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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which belongs to the vasoactive intestinal polypeptide/glucagon/secretin family. PACAP and its three receptor subtypes are expressed in neural tissues, with PACAP known to exert pleiotropic effects on the nervous system. This review provides an overview of current knowledge regarding the neuroprotective effects, mechanisms of action, and therapeutic potential of PACAP in response to ischemic brain injuries., (Copyright © 2015. Published by Elsevier Inc.)

Methylmercury (MeHg) is an environmental pollutant known to cause neurobehavioral defects and is especially toxic to the developing brain. With recent studies showing that fetal exposure to low-dose MeHg causes developmental abnormalities, it is therefore important to find ways to combat its effects as well as to clarify the mechanism(s) underlying MeHg toxicity. In the present study, the effects of MeHg on cultured neural progenitor cells (NPC) derived from mouse embryonic brain were investigated. We first confirmed the vulnerability of embryonic NPC to MeHg toxicity, NPC from the telencephalon were more sensitive to MeHg compared to those from the diencephalon. Buthionine sulfoximine (BSO) which is known to inhibit glutathione synthesis accelerated MeHg toxicity. Furthermore, antioxidants such as N-acetyl cysteine and alpha-tocopherol dramatically rescued the NPC from MeHg's toxic effects. Interestingly, a 12 hr delay in the addition of either antioxidant was still able to prevent the cells from undergoing cell death. Although it is now difficult to avoid MeHg exposure from our environment and contaminated foods, taking anti-oxidants from foods or supplements may prevent or diminish the toxicological effects of MeHg.

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.

Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike in rodents, suggesting that its anorexigenic action is mediated by alpha-melanocyte-stimulating hormone (alpha-MSH) but not corticotropin-releasing hormone. This led us to investigate whether MCH-containing neurons in the goldfish brain have direct inputs to alpha-MSH-containing neurons, using a confocal laser scanning microscope, and to examine whether the anorexigenic action of MCH is also mediated by other anorexigenic neuropeptides, such as cholecystokinin (CCK) and pituitary adenylate cyclase-activating polypeptide (PACAP), using their receptor antagonists. MCH- and alpha-MSH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. MCH-containing nerve fibers or endings lay in close apposition to alpha-MSH-containing neurons in the hypothalamus in the posterior part of the nucleus lateralis tuberis (NLTp). The inhibitory effect of ICV-injected MCH on food intake was not affected by treatment with a CCK A/CCK B receptor antagonist, proglumide, or a PACAP receptor (PAC(1) receptor) antagonist, PACAP((6-38)). ICV administration of MCH at a dose sufficient to inhibit food consumption also did not influence expression of the mRNAs encoding CCK and PACAP. These results strongly suggest that MCH-containing neurons provide direct input to alpha-MSH-containing neurons in the NLTp of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide via the alpha-MSH (melanocortin 4 receptor)-signaling pathway.

Chloride efflux is known to be involved in the progression of apoptosis in various cell types. We have recently shown that the volume-sensitive outwardly rectifying (VSOR) anion channel serves as the pathway for apoptotic chloride efflux in some cells. In the present study, we tested the neuroprotective effects of drugs that can block the VSOR anion channel, on delayed neuronal death (DND) induced by transient forebrain ischemia. The functional expression of the VSOR anion channel was first examined in hippocampal neurons in both primary culture and hippocampal slice preparations, by the whole-cell patch-clamp technique. We then tested the channel's sensitivity to an anion channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and a tyrosine kinase blocker, genistein. By histological examinations and cytochrome c release assessments, the protective effects of these drugs on the DND of hippocampal CA1 neurons in mice subjected to transient ischemia were examined. Drugs were administered via the jugular vein prior to ischemic treatment and into the peritoneal cavity after reperfusion. Hippocampal neurons were found to express the volume-sensitive Cl(-) channel, which exhibits outward rectification and is sensitive to DIDS and genistein. Administration of DIDS or genistein reduced cytochrome c release and the number of damaged neurons in the CA1 region after transient forebrain ischemia. This fact suggests that the DND induction mechanism involves the activity of the VSOR anion channel and that this channel may provide a therapeutic target for the treatment of stroke.

