Wakate Dojo

Wakate Dojo グリア、ミエリン、神経ネットワーク

2DJ1-1 Morphological analysis of dendritic spine in single neuron derived Atrx mice. Yamaguchi Kouya¹，Norifumi Shioda²，Kohji Fukunaga¹ ¹Dept Pharmacol. Grad Sch Pharm Sci, Tohoku Univ，²Dept Biofunctional Analysis, Gifu Pharm Univ ［Background］Dendritic spine is the protrusion that receives excitatory synaptic inputs in the brain. The morphological change in spine accompanying calcium dynamics and synaptic plasticity is associated with higher brain function such as cognitive and mental function. The Atrx gene is essential for chromatin remodeling and its gene mutations cause mental retardation. We previously reported that the Atrx gene mutative mice (Atrx mice) exhibit symptoms like mental retardation (J Neurosci 2011;31:346). Although the number of filopodia-like spines was increased, the number of mature spines was reduced in Atrx mice. To define the mechanism underlying spine morphological changes, we established method of spine morphological analysis in cultured single neuron. ［Method］Culture of cortical neurons were established using Banker methods with slight modifications. Briefly, cortical tissue was dissected out from embryonic day 18 mice and dissociated. Neurons were plated on coverslips coated with poly-L-lysine. After cell attachment, coverslips were transferred to dishes containing a glial cell monolayer and neurons were cultured until at DIV 5 (developmental stage) or 21 (mature stage). ［Result and Discussion］At DIV 5, the axonal length of cortical neurons from Atrx mice was shorter than in WT mice. The number of filopodia-like spine in Atrx mouse neurons was increased compared with WT mice. At DIV 21, the filopodia-like spines were observed in Atrx mice consistent with in vivo. These results suggest that the spine abnormality observed in Atrx mouse cortical neurons is reproduced in cultured neuron, which is useful to define its mechanism.		2DJ1-2 Abnormal amygdala-dependent fear memory in corticosterone-treated mice Ryo Inagaki，Shigeki Moriguchi，Kohji Fukunaga Dept Pharmacol. Grad Sch Pharm Sci, Tohoku Univ We here report that corticosterone-treated (CORT) mice exhibit impaired amygdala-dependent fear-related memory. CORT mice showed significant impairment of fear-related behaviors assessed by elevated-plus maze, light and dark, open-field and marble burying tasks. In addition, CORT mice were abnormal in tone-induced fear memory but normal in contextual memory. In immunohistochemical analyses, CORT mice revealed significant increase in the number of c-Fos positive cells in the lateral amygdala (LA) following fear-induced tone stimuli. The c-Fos expression peaked at 1 hr. Furthermore, enhancement of CaM kinase II or ERK activities in the LA were observed in CORT mice by immnoblot analyses. In addition, protein or mRNA expression levels of BDNF also significantly increased in the LA of CORT mice. Interestingly, CORT mice significantly potentiate long-term potentiation (LTP) compared with WT mice in the LA. Taken together, the increased CaM kinase II activitiy and in turn c-Fos expression likely account for the dysregulation of amygdala-dependent fear memory in CORT mice.

2DJ1-3 Mitochondrial dysfunction involved in autism-like behaviors in prenatally valproic acid-exposed rats Kazuya Matsuo，Yasushi Yabuki，Kohji Fukunaga Dept Pharmacol. Grad Sch Pharm Sci, Tohoku Univ Autism spectrum disorders (ASD) are a neurodevelopmental disease characterized by social deficits and learning disability. Mitochondrial dysfunction is related to brain pathology in ASD. It is known that prenatal exposure to valproic acid (VPA) increases a risk of pediatric ASD in human. We addressed whether autism-like behaviors in prenatally VPA-exposed rats are accompanied by mitochondrial dysfunction in the brain. Prenatal VPA exposure (600 mg/kg) at E12.5 was conducted and male pups were subjected to memory and social interaction tasks at 5-6 weeks of age. Prenatal VPA exposure elicited impairments in spatial reference memory, object recognition and social interaction. We then analyzed mitochondrial function in hippocampus, where is essential for learning and social skills. Enzymatic activities of mitochondrial electron transport chain complexes I and II were decreased, while complex IV activity was increased in VPA-exposed rats. These abnormal enzymatic activities caused reduction in ATP production. Finally, we assessed respective effects of oxytocin (OXT) and 5-aminolevulinic acid (ALA) on mitochondrial dysfunction in hippocampus and autism-like behaviors in prenatally VPA-exposed rats. Intranasal OXT administration (12 µg/kg), which could be a therapeutic neuropeptide for social deficits in ASD patients, improved autism-like behaviors, but not improved mitochondrial dysfunction in prenatally VPA-exposed rats. On the other hand, oral ALA administration (30 mg/kg), a biosynthesized amino acid in mitochondria, restored not only autism-like behaviors but also improved impaired enzymatic activities of mitochondrial electron transport chain and reduced ATP production. Taken together, ALA treatment ameliorates mitochondrial dysfunction in autism rats.		2DJ1-4 TRPV4 accumulates in astrocytic mitochondria, and regulates its metabolic states Shouta Sugio，Yasuki Ishizaki，Koji Shibasaki Dept Mol Cell Neurobiol. Gunma Univ Grad Sch Medicine We previously demonstrated that a multimodal cation channel, transient receptor potential vanilloid 4 (TRPV4), is expressed in a subset of astrocytes (approximately 30% of astrocytes in the CNS), and its activation triggered the release of gliotransmitters to enhance neuronal excitability (JBC, 2014). To further characterize the TRPV4 function in astrocytes, we examined the intracellular localization of TRPV4. Immunocytochemical analysis demonstrated that TRPV4 is abundantly present in endomembrane, especially in mitochondria, in addition to plasma membrane. As mitochondria synthesize ATP, mitochondrial TRPV4 localization raised the possibility that the channel regulates astrocytic cell metabolism. We demonstrated that TRPV4 activation depolarized mitochondrial membrane potential and resulted in both the significant reduction of cytosolic ATP and the increase of lactate release. As TRPV4 is a non-selective cation channel with high Ca²⁺ permeability (Ca²⁺ > Mg²⁺ > K⁺ > Na⁺ > Li⁺ > H⁺) and a part of enzymes involved in TCA cycle were depend on mitochondrial Ca²⁺ concentration (J Mol Cell Cardiol, 2015), TRPV4 activation might alter the mitochondrial Ca²⁺ concentration and influence the TCA cycle activities, which may in turn suppress the mitochondrial respiratory chain via the decrease of NADH production, and result in the change of astrocytic metabolic states from respiration to glycolysis. Recently it was reported that the lactate released from astrocytes enhances neuronal excitability (Science, 2015). Considering these findings, our results suggest that TRPV4-positive astrocytes can modulate neuronal excitability via not only gliotransmitter release but also lactate release.

