Effect of PACAP on Heat Exposure.

Heat stroke is a life-threatening illness caused by exposure to high ambient temperatures and relative humidity. The incidence of heat stroke is expected to increase due to climate change. Although pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in thermoregulation, the role of PACAP on heat stress remains unclear. PACAP knockout (KO) and wild-type ICR mice were subjected to heat exposure at an ambient temperature of 36 °C and relative humidity of 99% for 30-150 min. After heat exposure, the PACAP KO mice had a greater survival rate and maintained a lower body temperature than the wild-type mice. Moreover, the gene expression and immunoreaction of c-Fos in the ventromedially preoptic area of the hypothalamus, which is known to harbor temperature-sensitive neurons, were significantly lower in PACAP KO mice than those in wild-type mice. In addition, differences were observed in the brown adipose tissue, the primary site of heat production, between PACAP KO and wild-type mice. These results suggest that PACAP KO mice are resistant to heat exposure. The heat production mechanism differs between PACAP KO and wild-type mice.

The Pivotal Role of Neuropeptide Crosstalk from Ventromedial-PACAP to Dorsomedial-Galanin in the Appetite Regulation in the Mouse Hypothalamus.

We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) in the ventromedial hypothalamus (VMH) enhances feeding during the dark cycle and after fasting, and inhibits feeding during the light cycle. On the other hand, galanin is highly expressed in the hypothalamus and has been reported to be involved in feeding regulation. In this study, we investigated the involvement of the VMH-PACAP to the dorsomedial hypothalamus (DMH)-galanin signaling in the regulation of feeding. Galanin expression in the hypothalamus was significantly increased with fasting, but this increment was canceled in PACAP-knockout (KO) mice. Furthermore, overexpression of PACAP in the VMH increased the expression of galanin, while knockdown (KD) of PACAP in the VMH decreased the expression of galanin, indicating that the expression of galanin in the hypothalamus might be regulated by PACAP in the VMH. Therefore, we expressed the synaptophysin-EGFP fusion protein (SypEGFP) in PACAP neurons in the VMH and visualized the neural projection to the hypothalamic region where galanin was highly expressed. A strong synaptophysin-EGFP signal was observed in the DMH, indicating that PACAP-expressing cells of the VMH projected to the DMH. Furthermore, galanin immunostaining in the DMH showed that galanin expression was weak in PACAP-KO mice. When galanin in the DMH was knocked down, food intake during the dark cycle and after fasting was decreased, and food intake during the light cycle was increased, as in PACAP-KO mice. These results indicated that galanin in the DMH may regulate the feeding downstream of PACAP in the VMH.

Unbiased compound screening with a reporter gene assay highlights the role of p13 in the cardiac cellular stress response.

We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart.

Rivastigmine improves isolation rearing-induced prepulse inhibition deficits via muscarinic acetylcholine receptors in mice.

RATIONALE: The acetylcholinesterase inhibitors donepezil, galantamine, and rivastigmine are used for the treatment of Alzheimer's disease. We previously demonstrated that donepezil and galantamine differentially affect isolation rearing-induced prepulse inhibition (PPI) deficits and that this might be due to differential effects on brain muscarinic acetylcholine (mACh) receptor function in mice. OBJECTIVES: We examined the effects of rivastigmine on isolation rearing-induced PPI deficits, brain ACh levels, and mACh receptor function in mice. METHODS: Acoustic startle responses were measured in a startle chamber. Microdialysis was performed, and the levels of dopamine and ACh in the prefrontal cortex were measured. RESULTS: Rivastigmine (0.3 mg/kg) improved PPI deficits, and this improvement was antagonized by the mACh receptor antagonist telenzepine but not by the nicotinic ACh receptor antagonist mecamylamine. Rivastigmine increased extracellular ACh levels by approximately 2-3-fold, less than the increase produced by galantamine. Rivastigmine enhanced the effect of the mACh receptor agonist N-desmethylclozapine on prefrontal dopamine release, a marker of mACh receptor function, and this increase was blocked by telenzepine. In contrast, galantamine did not affect N-desmethylclozapine-induced dopamine release. Furthermore, rivastigmine did not affect cortical dopamine release induced by the serotonin1A receptor agonist osemozotan, suggesting that the effect of rivastigmine has specificity for mACh receptors. CONCLUSIONS: Taken together with our previous finding that marked increases in ACh levels are required for the PPI deficit improvement induced by galantamine, our present results suggest that rivastigmine improves isolation rearing-induced PPI deficits by increasing ACh levels and by concomitantly enhancing mACh receptor function.

Serotonin 5-HT(7) receptor blockade reverses behavioral abnormalities in PACAP-deficient mice and receptor activation promotes neurite extension in primary embryonic hippocampal neurons: therapeutic implications for psychiatric disorders.

