一般演題（ポスター）

一般演題（ポスター） Trophic factors・Cytokines

3P-14 The effects of accumbal BDNF overexpression on aversive memory Izumiseki Fumiya¹，Kimura Iku¹，Miyamoto Akiko²，Nabekura Junichi²，Mimura Masaru¹，Tanaka Kenji F.¹ ¹Dept. Neuropsychiatry, Keio Univ. Sch. Med.，²National Institute for Physiological Sciences The mesolimbic dopamine pathway, composed of dopaminergic neurons in the ventral tegmental area（VTA）and their projections to the nucleus accumbens（NAc）is affected by aversive stimuli. Abnormalities in this pathway are implicated in neuropsychiatric disorders including depression. It is known that Brain-derived neurotrophic factor（BDNF）in the NAc is increased by aversive stimuli, however, how accumbal BDNF affect aversive memory structure remains unclear.Here we examined the effects of BDNF protein overexpression in the NAc on acquisition, retrieval, and extinction of aversive memory by combining cell type-specific/time controllable BDNF mRNA overexpression technique with passive avoidance test. [Experiment 1]To investigate the effects of BDNF overexpression on aversive memory acquisition and retrieval, animals were first separated in BDNF overexpression and control groups. The animals were then exposed to a light compartment, and when they entered a dark compartment, an aversive foot shock（0.4 mA, 5 sec）was delivered（acquisition training）. The following day, animals were exposed to the same procedure as acquisition training without foot shock（retrieval test）. The latency to enter the dark compartment was not different between groups in acquisition and retrieval phases. [Experiment 2]To investigate the effects of BDNF overexpression on aversive memory extinction, animals first received acquisition training. Then, they were separated into BDNF overexpression and control groups. After 15 days from acquisition training, animals were exposed to the light compartment. Subsequently, animals received extinction training in the dark compartment for 5 min without foot shock. The following day, the animals were placed in the light compartment（extinction test）. The latency to enter the dark compartment decreased in overexpression group in extinction test but not in acquisition or retrieval phases.Taken together, our data suggest that accumbal BDNF overexpression enhances extinction but not acquisition or retrieval of aversive memory.		3P-15 Difference in a translation start site in BDNF exon I and exon IX. Saika Miyuki，Fukuchi Mamoru，Tabuchi Akiko，Tsuda Masaaki Dept. of Biol. Chem., Grad. Sch. of Med. ＆ Pharm. Sci., Univ. of toyama Brain-derived neurotrophic factor（BDNF）is a member of neurotrophin family and plays a crucial role in expressing various neuronal functions including neuronal survival, differentiation, and synaptic plasticity. Because of the multiple promoters and alternative splicing, multiple BDNF transcripts are produced. Although it is well known that these multiple BDNF transcripts possess a common translation start site in exon IX, another translation start site exists in 3’end of exon I. It is suggested that BDNF precursor protein（preproBDNF）with 8 additional amino acid residues at the N-terminus would be produced from BDNF exon I-IX mRNA. However, it is unclear that the translation start site in BDNF exon I is functional. In this study, therefore, we focused on the difference in a translation start site in BDNF exon I or exon IX. We constructed expression vectors of preproBDNF translated from BDNF exon I-AUG（termed ppBDNF exon I）and that from exon IX-AUG（termed ppBDNF exon IX）. We found that the expression level of ppBDNF exon I was highly than that of ppBDNF exon IX in NIH3T3 cells. This result suggests that the translation start site in BDNF exon I is functional, and the translation efficiency of the translation start site in BDNF exon I is higher than that in exon IX. We are now examining differences in intracellular localization of these BDNF protein in NIH3T3 and neuronal cells.

