ポスター発表 (Poster Sessions)

神経伝達物質、グリオトランスミッター、修飾物質 Neurotransmitters, Gliotransmitters, and Modulators
P3-1-1 ラット低体重出生モデル等を用いた中枢ニューロンにおける神経栄養因子の機能変化解析 Possible change in neurotrophin function in decreased weight rats model ○大島淑子¹, 沼川忠広¹, 安達直樹¹, 功刀浩¹ ○Yoshiko Ooshima¹, Tadahiro Numakawa¹, Naoki Adachi¹, Hiroshi Kunugi¹ 国立精神・神経医療研究センター　神経研　疾病3部¹ NCNP, Tokyo, Japan¹ Especially, in the onset of psychiatric disorders including schizophrenia, the developmental dysfunction in neuronal system is suggested to be involved. Importantly, a variety of studies indicate possible contribution of diminishment in expression levels of BDNF (one of the neurotrophins, which regulates neuronal survival and function) in the pathophysiology of psychiatric disorders. Indeed, we previously reported that cultured cortical neurons obtained from brains of IUGR (intrauterine growth restriction) have lower expression of TrkB receptor for BDNF, and that neuronal viability is also lower (Neurosci lett, 2010, 476, 104-9). Here, we observed significant decrease in weight of young rats after glucocorticoid exposure. As glucocorticoid is a well-known as a stress hormone and related to depressive disorder, we examined levels of receptors for glucocorticoid and for BDNF. We found decreased levels of glucocorticoid receptor, GR. Since we previously found that GR interaction with TrkB has a role in neuronal function (PNAS, 2009, 106(2), 647-52), such a GR downregulation in low weight young rats may influence brain function.	P3-1-2 テトラヒドロビオプテリン欠乏はドーパミン生合成の障害を介して生後発達に伴うチロシン水酸化酵素タンパク質量の増加を障害する Tetrahydrobiopterin deficiency impairs postnatal augmentation of tyrosine hydroxylase protein via insufficient dopamine biosynthesis ○一瀬宏¹, 本間大悟¹, 加藤節子² ○Hiroshi Ichinose¹, Daigo Homma¹, Setsuko Katoh² 東工大院・生命理工¹, 明海大学² Grad Sch Biosci & Biotech, Tokyo Inst Tech, Yokohama Japan¹, Meikai Univ, Sakado, Japan² Tyrosine hydroxylase (TH) is the rate-limiting enzyme for dopamine biosynthesis and requires tetrahydrobiopterin (BH4) as an essential cofactor. In human, the genetic defects in BH4 biosynthesis lead to psychomotor dysfunction in infant to childhood, while how BH4 deficiency affects the postnatal development of dopaminergic system is poorly understood. To address this issue, we analyzed a transgenic mouse lacking sepiapterin reductase (Spr) that catalyzes the final step of BH4 biosynthesis. The BH4 content in the brain of Spr knockout (KO) neonate was significantly lower (ca. 26%) than that of wild-type, and there was no significant age-related change in each genotype until postnatal day 14 (P14). The contents of dopamine and TH protein in Spr KO mouse were not significantly affected in neonatal period, whereas their developmental increases were totally suppressed. The levels of other dopaminergic marker proteins were unaltered in Spr KO mouse, indicating that the amount of TH protein was selectively affected by BH4 deficiency. To provide mechanistic insight into the loss of TH protein, we next investigated the effect of BH4 and dopa administration on the TH protein content in the brain. We found that BH4 administration persistently increased dopamine content in the brain and fully restored the TH protein level of Spr KO mouse. On the other hand, dopa administration less persistently increased the dopamine contents and partially restored the TH protein level of Spr KO mouse. Our results indicate that BH4 deficiency impairs the postnatal development of dopaminergic system and that the postnatal augmentation of TH protein requires its product dopamine. These findings provide a novel insight into the mechanism for the postnatal development of dopaminergic system and the pathogenesis of human BH4 deficiency.