Objective: Severe global ischemia often results in severe damage to the central nervous system of survivors. Hind-limb paralysis is a common deficit caused by global ischemia. Until recently, most studies of global ischemia of the central nervous system have examined either the brain or spinal cord, but not both. Spinal cord damage specifically after global ischemia has not been studied in detail. Because the exact nature of the neuronal damage to the spinal cord and the differences in neuronal damage between the brain and spinal cord after global ischemia are poorly understood, we developed a new global ischemia model in the rat and specifically studied spinal cord damage after global ischemia. Further, we compared the different forms of neuronal damage between the brain and spinal cord after global ischemia., Design: Randomized, controlled study using three different global ischemia models in the rat., Setting: University research laboratory., Subjects: Male, adult Sprague-Dawley rats (300 g)., Interventions: Animals were divided into three experimental groups, group A (n = 6, survived for 7 days), 12 mins of hemorrhagic shock; group B (n = 6, survived for 7 days), 5 mins of cardiac arrest; or group C (n = 6, each for 6 hrs, 12 hrs, 1 day, 3 days, and 7 days), 7 mins of hemorrhagic shock and 5 mins of cardiac arrest. Motor deficit of the hind limbs was studied 6 hrs to 7 days after resuscitation. Also, nonoperated animals (n = 6) were used as the control. Histologic analysis (hematoxylin and eosin, Fluoro-Jade B, terminal deoxynucleotidyl transferase- mediated dUTP end-labeling [TUNEL], Klüver-Barrera) and ultrastructural analysis using electron microscopy were performed on samples from the CA1 region of the hippocampus and lumbar spinal cord. Demyelination of the white matter of the lumbar spinal cord was analyzed semiquantitatively using Scion Image software., Main Results: No paraplegic animals were observed in either group A or B. All group C animals showed severe hind-limb paralysis. Severe neuronal damage was found in the CA1 region of the hippocampus in all groups, and the state of delayed neuronal cell death was similar among the three groups. Neuronal damage in the lumbar spinal cord was detected only in group C animals, mainly in the dorsal horn and intermediate gray matter. Demyelination was prominent in the ventral and ventrolateral white matter in group C. A significant difference was observed between control and group C rats with Scion Image software. Ultrastructural analysis revealed extensive necrotic cell death in the intermediate gray matter in the lumbar spinal cord in group C rats., Conclusion: The combination in the global ischemia model (i.e., hemorrhagic shock followed by cardiac arrest) caused severe neuronal damage in the central nervous system. Thereby, hind-limb paralysis after global ischemia might result from spinal cord damage. These results suggest that therapeutic strategies for preventing spinal cord injury are necessary when treating patients with severe global ischemia.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to decrease ischemic neuronal damage and increase IL-6 secretion in rats. However, the mechanisms underlying neuroprotection are still to be fully elucidated. The present study was designed to investigate the role played by PACAP and IL-6 in mediating neuroprotection after ischemia in a null mouse. Infarct volume, neurological deficits, and cytochrome c in cytoplasm were higher in PACAP(+/-) and PACAP(-/-) mice than in PACAP(+/+) animals after focal ischemia, although the severity of response was ameliorated by the injection of PACAP38. A decrease in mitochondrial bcl-2 was also accentuated in PACAP(+/-) and PACAP(-/-) mice, but the decrease could be prevented by PACAP38 injection. PACAP receptor 1 (PAC1R) immunoreactivity was colocalized with IL-6 immunoreactivity in neurons, although the intensity of IL-6 immunoreactivity in PACAP(+/-) mice was less than that in PACAP(+/+) animals. IL-6 levels increased in response to PACAP38 injection, an effect that was canceled by cotreatment with the PAC1R antagonist. However, unlike in wild-type controls, PACAP38 treatment did not reduce the infarction in IL-6 null mice. To clarify the signaling pathway associated with the activity of PACAP and IL-6, phosphorylated STAT (signal transducer and activator of transcription) 3, ERK (extracellular signal-regulated kinase), and AKT levels were examined in PACAP(+/-) and IL-6 null mice after ischemia. Lower levels of pSTAT3 and pERK were observed in the PACAP(+/-) mice, whereas a reduction in pSTAT3 was recorded in the IL-6 null mice. These results suggest that PACAP prevents neuronal cell death after ischemia via a signaling mechanism involving IL-6.

Orexins are neuropeptides that have a range of physiological effects including the regulation of feeding behavior and the sleep-wakefulness cycle. Recently, we reported that level of orexin A in spinal fluid was decreased in the patients of some neurodegenerative diseases and it is considered that orexin A and the receptors might be related to central nervous system disorders. However, the expression and localization of orexin receptors is not elicited well. Therefore, the purpose of this study is to investigate the time-dependent changes and the cellular localization of orexin receptor focusing on orexin-1 receptor (OX1R) in the mouse brain after transient common carotid artery occlusion (tCCAO) model by using immunohistochemical techniques. OX1R immunoreactivity dramatically increased and peaked in the hippocampus and cortex 2 days after tCCAO, but remained unchanged in the hypothalamus. Using double-immunohistochemistry, the OX1R immunopositive cells at 2 days after tCCAO were co-localized not only with neuronal marker, NeuN-immunoreactivity but also with astroglial and oligodendroglial markers, GFAP- and CNPase-immunoreactivities, respectively. These results suggested that OX1R is induced other cells in addition to the neurons during stress such as ischemia and orexins and its receptor might play an important role for ischemic insult.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in several physiological functions. Several lines of evidence from in vitro studies have shown that PACAP plays some important roles in development of nervous system such as neural proliferation and differentiation. Recently, mice lacking PACAP have been reported to show a higher mortality shortly after birth, impaired thermal adaptation, and altered psychomotor behaviors. Inasmuch as monoaminergic nervous systems are implicated in these phenotypes and a quite few data have been reported on the role of this peptide in nervous development in vitro, we studied early development [embryonic days 10.5 (E10.5) and 12.5 (E12.5)] of monoaminergic nervous systems in mice lacking PACAP. The fetuses lacking PACAP showed immunoreactivities (IRs) for tyrosine hydroxylase (TH) and serotonin (5-HT) similarly to the wild type. We observed TH-IR in the forebrain [striatal differentiating zone (dz) and hypothalamic dz], midbrain, hindbrain, neural-crest-derived sympathetic ganglionic primordia, ventral spinal cord dz, and bowel at E10.5 in both PACAP null and wild type with no difference. At E12.5, in the wild-type- and PACAP-gene-deficient mice, no differences of 5-HT- and TH-IRs were observed in several brain regions, including brainstem (midbrain and pons). Thus, the depletion of PACAP does not affect monoaminergic nervous systems in the early development.