The serotonin 5-HT(7) receptor has been linked to various psychiatric disorders, including schizophrenia, anxiety and depression, and is antagonized by antipsychotics such as risperidone, clozapine and lurasidone. In this study, we examined whether inhibiting the 5-HT(7) receptor could reverse behavioral abnormalities in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP), an experimental mouse model for psychiatric disorders such as schizophrenia. The selective 5-HT(7) antagonist SB-269970 effectively suppressed abnormal jumping behavior in PACAP-deficient mice. SB-269970 tended to alleviate the higher immobility in the forced swim test in PACAP-deficient mice, although SB-269970 reduced the immobility also in wild-type mice. In addition, we found that mutant mice had impaired performance in the Y-maze test, which was reversed by SB-269970. In the mutant mouse brain, 5-HT(7) protein expression did not differ from wild-type mice. In primary embryonic hippocampal neurons, the 5-HT(7) agonist AS19 increased neurite length and number. Furthermore, SB-269970 significantly inhibited the increase in neurite extension mediated by the 5-HT(1A/7) agonist 8-OH-DPAT. These results indicate that 5-HT(7) receptor blockade ameliorates psychomotor and cognitive deficits in PACAP-deficient mice, providing additional evidence that the 5-HT(7) receptor is a rational target for the treatment of psychiatric disorders.

Intranasal application of secretin, similarly to intracerebroventricular administration, influences the motor behavior of mice probably through specific receptors.

Secretin and its receptors show wide distribution in the central nervous system. It was demonstrated previously that intravenous (i.v.) and intracerebroventricular (i.c.v.) application of secretin influenced the behavior of rat, mouse, and human. In our previous experiment, we used a special animal model, Japanese waltzing mice (JWM). These animals run around without stopping (the ambulation distance is very limited) and they do not bother with their environment. The i.c.v. secretin attenuated this hyperactive repetitive movement. In the present work, the effect of i.c.v. and intranasal (i.n.) application of secretin was compared. We have also looked for the presence of secretin receptors in the brain structures related to motor functions. Two micrograms of i.c.v. secretin improved the horizontal movement of JWM, enhancing the ambulation distance. It was nearly threefold higher in treated than in control animals. The i.n. application of secretin to the left nostril once or twice a day or once for 3 days more effectively enhanced the ambulation distance than i.c.v. administration. When secretin was given twice a day for 3 days it had no effect. Secretin did not improve the explorative behavior (the rearing), of JWM. With the use of in situ hybridization, we have found very dense secretin receptor labeling in the cerebellum. In the primary motor cortex and in the striatum, only a few labeled cells were seen. It was supposed that secretin exerted its effect through specific receptors, mainly present in the cerebellum.

Neuroprotective effect of endogenous pituitary adenylate cyclase-activating polypeptide on spinal cord injury.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuroprotective peptide expressed in the central nervous system. To date, changes in the expression and effect of endogenous PACAP have not been clarified with respect to spinal cord injury (SCI). The aim of this study was to elucidate the expression pattern and function of endogenous PACAP on the contusion model of SCI using heterozygous PACAP knockout (PACAP(+/-)) and wild-type mice. Real-time polymerase chain reaction methods revealed that the level of PACAP mRNA increased gradually for 14 days after SCI and that PAC1R mRNA levels also increased for 7 days compared with intact control mice. PACAP and PAC1R immunoreactivities colabeled with a neuronal marker in the intact spinal cord. Seven days after SCI, PAC1R immunoreactivity was additionally co-expressed with an astrocyte marker. Wild-type mice gradually recovered motor function after 14 days, but PACAP(+/-) mice showed significantly impaired recovery from 3 days compared with wild-type mice. The injury volume at day 7 in PACAP(+/-) mice, and the number of single-stranded DNA-immunopositive cells as a marker of neuronal cell death at day 3 were significantly higher than values measured in wild-type mice. These data suggest that endogenous PACAP is upregulated by SCI and has a neuroprotective effect on the damaged spinal cord.

The melanocortin system is involved in regulating autonomic nerve activity through central pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a peptidergic neurotransmitter that is highly expressed in the nervous system. We have previously reported that a central injection of PACAP leads to changes in the autonomic nervous system tones including sympathetic excitation and parasympathetic inhibition. An anatomical study revealed that melanocortin and PACAP are colocalized in some hypothalamic nuclei. Here, we investigated the possible role of the melanocortin system in autonomic control by PACAP using SHU9119, an antagonist of the melanocortin receptors (MC3-R/MC4-R). Pretreatment with SHU-9119 did not affect the activating neural responses of adrenal, renal, and lumbar sympathetic nerves following a PACAP injection However, SHU9119 significantly eliminated the suppressing effect of a PACAP injection on gastric vagal nerve activity and excitation effects on liver and brown adipose tissue sympathetic nerve activities. These results suggest that the brain melanocortin system might play a key role in the control of thermogenic sympathetic outflows and digestive parasympathetic outflow by PACAP, but this system does not participate in the central effects of PACAP on cardiovascular function and neural activities of renal, adrenal, and lumbar sympathetic nerves.