P3-1-3 脂肪酸結合タンパク質 FABP3 欠損マウスにおける認知機能及び情動行動異常の神経機序 Impaired cognitive function and anxiety-like behaviors in FABP3 null mice ○山本由似¹, 福永浩司², 大和田祐二¹ ○Yui Yamamoto¹, Kohji Fukunaga², Yuji Owada¹ 山口大学医学系研究科　器官解剖学分野¹, 東北大学薬学研究科　薬理学分野² Dept. Organ Anatomy, Yamaguchi Univ. Grad. Sch. Med.¹, Dept. Pharmacology, Tohoku Univ. Grad. Sch. Pharm. Sci.² Brain lipid homeostasis is associated with neuronal development and higher brain functions. However, the mechanisms underlying the effects of long chain polyunsaturated fatty acids (PUFAs) on brain functions at cellular and molecular levels remain unknown. In this study, we found that fatty acid binding protein 3 (FABP3), an intracellular chaperone to transport PUFAs to appropriate intracellular compartments, is involved in the recognition memory and increased anxiety-like behavior. We used immunohistochemistry to show that FABP3 is localized in normal mice cortical neurons. To better understand the role of FABP3 in cognitive function and anxiety-like behavior, we analyzed learning memory and anxiety behaviors, including elevated plus maze test, passive avoidance test, hole-board test and novel object recognition test, in FABP3 null mice. To evaluate plasticity-related proteins expression and activity in the brain of FABP3 null mice, we measured CREB and BDNF expression and phosphorylation level, respectively, by Western blotting.In immunohistochemistry, FABP3 was localized predominantly to the neurons in the cingulate cortex. Behavioral analyses revealed that FABP3 null mice showed impaired cognitive function and increased anxiety-like behaviors. Interestingly, in Western blot analysis, CREB phosphorylation and BDNF expression, important indicators for synaptic plasticity, were significantly decreased in the cingulate cortex of FABP3 null mice.FABP3 regulates limbic nerve activities, and plays pivotal roles in cognitive function and anxiety-like behavior through the control of brain lipid homeostasis.	P3-1-4 Withdrawn
P3-1-5 新たなニコチン受容体の内在性修飾蛋白質LyPD2の同定と発現解析 Identification of LyPD2 as a novel endogenous modulator of the nicotinic acetylcholine receptor ○森脇康博¹, 篠塚恵理¹, 西田舞香¹, 石井智裕¹, 中村太洋¹, 稲葉翔¹, 三澤日出巳¹ ○Yasuhiro Moriwaki¹, Eri Shinozuka¹, Maika Nishida¹, Tomohiro Ishii¹, Taiyo Nakamura¹, Shou Inaba¹, Hidemi Misawa¹ 慶應大・薬・薬理¹ Dept Pharmacol, Fac Pharm, Keio Univ¹ The Ly-6/neurotoxin superfamily (Ly6SF) is a group of proteins that has a pattern of four to five disulfide bounds that are essential to build a unique three-dimensional structure, the so-called three-finger domain (TFD). These proteins are either secreted or found as GPI-anchored proteins. The TFD was first identified in the alpha-bungarotoxin, a snake neurotoxin that inhibits the function of nicotinic acetylcholine receptors (nAChR). Recently, it has been reported that some endogenously expressed Ly6SF proteins modulate the function of nAChR, just like the snake neurotoxins. In mammals, more than 30 proteins are known to contain one to three TFD and are categorized as Ly6SF proteins. As a paralog of SLURP-1, which is a positive allosteric ligand for α7 nAChR, we recently identified a Ly6SF protein, LyPD2 (Ly-6/PLAUR domain containing 2). Although LyPD2 mRNA is reported as one of the down-regulated proteins in the dorsal lateral geniculate nuclei in β2 nAChR deficient mice (Rubin et al., PLos One, 6, e18626, 2011), the LyPD2 protein localization is still unknown because no adequate antibody is yet available. In the present study, we firstly adopted a phylogenic tree analysis of Ly6SF proteins in humans, and found that LyPD2 is very similar to other Ly6SF nAChR modulating proteins. Secondly, to analyze the tissue distribution of LyPD2, we developed several rabbit polyclonal antibodies that specifically recognize the mouse LyPD2, and examined its histological localization in the mouse brain.	P3-1-6 側坐核におけるGABA作動性伝達に対するドパミンD₃受容体を介した調節機構 Dopaminergic modulation of GABAergic transmission via D₃ receptor activation in the nucleus accumbens shell ○鴻海俊太郎¹, 小西史朗¹ ○Shuntaro Kohnomi¹, Shiro Konishi¹ 徳島文理大学　香川薬学部　薬理学¹ Dept Neurophysiol, Kagawa School of Pharmaceutical Sci, Tokushima Bunri Univ, Kagawa, Japan¹ One of the main target of the mesolimbic dopaminergic pathway is the nucleus accumbens (NAc), where dopamine plays a crucial role in modulation of excitatory and inhibitory synaptic transmission. Both D₁-like (D₁) and D₂-like (D₂ and D₃) dopamine receptors have been reported to occur abundantly in the NAc. Using dual whole-cell patch-clamp recording we have previously demonstrated that IPSCs evoked by stimulation of one medium spiny neuron (MSN) and recorded in another MSN were inhibited by activation of either D₁- or D₂-like receptors in the rat NAc shell. However, it remains to be determined which of D₂-like receptor subtypes (that is, D₂ and D₃ receptors) can contribute to this inhibitory action. Because D₃ dopamine receptors have been shown to display specific localization patterns in particular brain regions including the NAc, we hypothesized that D₃ receptors could played a pivotal role in dopaminergic modulation of neurotransmission in this nucleus. In this study, we examined the effects of PD128907, a dopamine D₃ receptor preferential agonist, on spontaneous inhibitory postsynaptic currents (sIPSCs) in mouse MSNs. Synaptic responses were recorded in slices including the NAc cut from 2- to 3-weeks-old mice, and drugs were applied by perfusion. Application of PD128907 decreased both the amplitude and the frequency of sIPSCs recorded from MSNs (8 out of 10 cells tested at 30 nM) in a dose-dependent manner. Based on our previous and present studies, it appears highly likely that both D₂ and D₃ dopamine receptors are mainly involved in suppression of GABAergic inhibitory transmission onto MSNs. Moreover, it seems that D₃ receptor-mediated suppression of IPSCs is produced by both presynaptic and postsynaptic mechanisms, because D₃ receptors occur in both D₁- and D₂-receptor expressing MSNs. Overall, our data suggest that dopaminergic modulation via D₃ receptor activation play an essential role in the control of GABAergic transmission to MSNs in the NAc shell.