3P-16 FGF-1 release induced by oxidative stress enhances apoE/HDL generation of rat astrocytes in the autocrine manner Ito Jinichi，Nagayasu Yuko，Michikawa Makoto Department of Biochemistry, Nagoya City University Graduate School of Medical Sciences Jinichi Ito, Yuko Nagayasu, Makoto MichikawaDepartment of Biochemistry, Nagoya City University Graduate School of Medical Sciences FGF-1 release from rat astrocytes to enhance apoE/HDL generation under oxidative stress without inducing apoptosis We previously observed that the production and release of fibroblast growth factor（FGF-1）are increased in rat astrocytes during in vitro long-term culture, that FGF-1 enhances the generation of apoE-containing high density lipoproteins（apoE/HDL）. In this study, we examined effects of oxidative stress on release of FGF-1 from cultured rat astrocytes. The treatment of rat astrocytes with 100 μM hydrogen peroxide（H2O2）for 10 min enhanced FGF-1 release without inducing apoptosis. The conditioned medium prepared from the cells cultured in a fresh medium after the treatment with H2O2 had the FGF-1-like activities, which enhanced cholesterol synthesis, signalings to phosphorylate Akt and ERK, and apoE secretion. The oxidative stress induced by H2O2 enhanced the release of cytosolic proteins such as HSP70 and HSP90 in addition to FGF-1. The addition of lipoproteins such as low density lipoproteins（LDL）, furthermore, canceled H2O2-induced release of FGF-1 and cytosolic proteins. These findings suggest that oxidative stress is one of the candidates which triggers FGF-1 release from astrocytes in the brain accompanied with the release of cytosolic proteins.		3P-17 TNFα and IL-1β are differentially induced in microglia through distinct combination of MAP kinases Nakajima Kazuyuki¹，Sudo kenji¹，Takezawa Yosuke¹，Koshimoto Maasa¹，Kohsaka Shinichi² ¹Dept. of Science and Engineering, Soka University，²National Institute of Neuroscience Stimulation of rat microglia with endotoxin in vitro induces production of the inflammatory cytokines such as tumor necrosis factor alpha（TNFα）and interleukin 1beta（IL-1β）along with superoxide anion（O₂^-）and nitric oxide（NO）. In this study, we investigated the role of O₂^- and NO in the induction of TNFα and IL-1β in microglia. The lipopolysaccharide（LPS）-inducible TNFα was significantly suppressed by pretreatment with the O₂^- scavenger, but not by the NO scavenger, while the LPS-inducible IL-1β was strongly inhibited by pretreatment with the NO scavenger, but not by the O₂^- scavenger. On the other hand, an O₂^--donor and an NO-donor induced TNFα and IL-1β in microglia, respectively. These results suggested that O₂^- and NO activate each specific signaling cascade, and through which induce TNFα and IL-1β in microglia, respectively. LPS-dependent TNFα induction was significantly suppressed by c-Jun N-terminal kinase（JNK）and p38 inhibitors, whereas the IL-1β induction was significantly suppressed by extracellular signal-regulated kinase（ERK）and JNK inhibitors. These results indicated that TNFα and IL-1β are induced through the action of JNK/p38 and ERK/JNK, respectively. In fact, the O₂^--donor could activate JNK/p38 in microglia, and the NO-donor could activate ERK/JNK. Taken together, these results showed that TNFα and IL-1β are differentially induced through the different combination of mitogen-activated protein kinases（MAPKs）in endotoxin-stimulated microglia.

3P-18 Microglia regulate the cytokine/chemokine dynamics in the brain and enhance the functional maturation of blood-brain barrier. Shigemoto-mogami Yukari，Hoshikawa Kazue，Sekino Yuko，Sato Kaoru Lab of Neuropharmacol., Divi. of Pharmacol., Natl. Inst. Hlth. Sci. The blood-brain barrier（BBB）permeability is regulated by various cells comprised of neurovascular unit（NVU）. Microglia are already present in the brain prior to the brain vascular development at the embryonic stage and once the brain capillaries are formed, abundant microglia exist around the capillaries. However, microglial role on the functional maturation of the BBB is still unclear. In this study, we investigated the roles of microlia in the BBB maturation. We used in vitro BBB model comprised of endotherial cells, pericytes, and astrocytes（Pharmaco cell co）. When we added microglia on the astrocytes of the in vitro BBB model（brain side）during the maturation period（1-4 DIV）, significant increase in the transendothelial electrical resistance（TEER）and the expression levels of tight junction proteins（Claudine-5）were detected. On the other hand, when we added LPS-activated microglia, significant decrease in the TEER and the expression levels of tight junction proteins（Occludin, Claudine-5）were detected. We measured the amounts of cytokines/chemokines in the brain sides of these two situations comprehensively and quantitatively using MAGPIX system（millipore）. The dynamics of the cytokines/chemokines are totally different between two situations. We have detected two factors which exhibited the opposite dynamics in these two situations, suggesting that these factors are related to the microglia-induced maturation of BBB function. Currently we are examining the direct effects of these factors on the BBB functional maturation.		3P-19 Function of activated microglia following hypoglossal nerve axotomy. Tanaka Tatsuhide，Murakami Koichi，Nomura Taichi，Bando Yoshio，Yoshida Shigetaka Dept. of Functional Anatomy and Neuroscience, Asahikawa Med. Univ. Microglia, generally considered to be immune cells of the central nervous system（CNS）, are involved in many types of inflammatory processes in the brain. They are critical in developmental processes and are essential for the maintenance of neuronal homeostasis. Experimental axotomy such as hypoglossal nerve transection causes neurodegeneration and glial reactions. After nerve injury, microglia near the injured motor neurons are stimulated, migrate toward the injured neurons, and wrap up motor neuron cell bodies. This model appear to be suitable for studying microglia in the brain because there is no disruption of the blood brain barrier, and macrophage infiltration does not occur. It is thought that the perineuronal microglia protect axotomized motor meurons, whereas non-perineuronal microglia leads to gradual cell death of the injured motor neurons. Recent studies have demonstrated that under specific polarization conditions microglia develop into different phenotypes, termed M1 and M2. We analyzed the functional relevance of microglia in motor neurons, we examined the M1, M2 phenotype marker and growth factor expressions in hypoglossal nerve transection model. The M1 markers, neurotrophic factors and phagocytosis-related factors were induced after hypoglossal nerve axotomy, however, M2 markers were not changed.

