, VPAC antagonism in the medial pallium (putative prefrontal cortex homologue) significantly reduces gregariousness in both sexes, suggesting site-specific effects of VIP signaling. However, VPAC antagonism does not modulate novel-familiar social preferences in a familiar environment or general anxiety-like behaviors. The current results suggest that endogenous activation of VPAC receptors promotes social contact under novel environmental conditions, a function that may be accentuated in gregarious species. Moreover, endogenous VIP modulates gregariousness in both males and females.

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.

Multicellular Model for Intercellular Synchronization in Circadian Neural Networks

We developed a multicellular model characterized by a high degree of heterogeneity to investigate possible mechanisms that underlie circadian network synchronization and rhythmicity in the suprachiasmatic nucleus (SCN). We populated a two-dimensional grid with 400 model neurons coupled via γ-aminobutyric acid (GABA) and vasoactive intestinal polypeptide (VIP) neurotransmitters through a putative Ca2+ mediated signaling cascade to investigate their roles in gene expression and electrical firing activity of cell populations. As observed experimentally, our model predicted that GABA would affect the amplitude of circadian oscillations but not synchrony among individual oscillators. Our model recapitulated experimental findings of decreased synchrony and average periods, loss of rhythmicity, and reduced circadian amplitudes as VIP signaling was eliminated. In addition, simulated increases of VIP reduced periodicity and synchrony. We therefore postulated a physiological range of VIP within which the system is able to produce sustained and synchronized oscillations. Our model recapitulated experimental findings of diminished amplitudes and periodicity with decreasing intracellular Ca2+ concentrations, suggesting that such behavior could be due to simultaneous decrease of individual oscillation amplitudes and population synchrony. Simulated increases in Cl− levels resulted in increased Cl− influx into the cytosol, a decrease of inhibitory postsynaptic currents, and ultimately a shift of GABA-elicited responses from inhibitory to excitatory. The simultaneous reduction of IPSCs and increase in membrane resting potential produced GABA dose-dependent increases in firing rates across the population, as has been observed experimentally. By integrating circadian gene regulation and electrophysiology with intracellular and intercellular signaling, we were able to develop the first (to our knowledge) multicellular model that allows the effects of clock genes, electrical firing, Ca2+, GABA, and VIP on circadian system behavior to be predicted.

Differentiation of PC12 Cells Results in Enhanced VIP Expression and Prolonged Rhythmic Expression of Clock Genes

To examine for circadian rhythmicity, the messenger RNA (mRNA) amount of the clock genes Per1 and Per2 was measured in undifferentiated and nerve-growth-factor-differentiated PC12 cells harvested every fourth hour. Serum shock was needed to induce circadian oscillations, which in undifferentiated PC12 cultures lasted only one 24-h period, while in differentiated cultures, the rhythms continued for at least 3 days. Thus, neuronal differentiation provided PC12 cells the ability to maintain rhythmicity for an extended period. Both vasoactive intestinal polypeptide (VIP) and its receptor VPAC2 are expressed in the suprachiasmatic nucleus (SCN), and in agreement with VIP signaling being crucial for maintenance of rhythmicity, we found both VIP and VPAC2 mRNA increased after differentiation of PC12 cells. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts time- and concentration-dependent effects on Per gene expression in the SCN. We added 1 nM and 1 microM PACAP to oscillating PC12 cells at times corresponding to midday and early and late night to evaluate whether the effects were similar as in SCN. Induction of Per1 mRNA was found at all three times, which differs from results in SCN. Thus, PC12 cells seem more useful for studying mechanisms behind acquirement of rhythmicity of cell cultures than for resetting of circadian rhythm.

Metabolic rhythm abnormalities in mice lacking VIP-VPAC2 signaling

The circadian pacemaker in the suprachiasmatic nuclei (SCN) controls endogenous near 24-h physiological and behavioral rhythms in metabolism, neuroendocrine function, and locomotor activity. Recently, we showed that vasoactive intestinal polypeptide (VIP) and its receptor, VPAC(2) are critical to the intercellular communication between individual SCN neurons, and appropriate synchronization and phasing of these oscillatory cells. Mice defective in VIP signaling manifest grossly impaired circadian rhythms of SCN neuronal firing activity and are typically unable to maintain rhythmic wheel-running behavior in the absence of external time cues. Here we report that daily rhythms of metabolism and feeding behavior are also overtly altered in these animals. Under diurnal conditions (12:12-h light-dark; LD), metabolic and feeding rhythms are advanced in mice lacking either VIP or VPAC(2) receptor expression, peaking in the late day, rather than early night, as observed in wild-type mice. When placed in constant light (LL), both VIP-deficient and VPAC(2) receptor-knockout mice exhibit dampening of metabolic and feeding rhythms, which deteriorate after a few days. In addition, overall metabolic rate is greatly reduced in VPAC(2)-knockout mice, when compared with wild-type mice, regardless of lighting condition. The advancement of metabolic and feeding rhythms in these mice under LD suggests that these rhythms are less sensitive to masking by light. These results demonstrate that altering SCN function not only affects neuronal and wheel-running activity rhythms but also dramatically impairs temporal regulation of metabolism and feeding.

PKA-dependent activation of the vascular smooth muscle isoform of K ATP channels by vasoactive intestinal polypeptide and its effect on relaxation of the mesenteric resistance artery

Vasoactive intestinal polypeptide (VIP) is a potent vasodilator and has been successfully used to alleviate hypertension. Consistently, disruption of VIP gene in mice leads to hypertension. However, its downstream targets in the vascular regulation are still not well demonstrated. To test the hypothesis that the vascular smooth muscle isoform of K ATP channels is a downstream target of the VIP signaling, we performed the studies on the Kir6.1/SUR2B channel expressed in HEK293 cells. We found that the channel was strongly activated by VIP. Through endogenous VIP receptors, the channel activation was reversible and dependent on VIP concentrations with the midpoint-activation concentration ∼ 10 nM. The channel activation was voltage-independent and could be blocked by K ATP channel blocker glibenclamide. In cell-attached patches, VIP augmented the channel open-state probability with modest suppression of the single channel conductance. The VIP-induced Kir6.1/SUR2B channel activation was blocked by PKA inhibitor RP-cAMP. Forskolin, an adenylyl cyclase activator, activated the channel similarly as VIP. The effect of VIP was further evident in the native tissues. In acutely dissociated mesenteric vascular smooth myocytes, VIP activated the K ATP currents in a similar manner as in HEK293 cells. In endothelium-free mesenteric artery rings, VIP produced concentration-dependent vasorelaxation that was attenuated by glibenclamide. These results therefore indicate that the vascular isoform (Kir6.1/SUR2B) of K ATP channels is a target of VIP. The channel activation relies on the PKA pathway and produces mesenteric arterial relaxation.