P3-1-7 D2-D5-type receptors are activated by dopamine and full-field illumination in adult rat retinal ganglion cells D2-D5-type receptors are activated by dopamine and full-field illumination in adult rat retinal ganglion cells ○緒方元気¹ ○Genki Ogata¹, Tyler W. Stradleigh¹, Gloria J. Partida¹, Andrew T. Ishida^1,2 Dept of NPB, Univ of California, Davis¹, Dept of Ophthalmology and Vision Science, Univ of California, Davis² Dopamine can regulate signal generation and transmission by activating multiple receptors and signaling cascades, especially in striatum, hippocampus, and cerebral cortex. Dopamine modulates an even larger variety of cellular properties in retina, yet has been reported to do so by only D1 receptor-driven cyclic adenosine monophosphate (cAMP) increases or D2 receptor-driven cAMP decreases. We have tested the possibility that dopamine operates differently on retinal ganglion cells, because the ganglion cell layer binds D1 and D2 receptor ligands, and displays changes in signaling components other than cAMP under illumination that should release dopamine. In adult rat retinal ganglion cells, based on patch-clamp recordings, multielectrode recordings, Ca²⁺ imaging, and immunohistochemistry, we find that 1) spontaneous and activated spike firing are inhibited by dopamine, SKF 83959 (an agonist that does not activate homomeric D1 receptors or alter cAMP levels in other systems), and flickering full-field illumination; 2) D1 and D2 receptor antagonists (SCH 23390, eticlopride, raclopride) counteract these effects; 3) these antagonists also block light-induced rises in cAMP, light-induced activation of Ca²⁺/calmodulin-dependent protein kinase II, and dopamine-induced Ca²⁺ influx; and 4) the Ca²⁺ rise is markedly reduced by removing extracellular Ca²⁺ and by an IP3 receptor antagonist (2-APB). These results show that dopamine activates a receptor in adult mammalian retinal neurons that is distinct from classical D1 and D2 receptors, and that dopamine can activate mechanisms in addition to cAMP and cAMP-dependent protein kinase to modulate retinal ganglion cell excitability.	P3-1-8 GABAB Receptor-Mediated Tonic Inhibition of Locus Coeruleus Neurons in Developing and Citalopram-treated Rats ○Han-Ying Wang¹, Rei-Fen Chen^1,2, Ming-Yuan Min^1,2, Hsiu-Wen Yang³ Institute of Zoology, National Taiwan University, Taipei, Taiwan¹, Dept. Life Science, National Taiwan University, Taipei, Taiwan² GABA (γ- amino-butyric acid) has been reported to exert significant effect on excitability of locus coeruleus (LC) neurons; nevertheless, the underlying mechanisms remains unclear. Here we reported tonic inhibition of LC neurons mediated by postsynaptic GABAB receptors (GABABRs). Application of selective GABABR antagonists induced inward current and increased spontaneous firing rate (SFR) in LC neurons. These results show GABABR mediated a tonic inhibition and regulation of which could tune SFR in LC neurons. To further confirm this argument, we elevated ambient GABA by inhibiting GABA transporter 1 & 2 with bath application of (s)-SNAP5114 and NNC711, and found SFR of LC neurons was dramatically suppressed; subsequent application of CGP52466 not only reversed this effect, but also caused a rebound of SFR. In addition to ambient GABA, we also manipulated GABABR functionality in LC neurons by using development and chronic perinatal exposure of citalopram (CTM), a selective-serotonin-reuptake-inhibitor, as models. We observed a developmental increase in GABABR functionality and tonic inhibition in LC neurons, which, interestingly, was not associated with developmental increase in SFR, unless tonic inhibition was removed by blocking GABABR with CGP 54626. These observations indicate that the increasing tonic inhibition could keep SFR of LC neurons at constant level during development. In CTM treated male but not female rats, this developmental increase of GABABR functionality in LC neurons was retarded, corresponding to which was a reduction in the tonic inhibition. This impairment of tonic inhibition in LC neurons during development could partially account for abnormal SFR found in CTM treated male rats. In conclusions, our results show GABABR-mediated tonic inhibition could effectively regulate SFR of LC neurons, which is important for normal development and might be a major player in pathophysiological processes in chronic preinatal CTM exposure condition.
