Molecular mechanisms of exocytotic events in single ventral midbrain dopaminergic neurons : amprometric detection and characterization

单腹侧中脑多巴胺能神经元胞吐事件的分子机制：安培检测和表征

• 批准号: 16590205
• 负责人: SASAKAWA Nobuyuki
• 金额: $ 2.37万
• 依托单位: Sophia University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590205/
• 关键词: exocytosis; neuron; dopamine release; amperometric detection; ドパミンニューロン; 単一細胞; アンペロメトリー

Dopaminergic neurons were enzymatically dissociated from slices of ventral midbrain from postnatal rat pups (first 2 days after birth). The neurons were plated onto glial monolayers and maintained in culture for several weeks. Dopaminergic phenotype was confirmed by the immunocytochemistry of tyrosine hydroxylase. After 2-6 weeks in culture, dopaminergic neurons had cell bodies 20-40μm in diameter, displayed either fusiform or multipolar morphology, and had processes with varicosities of 3-5 pm in diameter. Amperometric detection of single exocytotic events was monitored with a carbon fiber microelectrode (5μm diameter). The applied voltage was +700mV versus the reference electrode. To characterize a single exocytotic event that is detected as a single current spike, the spike parameters (rise time, middle width and area) were analyzed. Exposure of the cells to 90 mM KC1 (1000 hps, 30 sec) evoked exocytotic events with distinctive kinetics. The values of rise time and middle width in dopaminergic neurons were approx. 10-and 20-fold less than those observed in cultured adrenal chromaffin cells, suggesting much faster exocytotic events in the neurons than adrenal chromaffin cells.

多巴胺能神经元通过酶法从出生后幼鼠（出生后前 2 天）的腹侧中脑切片中分离出来。将神经元铺在神经胶质单层上并在培养物中维持数周。通过酪氨酸羟化酶的免疫细胞化学证实多巴胺能表型。培养2-6周后，多巴胺能神经元的细胞体直径为20-40μm，呈梭形或多极形态，并具有直径为3-5μm的静脉曲张突起。使用碳纤维微电极（直径 5μm）监测单个胞吐事件的电流检测。施加的电压相对于参比电极为+700mV。为了表征被检测为单个电流尖峰的单个胞吐事件，分析了尖峰参数（上升时间、中间宽度和面积）。将细胞暴露于 90 mM KC1（1000 hps，30 秒）会引发具有独特动力学的胞吐事件。多巴胺能神经元的上升时间和中间宽度的值约为。比在培养的肾上腺嗜铬细胞中观察到的值低 10 倍和 20 倍，这表明神经元中的胞吐事件比肾上腺嗜铬细胞更快。

项目成果

Spatio-temporal regulation of neurotransmitter release by PKC;studies in adrenal chromaffin cells

PKC 对神经递质释放的时空调节；肾上腺嗜铬细胞的研究

• 发表时间: 2004
• 期刊: Critical Reviews in Neurobiol. 16(1&2)
• 作者: K.Kumakura

Myosin-Va regulates exocytosis through Ca^<2+> tdependent binding of syntaxin-1A.

肌球蛋白-Va 通过 Ca^2 依赖的突触蛋白-1A 结合来调节胞吐作用。

• 发表时间: 2005
• 期刊: Mol. Biol. Cell 16
• 作者: Watanabe;M;Nomura K;Ohyama A.;Ishikawa R;Komiya Y;Hosaka K Yamauchi E;Tarnguchi H;Sasakawa N;Kumakura K;Ushiki T;Sato;Ikebe M;Igarashi M.
• 通讯作者: Igarashi M.

Characterization of exocytotic events from single PC12 cells : Amerometric studies in native PC12h, DA-loadedPC12h cell and bovine adrenal chromaffin cells

单个 PC12 细胞胞吐事件的表征：天然 PC12h、负载 DA 的 PC12h 细胞和牛肾上腺嗜铬细胞的电流测量研究

• 发表时间: 2005
• 期刊: Mol.Cell.Neurobiol. 25
• 作者: Sasakawa;N.;Murayama;N.;Kumakura;K.
• 通讯作者: K.

Amperometric detection and characterization of exocytotic events in sinele ventral midbrain donaminereic neuron

正弦腹侧中脑多纳胺神经元胞吐事件的电流检测和表征

• 发表时间: 2006
• 期刊: J.Pharmacol.Sci., supplement I 100
• 作者: Yamada;S.;et. al.
• 通讯作者: et. al.

Myosin-Va regulates exocytosis through Ca2+-dependent binding of syntaxin-1A

肌球蛋白-Va 通过 Ca2 依赖的突触蛋白-1A 结合调节胞吐作用

• 发表时间: 2005
• 期刊: Mol. Biol. Cell 16
• 作者: Watanabe;M.;Nomura;K.;Ohyama;A.;Ishikawa;R.;Komiya;Y.;Hosaka;Y.;Yamauchi;E.;Taniguchi;H.;Sasakawa;N.;Ushiki;T,. Sato;O.;Kumakura;K.;Ikebe;M.;Igarashi M
• 通讯作者: Igarashi M