Tomoya Nakamachi

Direct Involvement of Orexinergic Systems in the Activation of the Mesolimbic Dopamine Pathway and Related Behaviors Induced by Morphine

In this study, we investigated the role of orexinergic systems in dopamine-related behaviors induced by the μ-opioid receptor agonist morphine in rodents. Extensive coexpression of tyrosine hydroxylase with orexin receptors was observed in the mouse ventral tegmental area (VTA). The levels of dopamine and its major metabolites in the nucleus accumbens were markedly increased by the microinjection of orexin A and orexin B into the VTA. The subcutaneous morphine-induced place preference and hyperlocomotion observed in wild-type mice were abolished in mice that lacked the prepro-orexin gene. An intra-VTA injection of a selective orexin receptor antagonist SB334867A [1-(2-methylbenzoxazol-6-yl)-3-[1.5]naphthyridin-4-yl urea] significantly suppressed the morphine-induced place preference in rats. Furthermore, the increased level of dialysate dopamine produced by morphine in the mouse brain was significantly decreased by deletion of the prepro-orexin gene. These findings provide new evidence that orexin-containing neurons in the VTA are directly implicated in the rewarding effect and hyperlocomotion induced by morphine through activation of the mesolimbic dopamine pathway in rodents.

Pleiotropic Functions of PACAP in the CNS

Abstract:  Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. PACAP prevents ischemic delayed neuronal cell death (apoptosis) in the hippocampus. PACAP inhibits the activity of the mitogen‐activated protein kinase (MAPK) family, especially JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia‐reperfusion, both pyramidal cells and astrocytes increased their expression of the PACAP receptor (PAC1‐R). Reactive astrocytes increased their expression of PAC1‐R, released interleukin‐6 (IL‐6) that is a proinflammatory cytokine with both differentiation and growth‐promoting effects for a variety of target cell types, and thereby protected neurons from apoptosis. These results suggest that PACAP itself and PACAP‐stimulated secretion of IL‐6 synergistically inhibit apoptotic cell death in the hippocampus. The PAC1‐R is expressed in the neuroepithelial cells from early developmental stages and in various brain regions during development. We have recently found that PACAP, at physiological concentrations, induces differentiation of mouse neural stem cells into astrocytes. Neural stem cells were prepared from the telencephalon of mouse embryos and cultured with basic fibroblast growth factor. The PAC1‐R immunoreactivity was demonstrated in the neural stem cells. When neural stem cells were exposed to PACAP, about half of these cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. This phenomenon was significantly antagonized by a PAC1‐R antagonist (PACAP6‐38), indicating that PACAP induces differentiation of neural stem cell into astrocytes. Other our physiological studies have demonstrated that PACAP acts on PAC1‐R in mouse neural stem cells and its signal is transmitted to the PAC1‐R‐coupled G protein Gq but not to Gs. These findings strongly suggest that PACAP plays very important roles in neuroprotection in adult brain as well as astrocyte differentiation during development.

Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury

BackgroundWe hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.MethodsUnilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNγ or IL-4.ResultsGp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNγ, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.ConclusionsClassical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

Regulation by Orexin of Feeding Behaviour and Locomotor Activity in the Goldfish

Orexin is a hypothalamic neuropeptide that is implicated in the regulation of feeding behaviour and the sleep‐wakefulness cycle in mammals. However, in spite of a growing body of knowledge concerning orexin in mammals, the orexin system and its function have not been well studied in lower vertebrates. In the present study, we first examined the effect of feeding status on the orexin‐like immunoreactivity (orexin‐LI) and the expression of orexin mRNA in the goldfish brain. The number of cells showing orexin‐LI in the hypothalamus of goldfish brain showed a significant increase in fasted fish and a significant decrease in glucose‐injected fish. The expression level of orexin mRNA in the brains of fasted fish increased compared to that of fed fish. We also examined the effect of an i.c.v. injection of orexin or an anti‐orexin serum on food intake and locomotor activity in the goldfish. Administration of orexin by i.c.v. injection induced a significant increase of food intake and locomotor activity, whereas i.p. injection of glucose or i.c.v. injection of anti‐orexin serum decreased food consumption. These results indicate that the orexin functions as an orexigenic factor in the goldfish brain.

Role of PACAP in Ischemic Neural Death

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that was first isolated from an ovine hypothalamus in 1989. Since its discovery, more than 2,000 papers have reported on the tissue and cellular distribution and functional significance of PACAP. A number of papers have reported that PACAP but not the vasoactive intestinal peptide suppressed neuronal cell death or decreased infarct volume after global and focal ischemia in rodents, even if PACAP was administered several hours after ischemia induction. In addition, recent studies using PACAP gene-deficient mice demonstrated that endogenous PACAP also contributes greatly to neuroprotection similarly to exogenously administered PACAP. The studies suggest that neuroprotection by PACAP might extend the therapeutic time window for treatment of ischemia-related conditions, such as stroke. This review summarizes the effects of PACAP on ischemic neuronal cell death, and the mechanism clarified in vivo ischemic studies. In addition, the prospective mechanism of PACAP on ischemic neuroprotection from in vitro neuronal and neuronal-like cell cultures with injured stress model is reviewed. Finally, the development of PACAP and/or receptor agonists for human therapy is discussed.

