古くて新しいGABAの機能～最新研究から見えてきた神経障害の新たな分子基盤～ GABA: An old player provides novel insights into molecular basis of neurological disorders
S2-3-1-1 慢性痛による大脳皮質一次体性感覚野の GABAA 受容体機能の変化 Changes of GABAA receptor function in the primary somatosensory cortex by persistent pain ○石橋仁1, 江藤圭1, 石川達也1,2, 鍋倉淳一1 ○Hitoshi Ishibashi1, Kei Eto1, Tatsuya Ishikawa1,2, Junichi Nabekura1 生理学研究所生体恒常機能発達機構研究部門1, 総合研究大学院大学2 Division of Homeostatic Development, National Institute for Physiological Sciences1, Graduate Univ for Advanced Studies, Hayama, Japan2 The amount of synaptic inhibition and excitation in neural networks needs to be balanced to maintain an appropriate range of neural activity. Inhibitory neurons in cortical areas play key roles in the maintenance of brain function. Experimental manipulations have shown that inhibitory neurotransmission is a critical determinant of neuronal network gain, suggesting that network properties are shaped by GABAergic function. We previously reported that the activity of excitatory layer 2/3 neurons (L2/3) in the primary somatosensory (S1) cortex is increased under persistent pain conditions, but it is not known how the local interneurons, nor the balance between excitation and inhibition, may change in the persistent pain. We report here that the response of layer 2/3 local inhibitory neurons to both sensory stimulation and to layer 4 electrical stimulation increases under inflammatory persistent pain conditions. In addition, local application into L2/3 of a GABAA receptor blocker reduced pain thresholds, while local application of the GABAA receptor modulators (muscimol and diazepam) transiently alleviated the allodynia, illustrating the importance of the local inhibitory pathways in chronic pain sensation. A reduction in the function of the potassium-chloride cotransporter, KCC2, occurred during chronic pain, which reduces the efficacy of the inhibitory outputs onto the L2/3 excitatory neurons. The present results suggest that both excitatory and inhibitory neuronal activities in the S1 are enhanced in the chronic pain model, but the increased inhibition is insufficient to completely counterbalance the increased excitation and alleviate the symptoms of chronic pain.	S2-3-1-2 GABA作動性トニックシグナルと病態 The role of GABAergic tonic signal in the pathophysiology ○山田順子1 ○Junko Yamada1 弘前大学大学院医学研究科　脳神経生理学講座1 Department of Neurophysiology, Hirosaki University Graduate School of Medicine1 The GABAergic system of the mammalian brain controls the inhibitory neuronal output, and it plays a pivotal role of local networks and functional coupling of different brain regions. The dysfunction of GABAergic system has been observed in a wide range of neuronal pathologies, including epilepsy. Synaptically released GABA acting on postsynaptic GABAA receptor produce not only phasic inhibition, but also tonic inhibition by persistent activation of extrasynaptic receptors by GABA spillover. Epilepsy comprises many syndromes, each of which is characterized by pathologic modifications of neuronal excitability and network synchrony. Here, we show two types animal model of epilepsy and their feature. Phospholipase C-related, but catalytically inactive protein (PRIP) was first identified as a novel inositol 1,4,5-triphosphate binding protein. The PRIP-1 subtype is expressed predominantly in the central nervous system and binds directly to the GABAA receptor subunit and several other proteins involved in the trafficking of GABAA-Rs to the plasma membrane. We found that the PRIP-1 knockout mouse (KO) showed an epileptic phenotype, confirmed by electroencephalogram.There was no difference the amplitude and frequency of miniature inhibitory postsynaptic currents between KO and wild-type (WT) neurons. In contrast, the amplitude of the tonic GABA current in KO neurons was markedly reduced compared with that in WT neurons. Dysfunction of extrasynaptic GABAergic transmission probably is involved in the epileptic phenotype of PRIP-1 KO mice.The transgenic rat strain S284L-TG harbors transgenes with the neuronal acetylcholine receptor subunit alpha-4 (Chrna4) mutation that corresponds to the human S284L CHRNA4 mutation identified in pedigrees of patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). S284L-TG rats showed epileptic seizure phenotypes same as human NFLE and exhibited the attenuation of synaptic and extrasynaptic GABAergic transmission.
