Wakate Dojo

Wakate Dojo 神経系の発生・再生、神経画像

2DJ2-1 Role of serotonin transporter phosphorylated by protein kinase C in depressive-like behaviors of the stressed mice Takahiro Ito¹，Yuka Hiramatsu¹，Mizuki Uchida¹，Hirotake Hida¹，Fumiya Yamamoto¹，Akira Yoshimi¹，Norio Ozaki²，Yukihiro Noda¹ ¹Div. Clin. Sci. Neuropsychopharmacol., Grad. Sch. Pham., Meijo Univ.，²Dept. Psychiatry, Grad. Sch. Med., Nagoya Univ. Selective serotonin reuptake inhibitors (SSRIs) are the first-line antidepressants in the treatment of major depressive disorders (MDD), and directly bind to the serotonin transporter (SERT). SERT activity is attenuated by the protein kinase C (PKC), which induced SERT phosphorylation and surface expression, thereby SERT is a key regulator of SERT-serotonergic functions. However, it is not clear about role of SERT-serotonergic functions regulated by PKC in depressive-like behaviors in the stressed mice. In the present study, we investigated whether PKC is related to depressive-like behaviors and functional change of SERT mediated by PKC is involved in expression of them in the forced swimming-stressed mice.Mice were forced to swim for 15 min (the 1st swimming: stressed mice) to get the immobility time stable at the 2nd swimming. On the next day, they were forced to swim again (the 2nd swimming: tested mice) and then immobility time was counted during the last 5 min of the 6 min period.The tested mice showing immobility were observed the significant decrease of phosphorylated PKC and SERT expression, or serotonin content, and increase of surface SERT expression, compared to those in the naive mice. Phorbol 12-myristate 13-acetate (PMA: a PKC activator) as well as sertraline (a SSRI) significantly attenuated the immobility and decrease of them in the tested mice. While, chelerythrine (a PKC inhibitor) was exacerbated the immobility in tested mice and decreased the phosphorylated PKC expression. These results suggest that PKC activator attenuates depressive-like behaviors to promote metabolism of SERT through PKC phosphorylation in the forced swimming-stressed mice. PKC activator might be a novel antidepressant in stressed disorders including MDD.		2DJ2-2 Analysis of corticostriatal pathway: approach from neural circuit-selective gene expression systems. Nozomu Yoshioka^1,2,3，Shigeki Kato¹，Masateru Sugawara¹，Kazuto Kobayashi¹ ¹Dept Mol Genetics. Inst of Biomed Sci, Fukushima Med Univ Sch Med，²Div Neurobiol and Anat. Grad Sch Med and Dent Sci, Niigata Univ，³Trans Res Prog, Niigata Univ Corticostriatal neurons provide the excitatory inputs to the striatum from the cerebral cortex. Corticostriatal neurons are classified into two types, according to distinct projection manners. One is an intratelencephalic (IT) type bilaterally projects to the striatum through the corpus callosum, and, the other is a pyramidal tract (PT) type ipsilaterally projects to the striatum while descending the pyramidal tract. In spite of the distinct projection manner of IT type and PT type corticostriatal neurons, their functional divergence has not been understood, due to lack of the method to manipulate each neural activity. In the present study, we have developed the subtype-selective gene expression system for corticostriatal neurons, by using double viral vector infection which induces the transgene expression only in dually virus-infected neurons. We have applied this gene expression system to the chemogenetic manipulation of each neural activity. In future, the neural network driven by IT type and PT type corticostriatal neurons will be revealed. In next, physiological roles of corticostriatal pathway arising from the primary motor cortex (M1) was evaluated by using immunotoxin-mediated cell targeting. To eliminate the corticostriatal pathway, the retrograde lentiviral vector encoding antigen for immunotoxin was infused into the dorsal striatum (DS) and immunotoxin was injected into the M1. Unexpectedly, elimination of corticostriatal pathway originating from the M1 did not affect motor functions and skill learning. However, elimination of the M1-DS pathway impaired the instrumental learning. Our results first revealed the role of the sensorimotor corticostriatal pathway in behaviors.