P3-1-9 海馬スライスにおけるL型カルシウムチャンネル依存的なアデノシン放出 The evoked increase of extracellular adenosine in rat hippocampus CA1 region depends on L-type Ca²⁺ channel ○森田光洋¹, 山城邦比古¹ ○Mitsuhiro Morita¹, Kunihiko Yamashiro¹ 神戸大学大学院理学研究科生物学専攻¹ Dept Biol, Kobe Univ¹ The extracellular concentration of adenosine in the brain fluctuates, and alters neuronal activities and blood flows. Even though the pivotal roles of extracellular adenosine in the brain physiology and pathology are well-established, the source and the release mechanism of adenosine in the brain are still far from to be determined. The identification of regulatory mechanisms underlying the fluctuation of extracellular adenosine concentration in the brain will provide important information for understanding synaptic plasticity, sleep and brain injury. Since the lack of appropriate method to measure extracellular adenosine has hampered the progress of this research filed, we developed a method for measuring extracellular adenosine in hippocampal slice preparation, and successfully measured the adenosine release following electrical stimulation or pharmacological treatment. HEK293 cells expressing adenosine A1 receptor and G_i/q fusion protein showed [Ca²⁺]_i increase in response to adenosine, and allowed to measure adenosine release from slices placed on this sensor cells by Ca²⁺ imaging. High frequency electrical stimulation evoked adenosine release in striatum radiatum of rat hippocampus, and both synaptic and antidromic stimulations were equally effective for the adenosine release. The pharmacological analysis of the antidromically-evoked adenosine release has revealed that Cd²⁺ and nimodipine block the release. These results suggest that the dendrites of CA1 pyramidal neuron release adenosine by a L-type Ca²⁺ channel dependent mechanism.	P3-1-10 発達期小脳からのATP放出の空間分布 Spatiotemporal ATP release to glutamate stimulation in developing rat cerebellar slices using a new ATP imaging system ○村本英樹¹, 原田太一¹, 古川和樹¹, 穂積直裕², 吉田祥子¹ ○Hideki Muramoto¹, Taichi Harada¹, Kazuki Furukawa¹, Naohiro Hozumi², Sachiko Yoshida¹ 豊橋技術科学大学　環境生命工学系¹, 豊橋技術科学大学　電気電子情報工学系² Dept Environ & Life Sci, Toyohashi Univ of Tech, Toyohashi¹, Electrical & Electronic Info Eng, Toyohashi Univ of Tech, Toyohashi² In the many kinds of neuronal transmitters, ATP is a newcomer. Extracellular ATP is known to play several roles as neurotransmitter, neuromodulator and trophic factor in the nervous system. Many functions of extracellular purines are reported in numerous brain areas, where they affect both glial cells and neurons. In developing cerebellar cortex, purinergic stimulation of the inhibitory input to Purkinje cells primarily mediated by activation of P₂X receptors, and the inhibitor of ATP-degrading enzymes suggested endogenous ATP would be released 2 weeks later after birth, however, little is known about the developmental modulation of the released ATP.Recently, we developed a new ATP imaging system using a ATP requiring redox enzyme, and observed distribution and transition of ATP release in the developing cerebellar cortex.ATP was released to 100 μM glutamate stimulation and observed in the lower molecular layer and granular layer. The ATP release was increased drastically at postnatal day 10 (P10), and decreased gradually to P14 with depending manner of extracellular [Ca²⁺]. Both AMPA and NMDA could induce ATP release, whereas tACPD couldn't. The reactivity of ATP release in the developing cerebellum was different from it in the adult spinal cord.Our new measurement system allowed observations of released ATP under several pharmaceutical conditions and developing transition. Using these methods, it is suggested that ATP would contribute to neuronal development in immature cerebellum.