3P-20 Possible involvement of the secretion-related protein CAPS2 in regulation of dynorphin, one of the endogenous opioids, secretion Katakai Momoko，Shinoda Yo，Furuichi Teiichi Dept. of Appl. Biol. Sci., Fac. of Sci. and Technol., Tokyo Univ. of Sci. Endogenous opioid dynorphin（Dyn）is known to be produced as a precursor protein pro-Dyn and packed into large dense-core vesicle（LDCV）, which is widely expressed in the brain including hippocampal dentate gyrus granule cells, and activity dependently secreted to CA3 region via mossy fiber axon terminals. It is relatively well known that their target receptor κ-opioid receptor and their physiological functions. However, the release machinery of Dyn and its related proteins have not been well understood. Calcium-dependent protein for secretion 2（CAPS2）is identified initially as a cytosolic protein associated with LDCV in endocrine cells and thought to be involved in LDCV secretion. We previously reported that CAPS2 is highly associated with secretion of neurotrophin BDNF and act as facilitator of their secretion. In the present study, we show that the possible involvement of CAPS2 in regulation of Dyn secretion, such as the localization of Dyn and CAPS2 at hippocampus, the alteration of Dyn distribution in CAPS2 KO mice and the alteration of Dyn release probability in the presence and absence of CAPS2.		3P-21 Effect of repetitive and transcranial near infrared irradiation on inflammatory processes in the brain of rats. Talahashi Taro¹，Yamada Kohei²，Suzuki Katsuaki¹，Mundalil Vasu Mahesh¹，Wakuda Tomoyasu¹，Mori Norio¹ ¹Department of Psychiatry, Hamamatsu University school of medicine，²Research Center for Child Mental Development,Hamamatsu University School of Medicine Immune activation processes including microglial activation and increased cytokine/chemokine expression in the brain have been implicated in the pathology of neurodevelopmental disorders such as schizophrenia and autism. Low-level laser therapy（LLLT）has been used in the treatment of inflammatory pathologies, reducing both pain and acute inflammatory process. In this study we examined possible therapeutic effects of LLLT on immune activations in the brain of rat offspring of dams exposed to gestational polyriboinosinic-polyribocytidilic acid（poly I：C）. Pregnant rat dams were given intraperitoneally either with poly I：C or saline at gestational day 13. At 8 weeks after birth, offspring of dams treated by poly I：C or by saline were subjected to LLLT. All the animals were photo-irradiated on the frontal cortex under pentobarbital anesthesia, once daily for 3 days. Energy densities were either 0（sham）, 45, 90, 180, 360, or 720 J/cm2. The wave length was 600-1600 nm and its output was 1800 mW. Twenty-four hours after the last irradiation, animals were sacrificed and their brains were subjected to measurement of mRNA expression of immunological markers by real time PCR. The body temperature of rats tended to elevated after photo-irradiation of the frontal cortex. Expression levels of several cytokine（IL-1beta, IL-6, IL-10）were shown to decrease in the prefrontal cortex of photo-irradiated animals in dose-dependent manner. The result suggests LLLT may affect brain function through modulation of expression of cytokines. Further investigations with regard to behavioral changes after LLLT are ongoing.