2DJ2-3 Ebf3 regulates neural differentiation of the developing neocortex. Ryota Iwai^1,2，Hidenori Tabata³，Mayuko Inoue²，Keiichiro Nomura²，Koh-ichi Nagata³，Ken-ichi Mizutani¹ ¹Graduate School of Pharmaceutical Sciences, Kobe Gakuin University，²Doshisha University，³Aichi Human Service Center During neocortical development, newborn neurons, generated from neural stem cells in the ventricular zone, undergo multiphasic radial process to reach their final position within the cortical plate (CP). During this process, postmitotic immature neurons exhibit a multipolar (MP) morphology with multiple neurites and migrate to random directions in the intermediate zone (IZ). Subsequently, they transform from MP to bipolar (BP) morphology with a leading process to migrate into the CP. Recently, the importance of the “MP phase” involved in neural differentiation has received much attention. In MP phase, many genes regulatory networks play key roles, but little is known about their networks. Previously, we demonstrated that PR domain transcription factor Prdm8 is expressed in the IZ, and that the knockdown of Prdm8 results in premature change from MP to BP morphology, whereas the overexpression of Prdm8 maintained the MP morphology. These results suggest that Prdm8 is involved in the control of the MP phase. Here, we performed DNA-microarray analysis and Chromatin immunoprecipitation (ChIP)-seq analysis to clarify Prdm8 downstream effector. We identified that early B-cell factor 3 (Ebf3), a transcription factor including bHLH motif, as Prdm8 target genes, and found that Ebf3 expression was specifically localized in the IZ. Furthermore, time-lapse imaging experiments showed that knockdown of Ebf3 resulted in impairment of invasion into the CP. Finally, we revealed that Ebf3 loss-of-function cells have aberrant leading process. These results indicate that the Prdm8 and Ebf3 mediated regulation of morphological change that normally occur during the MP phase plays a key role in neural differentiation of the developing neocortex.		2DJ2-4 In vivo imaging of AMPA receptor changes during epileptogenesis in drug induced Kindling model Yusuke Shibata^1,2，Chinatsu Ikeda¹，Yoko Kuroki¹，Tomoyuki Miyazaki^1,2，Makoto Higuchi²，Takuya Takahashi^1,2 ¹Dept Phisiol. Grad Sch Med Sci, Yokohama City Univ，²Mole Ima Cent, Natio Inst Radiol Sci The AMPA receptors(AMPARs) is a glutamate receptor responsible for excitatory neurotransmission of the central nervous system. Recent studies have reported abnormal quantitative changes in AMPARs in epilepsy. However, since diversity exists in the quantitative variation of AMPARs in such epilepsy pathology. In our laboratory, we developed AMPARs PET probe and follow AMPARs localization over time in whole brain. Therefore, in this study, we investigated whether quantitative fluctuation of AMPARs associated with epileptogenesis can be detected by this PET probe, and found that AMPARs therapy confirmed whether drugs are effective in identifying epilepsy. PTZ model and pilocarpine model as a drug epilepsy model in which fluctuation of AMPARs was reported and re-administered 5 weeks after drug withdrawal from acquisition of epilepsy to detect latency until seizure occurred. PET imaging was taken of the before acquisition of epilepsy, 3 weeks and 5 weeks. Furthermore, Talampanel, an antagonist of AMPARs was administered at the time of re-administration, and the effect was examined. Compared to the control, the latency to seizure was significantly shorter(p <0.05) in the PTZ, and shorter in the pilocarpine. From PET imaging, PTZ significantly increased the amount of AMPARs in the striatum and thalamus compared with the control, and significantly decreased in the thalamus in the Pilocarpine(P<0.05). Furthermore, Talampanel significantly suppressed the seizure latency in the PTZ group until the same time as the control group but not in the Pilocarpine group (p <0.05). in this results, it was shown that this PET drug is useful for discriminating patients effectively for therapeutic drugs targeting AMPA receptor in epilepsy.