P3-1-11 思春期オスラット海馬歯状回における神経ステロイドestradiolによるNMDA受容体双方向調節 Bidirectional modulatory effects of estradiol on NMDA receptors in the dentate gyrus of juvenile male rats ○田中基樹¹, 曽我部正博^1,2 ○Motoki Tanaka¹, Masahiro Sokabe^1,2 名古屋大学大学院　医学系研究科　細胞生物物理学¹, 名古屋大・革新ナノバイオデバイス研究センター² Dept Physiol, Nagoya Univ Grad Sch of Med, Nagoya, Japan¹, FIRST Research Center for Innovative Nanobiodevice, Nagoya Univ, Nagoya, Japan² The neurosteroid 17β-estradiol (E2) is synthesized by aromatase in both male and female hippocampus and believed to modulate the hippocampal synaptic functions. However, as there are some contradictory results regarding the modulatory effects of E2, the physiological and pathological roles of E2 in the hippocampus remain controversial. Our recent study showed that a low E2 level (1 nM) increases the amplitude of NMDA receptor-mediated EPSCs (NMDAR-EPSCs) and lowers the threshold for the induction of NMDA receptor-dependent long-term potentiation (NMDAR-LTP), while a high E2 level (7 nM) decreases the NMDAR-EPSCs and suppresses the NMDAR-LTP in the dentate gyrus of juvenile male rat hippocampal slices. The present study is a follow up to investigate the mechanisms of those effects of E2. We found that the ERα agonist PPT reproduced the effects of the low E2 level on the NMDAR-EPSCs and the NMDAR-LTP, and the ERβ agonist DPN reproduced the effects of the high E2 level on them in the same experimental conditions. Moreover, 4 nM E2 or PPT, but not DPN, restored the decrease in the NMDAR-EPSCs induced by the aromatase inhibitor letrozole, suggesting that E2 synthesized in the hippocampus under physiological conditions, enhances NMDA receptor activity via ERα. The PPT-induced enhancement of NMDAR-EPSCs was mediated by Src family kinase, while it was not caused by NR2B-specific modulation. These findings provide evidence that E2 exerts condition-dependent bidirectional effects on NMDA receptor activity and thus synaptic plasticity via ERα and ERβ in the dentate gyrus of juvenile male rats.	P3-1-12 統合失調症早期患者での脳内GABA, Glutamateおよびglutamine濃度の変化：MRS研究 Changes of brain gamma-aminobutylic acid (GABA) concentrations in early stage of schizophrenia patients: 3T Proton MRS study ○吉村玲児¹, 後藤直樹¹, 掛田伸吾², 森谷淳二², 興梠征典², 中村純¹ ○Reiji Yoshimura¹, Naoki Goto¹, Shingo Kakeda², Junji Moriya², Yukunori Korogi², Jun Nakamura¹ 産業医科大学・医・精神医学教室¹, 産業医科大学・医・放射線科学教室² Dept Psyciatry, UOEH, Kitakyushu¹, Dept Radiology, UOEH, Kitakyushu² Objective: Dysfunctions in gammma-aminobutyric acid (GABA) neurotransmission might be involved in the pathophysiology of schizophrenia. In the present study, we used MR spectoroscopy at 3T to measure brain concentrations of GABA, and a composite measure of glutamate (glu) and glutamine (gln) in patients with early-stage of schizophrenia. Methods: Enrolled in the study were 18 patients in the early stage of schizophrenia (9 males, 9 females; age range 13-52 y) who fulfilled DSM-IV-TR criteria A, B, D, E, and F, but who were within 6 months from the onset, and finally diagnosed as schizophrenia. Also enrolled were 18 sex- and age-matched 18 healthy controls (9 males, 9 females; age range; 15-49 y). [1H]-MRS was used to measure GABA, and glu+gln in the frontal lobe, basal ganglia, and parieto-occipital lobe. Results: The concentrations of GABA/Cr and glu+gln/Cr in the left basal ganglia, but not in the frontal or parieto-occipital lobe, differed significantly between the early-stage schizophrenia patients and the healthy controls. Conclusions: These preliminary data suggest that dysfunction of GABAergic and glutaminergic neurons exist even in early stage of schizophrenia.
P3-1-13 扁桃体におけるセロトニンおよびカンナビノイドシグナル伝達系の相互調節機構 The interaction between serotonergic and cannabinoidergic neurotransmissions in the basolateral amygdala ○吉田隆行¹, 今野幸太郎², 内ヶ島基政², 大村優¹, 泉剛¹, 渡辺雅彦², 吉岡充弘¹ ○Takayuki Yoshida¹, Kohtaro Konno², Motokazu Uchigashima², Yu Ohmura¹, Takeshi Izumi¹, Masahiko Watanabe², Mitsuhiro Yoshioka¹ 北海道大学大学院医学研究科神経薬理学分野¹, 北海道大学大学院医学研究科解剖発生学分野² Dept Neuropharmacol, Hokkaido Univ Grad Sch Med, Sapporo, Japan¹, Dept Anat, Hokkaido Univ Grad Sch Med, Sapporo, Japan² In the amygdala, it has been demonstrated that cannabinoid CB₁ receptor is particularly enriched in GABAergic axon terminals and moderately expressed on glutamatergic fibers, and involved in the passive or active fear coping strategies. In this study, we found predominant neuronal projections of serotonergic, dopaminergic and cholinergic fibers in the basolateral amygdala (BLA), and CB₁ receptor was frequently localized on serotonergic axon but not dopaminergic, noradrenergic or cholinergic fibers. Furthermore, extracellular release of serotonin was significantly reduced by WIN55212-2, CB agonist, whereas dopamine concentration was not altered, indicating presynaptic serotonin release is modulated by CB₁ receptors. On the other hand, α-methyl-5-HT, 5-HT₂ receptor agonist decreased evoked IPSC and EPSC amplitude accompanied with enhancement of paired pulse ratio and induced inward currents from patch-clamp recording BLA pyramidal neurons, these are parameter of presynaptic effect and postsynaptic localization of 5-HT₂ receptors, respectively. This suppression of IPSC amplitude was completely blocked by MDL100907, 5-HT_2A antagonist, or AM251, CB₁ antagonist. These findings suggest that endocannabinoid is synthesized via activation of postsynaptic 5-HT_2A receptors, and regulates not only inhibitory presynaptic GABA release but also local serotonergic transmission around invaginating synapses in the BLA.	