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3O3-1 A Flp-dependent G-CaMP9a transgenic mouse for neuronal imaging in vivo Masayuki Sakamoto¹，Masatoshi Inoue¹，Kazuki Sakai¹，Shigetaka Kobari¹，Sayaka Takemoto-Kimura¹，Manabu Abe²，Kenji Sakimura²，Haruhiko Bito¹ ¹Dept Neurochem. Grad Sch Med, The Univ. of Tokyo，²Dept Cell Neurobiol, Brain Res. Inst, Niigata Univ. Genetically encoded calcium indicators (GECIs) are widely used for detecting calcium transients in somata and processes that are triggered by neuronal activities. In vivo calcium imaging with two-photon microscopy is usually achieved by viral gene delivery using adeno associated virus (AAV). However, viral injection produces heterogeneous and nonstationary expression in neighboring neurons. In addition, current GECI expressing transgenic mice are regulated by Cre and tTA. Here, we generated a novel transgenic line expressing an improved green GECI, named G-CaMP9a, in Flp-dependent manner in various neuronal subpopulations from the Rosa26 locus, driven by a strong CAG promoter. Combining this reporter mouse with appropriate Flp delivery methods (Flp-expressing transgenic mice, AAV, or in utero electroporation) produced robust and stable G-CaMP9a expression in defined neuronal populations in the cortex. In vivo imaging with two-photon microscopy revealed that this transgenic mouse can detect spontaneous and sensory-evoked Ca²⁺ transients in both excitatory and inhibitory neurons with cellular resolution. Our results show that this new reporter line allows investigation of neuronal activity in defined populations in vivo and will notably facilitate dissecting functional relationships of neural networks.		3O3-2 Mitochondrial rescue therapy for Parkinson’s disease model mice Hidaka Haga，Yasushi Yabuki，Kohji Fukunaga Dept Pharmacol. Grad Sch Pharm Sci, Tohoku Univ [Background] Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor dysfunction. About 30 % of PD patients also exhibit cognitive dysfunction, which is no appropriate therapy. We have developed novel mitochondrial rescue therapy using methyl pyruvate and sigma-1 receptor agonist in neurodegenerative diseases (Biochem Biophys Acta 2014;1840:3320). We here focused mitochondrial rescue therapy in MPTP-induced Parkinson’s disease (PD) model mice. [Methods] Animals were treated with MPTP (25 mg/kg, i.p.) for consecutive 5 days. One day after the final injection of MPTP, mice were treated once a day with ethyl pyruvate (EP) (25, 50, 100 mg/kg, i.p.) or sigma-1 receptor agonist, SA4503 (1 mg/kg, i.p.) for consecutive 4 weeks. Mice were then subjected to motor function and cognitive behavioral tests. [Results] Chronic administration of EP (100 mg/kg i.p.) or SA4503 improved not only motor deficits but also cognitive dysfunctions seen in PD model mice. When tissue ATP and tyrosine hydroxylase (TH) protein levels were measured in VTA, striatum and hippocampal CA1 region, decreases in both ATP and TH levels of all regions in PD model mice were restored by EP (100 mg/kg i.p.) or SA4503 administration. [Discussion] Mitochondrial rescue with EP or SA4503 administration is attractive therapy to improve both motor and cognitive dysfunction in PD model mice. The enhancement of ATP may be one of the mechanism underlying neuroprotection of dopaminergic neurons. The rescue of dopaminergic neurons possibly accounts for both improvements in motor dysfunction and cognitive impairment in PD model mice.

3O3-3 Inhibition of Αβ accumulation by T-type calcium channel enhancer and its in vivo pharmacokinetics Hisanao Izumi，Yasuharu Shinoda，Keita Sato，Kohji Fukunaga Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University As Alzheimer disease therapeutics candidate, we have developed SAK3 (Ethyl 2’,3’-dyhydro-8-methyl-2’,4-dioxo-2-peperidinospiro[2-cyclopentene-1,3’-imidazo[1,2-a]-pyridine]-3-carboxylates) (PCT/JP2013/051388). SAK3 stimulates T-type voltage-gated Ca2+ channels (T-VGCC) in Neuro2A cells transfected Cav3.1 and Cav3.3 genes (Yabuki et al., Neuropharmacology 2017;117:1-13). We also reported that SAK3 stimulates acetylcholine release and promotes long-term potentiation in mouse hippocampus. We here tested whether SAK3 reduces amyliod beta (1-42) accumulation in Alzheimer model (APP23) mice. APP23 mice aged 6 and 9 months were treated for two or three months, respectively, with SAK3 (0.5mg/kg, p.o.) and measured amyliod beta (1-42) levels in both soluble and insoluble fractions from APP23 mouse cortex. The chronic administration significantly reduced the amyliod beta (1-42) levels in both fractions. Consistent with reduced amyliod beta (1-42) levels, the number of amyloid plaques assessed by thioflavin-S staining were also significantly reduced by chronic SAK3 treatment. Furthermore, the cognition assessed by novel object recognition task was improved by the chronic SAK3 administration. Using LC-MS/MS systems, we demonstrated a pharmacokinetics of SAK3-d10 to determine the concentration in blood and brain of mice. Taken together, the novel T-type calcium channel enhancer SAK3 have potential pharmacodynamics to reduce amyliod beta (1-42) accumulation/aggregation in APP23 mice.