Galantamine and donepezil differently affect isolation rearing-induced deficits of prepulse inhibition in mice.

RATIONALE: Previous studies have shown that alterations in acetylcholine (ACh) receptor subtypes might contribute to cognitive impairment observed in schizophrenia and that choline acetyltransferase activity in the parietal cortex is negatively correlated with the severity of such cognitive impairment. However, clinical data suggest that the acetylcholinesterase (AChE) inhibitors galantamine and donepezil have different effects on negative and cognitive symptoms in schizophrenia. Prepulse inhibition (PPI) deficits--sensory information-processing deficits observed in schizophrenia--may be useful models for studying the efficacy of AChE inhibitors as cognitive enhancers. OBJECTIVES: The present study examined the effects of galantamine and donepezil on PPI deficits induced by an environmental factor and drugs. MATERIALS AND METHODS: In the isolation-rearing model, 3-week-old male ddY mice were housed either in groups of five or six per cage or isolated in cages of the same size for more than 6 weeks. In the drug-induced model, apomorphine 1 mg/kg and MK-801 0.2 mg/kg were administered to 9- to 10-week-old male ddY mice. RESULTS: In isolation-reared mice, galantamine attenuated PPI deficits, while donepezil did not. Galantamine and donepezil both attenuated PPI deficits induced by apomorphine, but not by MK-801. The galantamine-induced improvements in PPI deficits were not prevented by the nicotinic ACh receptor antagonists mecamylamine and methyllycaconitine. CONCLUSIONS: These observations suggest that galantamine and donepezil have different effects on the environmentally induced PPI deficits and that these observations may be relevant to the different effects of these drugs observed clinically in schizophrenia.

Psychostimulant-induced attenuation of hyperactivity and prepulse inhibition deficits in Adcyap1-deficient mice.

Psychostimulants, including amphetamine, act as antihyperkinetic agents in humans with hyperkinetic disorder such as attention-deficit hyperactivity disorder and are known to be effective in enhancing attention-related processes; however, the underlying mechanisms have not been adequately addressed. Mice lacking the Adcyap1 gene encoding the neuropeptide pituitary adenylate cyclase-activating polypeptide (Adcyap1(-/-)) display psychomotor abnormalities, including increased novelty-seeking behavior and hyperactivity. In this study, Adcyap1(-/-) mice showed sensory-motor gating deficits, measured as deficits in prepulse inhibition (PPI), and showed normal PPI in response to amphetamine. Amphetamine also significantly decreased hyperlocomotion in Adcyap1(-/-) mice, and this paradoxical antihyperkinetic effect depended on serotonin 1A (5-HT(1A)) receptor signaling. c-Fos-positive neurons were increased in the prefrontal cortex in amphetamine-treated Adcyap1(-/-) mice, suggesting increased inhibitory control by prefrontal neurons. Additionally, amphetamine produced an antihyperkinetic effect in wild-type mice that received the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin. These results indicate that Adcyap1(-/-) mice act as a model of hyperlocomotion and PPI deficits and suggest that 5-HT(1A)-mediated pathways are important determinants of the psychostimulant-elicited, rate-dependent effects that are in a negative function of the baseline rate of activity.

Pituitary adenylate cyclase-activating polypeptide does not colocalize with vasoactive intestinal polypeptide in the hypothalamic magnocellular nuclei and posterior pituitary of cats and rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) immunoreactive cells were demonstrated in the hypothalamic magnocellular nuclei in cats and rats. In cats these immunoreactive cells were stained without any treatment or intervention; however, in rats we had to use the pituitary stalk section to enhance the amount of PACAP and VIP for successful immunostaining. In both species the regions occupied by PACAP and VIP immunoreactive cells partially overlap each other in the paraventricular and supraoptic nuclei. Nevertheless, in either cats or rats PACAP and VIP immunoreactivities do not colocalize in the same cells studied by double labeling immunohistochemistry (IHC) or by the combination of immunohistochemistry and in situ hybridization. As was expected, PACAP and VIP immunoreactive materials were stored in different fibers of the posterior pituitary where the distribution of PACAP and VIP fibers also showed different patterns: PACAP fibers form a dense plexus at the periphery of the posterior lobe, in the vicinity of the intermediate lobe; however, the VIP fibers were evenly distributed mainly in the center of the posterior lobe. In spite of the high sequence homology of PACAP and VIP, the two peptides are synthesized in different subpopulations of hypothalamic neurons. This different distribution correlates well with the different role of the hypothalamic PACAP and VIP in the biologic clock and in the functions of the anterior and posterior pituitary.