P3-1-14 小胞型GABAトランスポーター (VGAT)-Venusトランスジェニックマウスを用いた扁桃体基底外側群GABA作動性ニューロンの免疫組織化学的分類 Immunohistochemical characterization of GABAergic neurons in the basolateral amygdala complex using vesicular GABA transporter (VGAT)-Venus transgenic mice ○三輪秀樹^1,2, 柳川右千夫^1,2 ○Hideki Miwa^1,2, Yuchio Yanagawa^1,2 群馬大学大学院　医学系研究科　遺伝発達行動学¹, 独立行政法人　科学技術振興機構 CREST² Dept of Genetic and Behavioral Neurosci, Gunma Univ Grad Sch Med, Maebashi, Japan¹, Japan Science and Technology Agency, CREST, Tokyo, Japan² The amygdala plays a critical role in emotional learning and emotional behaviors. The basolateral amygdala complex (BLA) is a key site of neuronal plasticity of fear learning and inhibitory GABAergic neurons in the BLA play an important role in acquisition and extinction of fear memory by controlling projection neuron activities. GABAergic neurons in the BLA, like the cerebral cortex, are morphologically, connectionally, electrically and chemically heterogeneous. However, little is known how each subclass of GABAergic neurons serves its functions for the amygdaloid nuclei. Here we provide the immunohistochemical characterization of calcium-binding protein (parvalbumin, calretinin, and calbindin-D28k)-containing and peptides (somatostatin, vasointestinal polypeptide, cholecystokinin, and neuropeptide Y)-containing GABAergic neurons in the BLA, using vesicular GABA transporter (VGAT)-Venus transgenic mice, in which GABAergic neurons are labeled with Venus fluorescent protein. Our results show that each subclass of calcium-binding protein-containing and peptide-containing GABAergic neurons in the amygdala might have unique properties for regulating amygdaloid functions.
P3-1-15 胎生期・授乳期低用量ビスフェノールA曝露が、生後ラット脳の神経伝達物質量に与える影響 Effects of prenatal and lactation period exposure to low-dose bisphenol A on the levels of brain neurotransmitters in the rat ○新田晃啓¹, 小栗清美¹, 金松知幸¹ ○Akihiro Nitta¹, Kiyomi Oguri¹, Tomoyuki Kanamatsu¹ 創価大学大学院工学研究科環境共生工学専攻¹ Department of Environmental Engineering for Symbiosis, Graduate School of Engineering, Soka University, Tokyo¹ Bisphenol A (BPA) is one of the endocrine disruptor chemicals, which is widely used for industrial product such as polycarbonate plastics and a component of epoxy resins. It has been reported that BPA has an estrogenic activity and is transported to fetus and neonatal infant through placenta and breast milk. It has been also reported that the neurotransmitters such as serotonin (5-HT) and dopamine (DA) were involved in emotional behavior. Recently, we found that the anti-disturbed behavior tested by elevated plus-maze experiment increased in the offspring exposed to BPA during prenatal and lactation periods. The purpose of this study is to clarify the effects of BPA exposure on the levels of neurotransmitters in the brain of offspring. Adult female Wistar rats were treated with 0 (control), 0.1 and 1.0 ppm BPA in drinking water during prenatal and lactation periods. The crania of the male-rat offspring at 19-week-old were treated with microwave and the brains were removed. The coronary sections of 2 mm thickness were dissected out from the brains using rat brain matrix (RBM-4000C). Each section was divided into several regions, such as frontal cortex, striatum and hippocampus. Monoamine levels of the several regions were measured with HPLC. The levels of DA, 3, 4-Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in a part of striatum decreased significantly and that of septum and raphe nucleus increased in BPA exposed group compared with those in control group. Moreover, the levels of 5-HT in a part of striatum decreased and that of 5-hydroxyindoleactic acid (5-HIAA) increased. These results suggest that prenatal and lactation period exposure to low-dose BPA may alter the metabolic dynamics of neurotransmitters, especially in the dopaminergic and serotoninergic neuron, and it may change the emotional behavior.	P3-1-16 中脳とは異なる線条体でのドーパミン量の調節機構 Distinct regulation mechanism of the dopamine content in the striatum from that in the midbrain ○岩下由佳¹, 徳岡宏文¹, 宗実悠佳¹, 村松慎一², 一瀬宏¹ ○Yuka Iwashita¹, Hirofumi Tokuoka¹, Haruka Munezane¹, Shin-ichi Muramatsu², Hiroshi Ichinose¹ 東工大・院・生命理工¹, 自治医科大・医・神経内科² Grad Sch of Biosci and Biotech, Tokyo Inst of Tech, Yokohama, Japan¹, Div of Neurol, Dep of Med, Jichi Med Univ, Tochigi, Japan² Tyrosine hydroxylase (TH) is a rate-limiting enzyme in dopamine synthesis. Tyrosine is converted into L-DOPA by TH, then into dopamine by aromatic amino acid decarboxylase (AADC). Nigrostriatal dopaminergic neurons regulate voluntary movements and reward-dependent behaviors. We previously showed that the reduction of dopamine content was much less than that of TH protein level in the partial Th gene knock-out mice. The results suggest the presence of compensatory regulation to keep dopamine level constant in the