Isolation of Peptide Transport System-6 from Brain Endothelial Cells: Therapeutic Effects with Antisense Inhibition in Alzheimer and Stroke Models:

By isolating for the first time ever a peptide transporter from the blood—brain barrier (BBB) and developing an antisense that selectively targets the brain-to-blood efflux component, we were able to deliver a therapeutic concentration of the neurotrophic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) 27 to brain in animal models of Alzheimers and stroke. Efflux pumps at the BBB are major causes of BBB impermeability to peptides. PACAP is neuroprotective in vitro in femtomole amounts, but brain uptake of PACAP27 is limited by an efflux component of peptide transport system-6 (PTS-6). Here, we characterized, isolated, and sequenced this component of PTS-6, identifying it as β-F1 ATPase, and colocalized it with PACAP27 on BBB endothelial cells. Antisenses targeting the BBB inhibited PACAP27 efflux, thus increasing brain uptake of PACAP27. Treatment with antisense +PACAP27 improved cognition in a mouse model of Alzheimers disease and reduced infarct size after cerebral ischemia. This represents the first isolation from BBB tissue of a peptide transporter and shows that inhibition of peptide efflux pumps is a potential strategy for drug delivery to brain.

Inhibitory effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) on food intake in the goldfish, Carassius auratus

Pituitary adenylate cyclase-activating polypeptide (PACAP) has a similar structure to that of vasoactive intestinal peptide (VIP) and both the polypeptides belong to the same molecular group, the secretin-glucagon superfamily. PACAP and VIP have possible potency as hypothalamic factors mediating the release of pituitary hormones in the fish pituitary. However, the roles of PACAP and VIP in the central nervous systems of fish have not yet been made clear. Recently, it was reported that PACAP and/or VIP are involved in the feeding behavior of the mouse and chick. Therefore, we investigated the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic PACAP and VIP on food intake in the goldfish, Carassius auratus. Cumulative food intake was significantly decreased by ICV injection of PACAP (11 or 22 pmol/g body weight) or VIP (11 or 22 pmol/g) during a 60-min observation period after treatment. IP administration of PACAP (44 or 88 pmol/g) or VIP (22 or 44 pmol/g) induced a significant decrease in food intake during a 60-min observation period after treatment. These results suggest that PACAP and VIP may be involved as feeding regulators in goldfish.

Stroke upregulates TNFα transport across the blood–brain barrier

Abstract To determine how cytokine transport systems at the blood–brain barrier (BBB) participate in stroke progression and recovery, we generated a mouse model of transient middle cerebral artery occlusion (tMCAO). After 1 h of occlusion followed by nearly complete reperfusion, the neurological deficits lasted more than a week as shown by several behavioral tests. Despite the prominent infarct area indicated by reduced cerebral perfusion and confirmed by vital staining, the volume of distribution of 131 I-albumin in various brain regions was not significantly altered over time (12 h to 14 days). In sharp contrast, the blood-to-brain permeation of 125 I-TNFα was significantly increased 5 days after tMCAO. Furthermore, excess unlabeled TNFα abolished this enhanced 125 I-TNFα uptake. Thus, not only did the known saturable transport system for TNFα persist, but it functioned at a higher capacity in tMCAO mice. Upregulation of TNFR1 and TNFR2 partially explains the increased transport, as mRNA for both receptors showed the most pronounced increase (15-fold and 30-fold, respectively) in the ischemic hemisphere 5–7 days after tMCAO. However, even in the hemisphere contralateral to the ischemia induced by stroke, there was increased TNFα transport. The bilateral increase in 125 I-TNFα entry from blood to brain suggests that TNFα trafficking in cerebral endothelial cells is influenced by global mediators in addition to the transporting receptors. Given the known multiple modulatory effects of TNFα after stroke, the results indicate that the TNFα transport system at the BBB facilitates neuroplasticity and plays an important role in stroke recovery.

Localization, characterization and function of pituitary adenylate cyclase-activating polypeptide during brain development.

Neural development is controlled by region-specific factors that regulate cell proliferation, migration and differentiation. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts a wide range of effects on different cell types in the brain as early as the fetal stage. Here we review current knowledge concerning several aspects of PACAP expression in embryonic and neonatal neural tissue: (i) the distribution of PACAP and PACAP receptors mRNA in the developing brain; (ii) the characteristic generation of neurons, astrocytes and oligodendrocytes in brain areas where the PACAP receptor is expressed and (iii) the role of PACAP as a regulator of neural development, inducing differentiation and proliferation in association with other trophic factors or signal transduction molecules.

Role of PACAP in Neural Stem/Progenitor Cell and Astrocyte: from Neural Development to Neural Repair

After central nervous system (CNS) injury, reactive astrocytes display opposing functions, inducing neural repair and axonal regeneration via the release of growth factors, or forming a glial scar which acts as a barrier to axonal regeneration. Endogenous neural stem/progenitor cells have also recently been identified at the site of CNS injury, where they have been shown to differentiate into mature neurons in an animal model of ischemia. However, the pathophysiological mechanisms underpinning the contribution of reactive astrocytes and neural stem/progenitor cells to neural repair are still to be fully elucidated. Pituitary adenylate cyclase activating polypeptide (PACAP) is widely expressed in the CNS, where it has been shown to exert numerous biological effects. This review will summarize the current state of knowledge regarding the expression of PACAP and its receptors during neural development, as well as the involvement of PACAP in astrocytes and neural stem/progenitor cell biology. In addition, we will also discuss emerging evidence that implicates PACAP in neurogenesis and neural repair in response to brain pathophysiology.