S2-3-1-3 Angelman症候群モデル（Ube3a機能欠失マウス）小脳顆粒細胞におけるGAT1分解減少によるトニックGABA抑制低下 Diminished tonic GABAA inhibition attributable to decreased GAT1 degradation in cerebellar granule cells in a model of Angelman syndrome (Ube3am-/p+ mice) ○福田敦夫1, 江川潔1 ○Atsuo Fukuda1, Kiyoshi Egawa1 浜松医科大学医学部神経生理学講座1 Department of Neurophysiology, Hamamatsu University School of Medicine1 Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss-of-function of UBE3A gene encoding the E3 ubiquitin ligase Ube3a (also known as E6-AP), which is characterized by mental retardation, epilepsy, speech disorder, frequent laughter/smiling and movement or balance disorder. Ataxia of gait is one of the most common symptoms and disturbs quality of life of AS patients. However, mechanisms of which are still unclear. Ube3a maternal-deficit mice (Ube3am-/p+, AS mice) exhibit phenotypes those seen in human AS. We have evaluated GABAergic functions of AS mice using patch-clamp recordings from acute cerebellar slices. There were no differences in any parameters for spontaneous inhibitory post synaptic current (IPSC). Miniature IPSC, paired pulse ratio and reversal potentials for GABA were also unchanged. In contrast, tonic inhibition was significantly decreased in cerebellar granule cells of AS mice. By immunoprecipitation and degradation assays we show that GABA transporter (GAT) 1 could be a substrate of Ube3a which controls degradation of GAT1. As a mechanism of the decrease of tonic inhibition, Ube3a deficiency induces a surplus of GAT1 and hence of synaptic GABA uptake, resulting in a decrement of ambient GABA levels. Administration of low dose 4,5,6,7-tetrahydroisothiazolo-[5,4-c] pyridin-3-ol (THIP), a selective agonist for extrasynaptic GABAA receptors, e.g. δ subunit, could improve the abnormal firing properties of a population of Purkinje cells in cerebellar brain slices and reduces cerebellar ataxia in AS mice in vivo. These results indicate that decreased tonic inhibition in cerebellar granule cells may underlie ataxia and pharmacologically increasing tonic inhibition can be a pragmatic strategy for alleviating motor problems in AS.	S2-3-1-4 Intracellular Trafficking of Neuronal GABAB Receptors ○Andrés Couve1 Physiology and Biophysics, Instituto de Ciencias Biomédicas and Biomedical Neuroscience Institute, Facultad de Medicina, Universidad de Chile1 The availability of neurotransmitter receptors, which is a critical determinant of synaptic strength, is regulated by coordinated mechanisms that deliver newly synthesized receptors to the plasma membrane and remove them for recycling or degradation. Metabotropic GABABRs are obligatory heteromers responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Deficits in GABABR activity are believed to contribute to the pathology of Fragile X syndrome and Autism. This issue has gained heightened importance as the GABABR agonist arbaclofen has been reported to improve social functioning in Fragile X patients. GABABRs are constituted by two subunits, GABABR1 and GABABR2. Their progression through the secretory pathway is regulated by an endoplasmic reticulum (ER) retention motif present in GABABR1, but the control of their plasma membrane availability still remains unclear. To address these issues we have combined conventional biochemistry with fixed and live-cell imaging in primary cultures of rat central neurons. Studying endocytic trafficking we have demonstrated that prolonged activation of NMDA receptors leads to endocytosis and the activation of a phosphorylation-dependent post-endocytic sorting event that results in the diversion from a recycling route towards lysosomal degradation. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors. Studying biosynthetic trafficking we have demonstrated that in axons GABABRs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABABR1a operate in a pre-Golgi compartment. GABABR1a subunits are enriched in the ER, and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABABR1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. In dendrites endogenous and recombinant GABABR1 subunits are also abundant in the dendritic ER. GABABR1 is transported long distances through the dendritic ER, traffics through satellite Golgi outposts and inserts throughout the somatodendritic plasma membrane. Our studies uncover the role of glutamate receptor activation in regulating the function and surface availability of GABAB receptors in central neurons. Additionally, considering that progression of GABABRs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal and dendritic ER in non-canonical sorting and targeting of neurotransmitter receptors.
S2-3-1-5 GABAB受容体による長期記憶制御のメカニズム The role of postsynaptic GABAB receptors in long-term memory formation ○照沼美穂1 ○Miho Terunuma1 Department of Neuroscience, Tufts University, Boston, MA, USA1 GABAB receptors (GABABRs) are negative regulators of cAMP levels but the significance of this regulation as a long-term determinant of neuronal activity and cognitive function remains unknown. To explore this we created mice with enhanced postsynaptic GABABR signaling by mutating the serine 783 (S783) in the receptor R2 subunit (S783A), which caused modest decreases in PKA activity. Enhanced GABABR activity resulted in deficits in CREB phosphorylation, effector which is critical for memory formation. Consistent with this, S783A mice had selective deficits in long-term spatial memory. Our results revealed a novel and unappreciated role for postsynaptic GABABRs in determining spatial memory via their ability to regulate cAMP signaling and CREB phosphorylation.

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