striatum. Here, to further understand the mechanisms regulating dopamine levels in the nerve terminals, we took advantage of an adeno-associated virus vector expressing the mouse TH gene (AAV-mTH) to investigate how striatal dopamine levels change when the TH protein is overexpressed. AAV-mTH injection into the substantia nigra of adult mice successfully increased the TH protein levels in both the ventral midbrain and the striatum in the nigrostriatal projection. Accordingly, TH activity in the brain homogenate was increased up to 8-fold in the striatum. However, the striatal dopamine content was unchanged, whereas the dopamine content in the midbrain was significantly increased. Furthermore, in vivo tyrosine hydroxylation activity in the striatum, measured by accumulation of L-DOPA after administration of an AADC inhibitor, was unchanged by AAV-mTH injection. These data suggest that even a substantial increase in TH protein level in the nigrostriatal projection in the adult brain does not directly lead to an increase in dopamine content in nerve terminals, primarily because in vivo tyrosine hydroxylation activity is tightly controlled independent of the TH protein level to maintain a constant dopamine level.
P3-1-17 内因性カンナビノイド2-AGのカンナビノイド受容体を介さない神経活動促進作用 The endocannabinoid 2-arachidonylglycerol increases the frequency of spontaneous synaptic currents in a CB1/CB2-independent manner ○少作隆子¹, 菅原優翔¹, 熊切幸乃¹, 若林優衣¹, 米田貢¹ ○Takako Ohno-Shosaku¹, Yuto Sugawara¹, Yukino Kumakiri¹, Yui Wakabayashi¹, Mitsugu Yoneda¹ 金沢大学・保健・リハビリテーション科学¹ Fac Health Sci, Kanazawa Univ, Kanazawa, Japan¹ Endocannabinoids, endogenous ligands for CB1 and CB2 cannabinoid receptors, are lipid mediators and play an important role in modulatrion of synaptic transmission in the brain. The major endocannabinoid 2-arachidonylglycerol (2-AG) is produced and released from postsynaptic neurons in an activity-dependent manner, and suppresses the transmitter release. In some neurons, it also induces hyperpolarization and inhibits neuronal firing. These inhibitory effects of 2-AG are mediated by CB1 cannabinoid receptors. Here we report a novel excitatory action of 2-AG (or its metabolites), which is independent of CB1/CB2 cannabinoid receptors. Effects of 2-AG and the synthetic cannabinoid agonist WIN55,212-2 on spontaneous synaptic currents were examined in cultured hippocampal neurons. Application of WIN55,212-2 always decreased the frequency of synaptic currents. In contrast, 2-AG application decreased the frequency of synaptic currents in some neurons, but increased it in some other neurons. When neurons were pretreated with the CB1 antagonist AM281, 2-AG increased the frequency of synaptic currents constantly. This CB1-independent excitatory effect of 2-AG was partially suppressed by the blockade of the 2-AG degradation enzyme ABHD6, but not affected by the CB2 antagonist AM630. In AM281-treated neurons, neither resting membrane potential nor action potential was changed by 2-AG application. These data show that 2-AG has dual effects on spontaneous synaptic events. The nhibitory effec is caused by activation of CB1 receptors by 2-AG, and the excitatory effect is caused by 2-AG or its metabolites via CB1/CB2-independent as yet unknown mechanisms.	P3-1-18 胎児期バルプロ酸の投与は発達期小脳の分化とGABA放出を変化させる Valproate application to rat fetus changes developing GABA release and neuronal differentiation ○吉田祥子¹, 田野崎真¹, 福田敦夫², 笛田由紀子³, 関野祐子⁴ ○Sachiko Yoshida¹, Makoto Tanozaki¹, Atsuo Fukuda², Yukiko Fueta³, Yuko Sekino⁴ 豊橋技術科学大学　環境生命工学系¹, 浜松医科大学², 産業医科大学³, 国立医薬品食品衛生研究所⁴ Dept Environment. and Life Sci, Toyohashi Univ of Technology, Toyohashi¹, Hamamatsu Univ. Sch. of Med. Hamamatsu², Univ of Occupational and Environmental Health, Kitakyushu³, National Institute of Health Sciences, Tokyo⁴ γ-amino butyric acid (GABA) plays important roles in cerebellar differentiations and functions. Using the neurotransmitter photo assay device that we developed, GABA was much released from the glial cells in developing cerebellar external granular layer (EGL) from P3 to P7, and dropped down suddenly. Inhibition of GABA transmission by bicuculine, GABA receptor inhibitor, depressed granule cell proliferation in developing cerebellar cortex. Glial GABA would act as excitatory transmitter and/or an inducer for proliferation in developing cerebellar cortex, whereas GABA acts as inhibitory transmitter in mature brain. GABA concentration in the developing EGL was higher than under the developing Purkinje layer, however, GABA release in the molecular layer was decreased to be less than in the mature granular layer. Recently we investigated the effects of application of sodium Valproate to fetus. Valproate, which is the popular antidepressant, and inhibits GABA transaminase, increased GABA release even from early developing periods, and GABA releasing spatial pattern was changed. In addition, GABA_AR α6 expression was observed earlier and its distribution was changed. The application of Valproate caused precocious development of cerebellar cortex, whereas their health and behavior changed slightly. We suggested that GABA conditioned drugs would have some effect to cerebellar development.
