Chihiro Kawaguchi

Defects in reproductive functions in PACAP-deficient female mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved neuropeptide and widely expressed in both brain and peripheral tissues, including several reproductive organs (e.g., testis and ovary). PACAP stimulates syntheses of several sexual hormones and steroids, suggesting it has possible roles in reproductive function. In this study, the role of PACAP in female reproductive functions such as fertility, mating behavior and maternal behaviors were investigated by using mice lacking PACAP (PACAP(-/-)). PACAP(-/-) females showed reduced fertility (the number of parturitions relative to the number of pairings). Mating experiments using vasectomized males revealed that mating frequency and its intervals in some PACAP(-/-) females were quite different (zero to eight times/4 weeks), whereas the frequency was relatively constant (two to three times/4 weeks) in wild-type females. In PACAP(-/-) females, maternal crouching behavior tended to decrease compared to wild-type females, although the influence of litter size on maternal behavior needs to be considered. These data suggest a role for endogenous PACAP in female reproductive processes.

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-HT1A) 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-HT1A 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-HT1A-mediated pathways are important determinants of the psychostimulant-elicited, rate-dependent effects that are in a negative function of the baseline rate of activity.

Neuroprotective action of endogenous PACAP in cultured rat cortical neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic effects both in vitro and in vivo. Here we demonstrate the upregulation of PACAP mRNA expression in cultured rat cortical neurons after excitotoxic glutamate exposure, and the exacerbating effect of the PACAP receptor antagonist, PACAP(6-38), on neuronal viability. PACAP mRNA levels were increased up to 3.5-fold 8 h after glutamate exposure. PACAP(6-38) decreased the viability of cortical neurons, irrespective of whether the cells were exposed to glutamate or not. PACAP(6-38) also inhibited glutamate-induced expression of PACAP mRNA, suggesting that PACAP acts via an autocrine or paracrine mechanism to enhance PACAP expression itself. Glutamate exposure is known to increase brain-derived neurotrophic factor (BDNF) mRNA expression. This increased expression was markedly suppressed by PACAP(6-38). Our previous study has shown that PACAP stimulates the PACAP gene transcription in PC12 cells. Taken together, these data may suggest that endogenous PACAP regulates the expression of PACAP itself and BDNF. Although it may also be possible that PACAP(6-38)-induced death of PACAP and BDNF mRNA-expressing cells, per se, results in reduced levels of these mRNAs, the present results support the idea that endogenous PACAP has a neuroprotective action.

PACAP-Deficient Mice Exhibit Light Parameter–Dependent Abnormalities on Nonvisual Photoreception and Early Activity Onset

Background The photopigment melanopsin has been suggested to act as a dominant photoreceptor in nonvisual photoreception including resetting of the circadian clock (entrainment), direct tuning or masking of vital status (activity, sleep/wake cycles, etc.), and the pupillary light reflex (PLR). Pituitary adenylate cyclase-activating polypeptide (PACAP) is exclusively coexpressed with melanopsin in a small subset of retinal ganglion cells and is predicted to be involved extensively in these responses; however, there were inconsistencies in the previous reports, and its functional role has not been well understood. Methodology/Principal Findings Here we show that PACAP-deficient mice exhibited severe dysfunctions of entrainment in a time-dependent manner. The abnormalities in the mutant mice were intensity-dependent in phase delay and duration-dependent in phase advance. The knockout mice also displayed blunted masking, which was dependent on lighting conditions, but not completely lost. The dysfunctions of masking in the mutant mice were recovered by infusion of PACAP-38. By contrast, these mutant mice show a normal PLR. We examined the retinal morphology and innervations in the mutant mice, and no apparent changes were observed in melanopsin-immunoreactive cells. These data suggest that the dysfunctions of entrainment and masking were caused by the loss of PACAP, not by the loss of light input itself. Moreover, PACAP-deficient mice express an unusually early onset of activities, from approximately four hours before the dark period, without influencing the phase of the endogenous circadian clock. Conclusions/Significance Although some groups including us reported the abnormalities in photic entrainments in PACAP- and PAC1-knockout mice, there were inconsistencies in their results [1], [2], [3], [4]. The time-dependent dysfunctions of photic entrainment in the PACAP-knockout mice described in this paper can integrate the incompatible data in previous reports. The recovery of impaired masking by infusion of PACAP-38 in the mutant mice is the first direct evidence of the relationship between PACAP and masking. These results indicate that PACAP regulates particular nonvisual light responses by conveying parametric light information—that is, intensity and duration. The “early-bird” phenotype in the mutant mice originally reported in this paper supposed that PACAP also has a critical role in daily behavioral patterns, especially during the light-to-dark transition period.