2DJ2-5 A novel PET imaging technique to assess the AMPA receptor dynamics after cortical injury. Yuki Katsuno，Susumu Jitsuki，Hiroki Abe，Tomoyuki Miyazaki，Waki Nakajima，Aoi Jitsuki，Yusuke Shibata，Akane Sano，Kumiko Suyama，Takuya Takahashi Dept Physiology, Yokohama city univ, Yokohama Acute damage to central nervous system such as stroke is a leading cause of serious functional disability. Restoration of functional disability is considered to be the result of compensative neural plasticity in the intact brain regions. Synaptic AMPA receptor (AMPAR) delivery is a fundamental mechanism underlying behaviors that requires neural plasticity. Facilitation of experience-dependent synaptic AMPAR delivery could result in rehabilitative training-dependent motor cortical reorganization after brain damage. However, due to the lack of molecular imaging techniques in vivo, the molecular mechanisms underlying cortical reorganization during functional recovery remain poorly understood. To this end, we developed a novel PET probe to detect the AMPARs in the living brain. Here, we demonstrated that higher level of AMPAR accumulation in the peri-injured region of cortical cryogenic injury after rehabilitative training of forelimb reaching in ‘recovered’ rats, but not ‘non-recovered’ rats. Furthermore, pharmacologically blockade of AMPAR in the AMPAR accumulated region inhibited forelimb reaching performance in ‘recovered’ rats. These results indicate that AMPAR accumulation in the peri-injured region contribute functional recovery after cortical damage. PET imaging of AMPAR will provide the evaluation of degree of recovery in living human brain.		2DJ2-6 Dopamine D1 receptor mediates social defeat stress-induced dendritic growth in the mPFC and regulates stress-induced behavioral resilience in mice Masayuki Taniguchi¹，Ryota Shinohara¹，Aliza T. Ehrlich²，Kentarou Yokogawa²，Yuichi Deguchi²，Atsubumi Ogawa²，Shiho Kitaoka¹，Akira Sawa³，Shuh Narumiya²，Tomoyuki Furuyashiki¹ ¹Div Pharmacol. Grad Sch Med, Kobe Univ，²MIC. Grad Sch Med, Kyoto Univ，³Dept Psychiatry. Sch Med, Johns Hopkins Univ It has been proposed that stress may not only induce emotional and cognitive abnormalities but also promote behavioral resilience to stress, depending on its time and severity. Using social defeat stress in mice, we previously reported that social defeat stress preferentially activates the mesocortical dopaminergic pathway projecting to the medial prefrontal cortex (mPFC), and that repetition of social defeat stress attenuates this activation, leading to social avoidance. However, the mechanisms of action of dopamine in the mPFC in regulating stress-induced behavioral resilience remain elusive. Here we show a role of dopamine D1 receptor in mPFC excitatory neurons for regulating behavioral resilience to social defeat stress. Repeated social defeat stress selectively reduced expression of D1 receptor in mPFC of mice susceptible to repeated social defeat stress. Knockdown of D1 receptor in whole neuronal populations or excitatory neurons in mPFC facilitated induction of social avoidance by social defeat stress. Single social defeat stress induced D1 receptor-mediated ERK phosphorylation and c-Fos expression in mPFC neurons. Whereas repeated social defeat stress reduced dendritic lengths of mPFC layer II/III pyramidal neurons, single social defeat stress increased arborization and spines of apical dendrites of these neurons in a D1 receptor-dependent manner. Collectively, our findings show that D1 receptor and related signaling in mPFC excitatory neurons mediate social defeat stress-induced dendritic growth, and contribute to behavioral resilience to social defeat stress. Therefore, we propose that D1 receptor-mediated dendritic growth in mPFC excitatory neurons confers stress-induced behavioral resilience.

2DJ2-7 Coffee reduces BACE1 expression by activation of proteasomal degradation in human neuroblastoma SH-SY5Y cells. Kazuya Fukuyama¹，Shota Kakio¹，Kenji Kobata²，Toshiharu Suzuki³，Megumi Tago¹，Hiroomi Tamura¹ ¹Dept Hygenic Chem., Grad Sch Pharm Sci, Keio Univ，²Dept Clinical Dietetics and Human Nutrition, Grad Sch Pharm Sci, Josai Univ，³Dept Neuronal Sci, Grad Sch Pharm Sci, Hokkaido Univ ObjectivesCoffee is one of the most world-widely consumed beverage on a daily bases, and recent epidemiological studies have reported that 3-5 cups of coffee per day can reduce the risk of AD. However, the precise molecular mechanisms of the effects of coffee are not fully understood. Therefore, we investigated possible effects of coffee on BACE1 expression using human neuroblastoma SH-SY5Y cells.Methods The cells were exposed to coffee or coffee extracts up to at 2.0% (v/v). After 15 hours, the whole cell lysates were isolated and subjected to immunoblotting for BACE1. Cells were treated with either 25 μM lysosomal inhibitor chloroquine, 10 μM  proteasome inhibitor MG132, or DMSO 1 hr before the coffee treatment. The whole cell lysates were isolated from the cells after 12 hr coffee treatment. Cells were cultured at 96-well plate co-treated with proteasome substrate LLVY-R110 and coffee. Proteasome activity was then measured each 3 hr after the treatment by monitoring the fluorescence intensity.ResultsCoffee reduced BACE1 expression at the protein levels in a dose dependent manner. However, no change was observed in the gene expression of BACE1. A significant accelerated reduction of BACE1 was observed 12hr after coffee treated cells. The coffee-mediated reduction in the BACE1 levels was blocked by the treatment with MG132 but not with CQ. Coffee significantly increase proteasome activity.conclusionCoffee reduced BACE1 protein expression. These activities in coffee may contribute to the preventive effects of coffee on AD. Further study to identify the active components and to elucidate the mechanism of the effects is needed to clarify the molecular basis of prevention of the disease associated with daily coffee consumption.