P3-1-19 脳血管拡張によるアストロサイトのカルシウム応答 Responses of cortical astrocytes to systemic vasoactivation ○浅田晶子¹, 松木則夫¹, 池谷裕二¹ ○Akiko Asada¹, Norio Matsuki¹, Yuji Ikegaya¹ 東京大学大学院　薬学系研究科　薬品作用学教室¹ Lab. Chem Pharmacol, Grad Sch Pharm, Univ Tokyo, Tokyo, Japan¹ Astrocytes interact with cerebral blood vessel. Most studies have focused on the change in blood vessel diameter triggered by astrocytic calcium activity and release of gliotransmitters. In contrast, less attention is paid to the effect of the change of blood vessel on astocytic activity, although the mechanical sensors are abundantly expressed in astrocytes. To investigate this possibility, we utilized in vivo large-scale imaging and simultaneously recorded cerebrovascular diameter and astrocytic calcium activity from the somatosensory cortex layer 1. To induce diameter change without affecting astrocytes or neurons, mice were intravenously injected norepinephrine, which is known not to pass the blood-brain barrier. In response to norepinephrine, the majority of arterioles and veins exhibited dilation. Cortical astrocytes showed intracellular calcium elevation immediately after dilation of the blood vessel. Calcium activity propagated from astrocytes near artery to those near the capillary in wave-like form. The number of astrocytes that showed calcium activity and the intensity of calcium elevation increased depending on the drug concentration. These results show that peripheral norepinephrine application evokes complex responses of cortical astrocytes and implies novel form of the glio-vascular interaction through mechanical sensation. Further properties and pharmacological profiles are now under investigation.	P3-1-20 アストロサイト株GL261細胞のチャネルロドプシン2活性化によるグルタミン酸放出 Glutamate release from astrocyte cell-line GL261 by photo-activated channelrhodopsin-2 ○小野健治¹, 比嘉円¹, 田畑香織¹, 鈴木弘美¹, 澤田誠¹ ○Kenji Ono¹, Madoka Higa¹, Kaori Tabata¹, Hiromi Suzuki¹, Makoto Sawada¹ 名古屋大環医研脳機能¹ Dept Brain Funct, Res Inst Environ Med, Nagoya Univ, Nagoya¹ Channelrhodopsin-2 (ChR2), derived from Chlamydomonas reinhardtii, is an ion channel activated by blue light. As ChR2-expressing neurons are induced action potentials by blue light exposure, ChR2 is useful for the control of neuronal spiking and synaptic transmission with blue light exposure; however it is unclear whether ChR2 functions in glial cells such as astrocytes, which cannot generate action potentials unlike neurons. Functions of glial cells are also regulated via ion fluxes, some of which are involved in communication between glial cells and neurons and are essential for brain functions. In this study, ChR2-EYFP gene was transferred in GL261 cells, clonal astrocytes, and the effects of blue light exposure was investigated on properties of ChR2-expressing GL261 (GLChR2) cells. Increased intracellular sodium and calcium ion concentrations and an altered membrane potential were observed in GLChR2 cells with blue light exposure. Alterations in the intracellular ion environment caused intracellular acidification and the inhibition of proliferation. In addition, it triggered glutamate release from GLChR2 cells. Glutamate from photo-activated GLChR2 cells acted on N18 cells, clonal neuronal cells, as both a transmitter and neurotoxin depending on its concentration. Our results show that the properties of ChR2-expressing astrocytes can be controlled by blue light exposure, and cation influx through photo-activated ChR2 might trigger functional cation influx via endogenous channels and result in the increase of glutamate release. Further our results suggest that ChR2-expressing glial cells could become a useful tool in understanding the roles of glial cell activation and neural communication in the regulation of brain functions.

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