2DJ1-5 Pathological study of cuprizone-induced demyelination in the cerebellum Taichi Nomura，Yoshio Bando，Shigetaka Yoshida Dept Functional Anat. and Neurosci, Asahikawa Medical Univ Multiple sclerosis (MS) is an inflammatory demyelinating disease in the central nervous system. Demyelination causes neuronal damage and this is critical for MS prognosis. However, how demyelination affects the neuronal cell bodies has not been fully understood. Cuprizone, copper chelator, induces toxic demyelination in the corpus callosum and the cerebellum. While demyelinated axons in the corpus callosum are apart from their neuronal cell bodies, demyelinated axons and their neuronal cell bodies are close in the deep cerebellar nuclei. Neuronal damage may differ in these two regions. We therefore examined how demyelination affects the deep cerebellar nuclei in the cerebellum in the cuprizone-induced demyelination. Female C57BL/6 mice aged 6 week were fed 0.2% cuprizone diet for 12 weeks. Demyelination was induced in the deep cerebellar nuclei shown by Luxol fast blue staining. Then we investigated the glial and neuronal responses in the deep cerebellar nuclei by immunohistochemical analysis. Three weeks of cuprizone exposure decreased the number of APC-positive oligodendrocytes in the deep cerebellar nuclei compared to control mice. At 4 weeks of cuprizone exposure, microglial cells and astrocytes were accumulated in the deep cerebellar nuclei. In addition, the immunoreactivity of parvalbumin-positive neuronal cell bodies in the deep cerebellar nuclei decreased compared to control mice. Thus, it is a possible hypothesis that cuprizone-induced demyelination lead to impair the neuronal activity in the deep cerebellar nuclei.		2DJ1-6 Phagocytic astrocytes after brain ischemia Yosuke Morizawa¹，Schuichi Koizumi² ¹Super-network Brain Physiology. Grad Sch Life Sciences, Tohoku Univ，²DeptNeuropharmacol. Grad Fac Interdisciplinary Research, Univ. Yamanashi Astrocytes are highly responsive to changes in brain environments including various brain diseases, and dramatically change their genetical and morphological features. However, our understandings of their functions are inadequate. Recent gene profiling study revealed that astrocytes have several phagocytic molecules even in physiological conditions. However, whether astrocytes are able to phagocytose cellular debris or not, moreover, molecular pathways how astrocytes engulf them remain largely unknown. In this study, we show that a subset of reactive astrocytes become phagocytic after transient ischemic injury in limited spatiotemporal pattern. Within the penumbra region, many reactive astrocytes had phagocytic inclusions and expressed phagocytic markers after middle cerebral artery occlusion. Gene and protein expression analysis showed that ABCA1 (ced-7) and its pathway molecules, MEGF10 (ced-1), GULP1 (ced-6), genes implicated in engulfment, were increased in reactive astrocytes. Also, we identified that ABCA1 is responsible for astrocytic phagocytosis in vitro and in vivo. Overall we propose a model in which not only microglia but also astrocytes cooperatively to mediate engulfment of neuronal debris, which in turn lead to recovery of brain microenvironment after brain insult.

2DJ1-7 Study on the neural mechanisms underlying the preference and aversion to environmental temperature Daiki Tachibana，Airi Ido，Hirosi Nomura，Masabumi Minami Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University Selection of ambient temperature is an important behavior conserved in many species to control body temperature. Preference/aversion to ambient temperature is based on pleasant/unpleasant emotion induced by environmental temperature. However, the neural mechanisms by which environmental temperature induces the pleasant/unpleasant emotion remain poorly understood. In order to investigate the neural mechanisms underlying the temperature preference/aversion, we established a new temperature preference/aversion test in which three temperatures (ambient temperature and temperatures of two floor plates) can be controlled independently. Male C57Bl/6J mice (8-10 weeks old) stayed for a longer time on 35ºC plate than on 25ºC plate under the ambient temperature of 28ºC. In contrast, they stayed for a longer time on 25ºC plate than on 35ºC plate under the ambient temperature of 40ºC. Using this temperature preference/aversion test, we examined the involvement of the paraventricular thalamic nucleus (PVT) in the temperature preference/aversion. We focused on the PVT because exposure to the ambient temperature of 38ºC increased c-Fos expression in the PVT of rats. Both PVT-lesioned and control mice preferred 25ºC plate rather than 35ºC plate under the ambient temperature of 40ºC. These results suggest the two possibilities. That is, 1) the PVT is not involved in temperature preference/aversion, 2) temperature preference/aversion is controlled by multiple brain regions, so that the lesion of one of such brain regions is not sufficient to affect temperature preference/aversion. We are conducting further experiments to assess these possibilities.