Serotonergic Inhibition of Intense Jumping Behavior in Mice Lacking PACAP (Adcyap1

Abstract:  Genetic manipulation of pituitary adenylate cyclase‐activating polypeptide (PACAP) in mice has uncovered its involvement in psychomotor function. We previously observed that mice lacking the Adcyap1 gene encoding the neuropeptide PACAP (Adcyap1−/−) displayed intense jumping behavior when placed in a novel environment such as an open field. Here, we show that Adcyap1−/− mice manifest jumping behavior as early as at least 6 weeks of age when compared with wild‐type mice and that the selective serotonin (5‐HT) reuptake inhibitor, fluoxetine, as well as the serotonin precursor, 5‐hydroxytryptophan, suppress jumping behavior. Our previous study showed a slight decrease in 5‐HT metabolite, 5‐hydroxyindoleacetic acid (5‐HIAA) in Adcyap1−/− mouse brain. Taken together, these results suggest that there is a developmental aspect to the jumping behavior seen in Adcyap1−/− mice, and that jumping behaviour may involve the serotonergic system.

Electrical detection of single pollen allergen particles using electrode-embedded microchannels

We report on the development of a microfluidic system for the electrical detection of single pollen allergen particles. Our device consists of 500 nm electrode gaps fabricated in an 800 nm wide fluidic channel. We flowed pollen allergen particles of average size 330 nm along the channel via fluid pumping and simultaneously monitored temporal change in dc current flowing through the sensing electrodes. Current spikes were detected, which can be attributed to a capacitance discharging upon trapping/detrapping of single allergens in the electrode gap. This sensing mechanism may open new avenues for a highly sensitive pollen allergen sensor.

Overexpression of PACAP in the pancreas failed to rescue early postnatal mortality in PACAP-null mice.

PACAP exerts multiple activities as a hormone and neurotransmitter, and has been proposed to play vital roles in a variety of neuronal functions. PACAP is also involved in insulin secretion from pancreatic beta-cells. Recently, we and other groups demonstrated that PACAP-deficient mice (PACAP(-/-)) are viable, but suffer from increased postnatal mortality. To ascertain whether this high mortality is rescued by overexpression of PACAP in peripheral tissue (such as pancreas), we performed a genetic cross between PACAP(-/-) and our recently developed transgenic mice overexpressing PACAP in pancreatic beta-cells; and then examined the survival rate of their F2 progeny. PACAP(-/-) mice were segregated into two groups based on mortality as well as body weight gain: PACAP(-/-) that survived >20 days of age with normal weight gain and PACAP(-/-) that died before 20 days with a marked weight loss. Kaplan-Meier survival analysis demonstrated that PACAP(-/-) mice and those carrying the PACAP transgene have similarly lower survival probability compared with their heterozygous littermates that served as positive controls. Further study using additional tissue-specific transgenic or knockout mouse models will be required to determine the causative defects underlying the high mortality of PACAP(-/-) mice.

Electrical Detection of Pollen Allergen Using Electrode-Embedded-Micro-Channel

Hay fever is a disease that approximately 20 percent of the world population develop. Providing appropriate treatment for this plant-specific pollen allergy requires a method to identify various types of pollen. Here we introduce a technique that can be used for direct detections of single biological macromolecules such as pollen. Our method is based on two-probe current measurements using a microfluidic-channel-embedded-electrode system. A fabrication procedure of the device is as follows. First, we formed two gold nanoelectrodes by standard electron beam lithography and lift-off processes. The interelectrode gap distance was designed to be 500 nm. We then deposited a SiO2 layer. After that, we fabricated Cr etching mask. Finally, we dry-etched the sample by reactive ion etching and obtained a microfluidic-channel-embedded-electrode structure. We flowed HEPES(2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid) solution containing pollen of 500 nm size into the micro-channel and simultaneously measured current flowing through the two electrodes. Characteristic spike-like signals were observed; sharp rise of the current followed by a rather smooth decrease to the base current level. We exhibited control experiments in a HEPES solution wherein no pollen was added and observed only featureless current traces. We anticipate that the sharp current rise is associated with trapping of a single cedar pollen between the electrodes whereas the gradual current decrease represents the pollen detrapping. We propose the technique for identifying several kinds of pollens based on the height and the width of the current spikes.Copyright

PACAP deficient mice exhibit light parameter dependent abnormalities of nonvisual photoreception

P2-g35 PACAP deficient mice exhibit light parameter dependent abnormalities of nonvisual photoreception Yasushi Isojima1,2, Chihiro Kawaguchi3, Norihito Shintani3, Michiyoshi Hatanaka3, Xiaohong Guo3, Katsuya Nagai2, Hitoshi Hashimoto3,4, Akemichi Baba3 1 Genomic Science Center, RIKEN, Japan; 2 Institute of Protein Research, Osaka University, Japan; 3 Graduate School of Pharmaceutical Science, Osaka University, Japan; 4 Center for Child Mental Development, Graduate School of Medicine, Osaka University, Japan