Chiodo L A, Kapatos G
Department of Psychiatry, Wayne State University School of Medicine, Detroit, Michigan 48201.
Synapse. 1992 Aug;11(4):294-309. doi: 10.1002/syn.890110405.
Dopamine (DA)-containing neurons in primary dissociated cell cultures derived from the embryonic mouse mesencephalon (day E13) were studied by histochemical and electrophysiological techniques. DA neurons exhibited two distinct morphologies, fusiform and multipolar, tended to reside in groups and organize dendrites into common fascicles. While these neurons expressed the cell-surface marker acetylcholinesterase, the presence of this enzyme could not be used to identify DA neurons unequivocally, since it was also observed in nondopaminergic cells. Neurons were therefore identified as DA by their distinct morphology, and this identification was validated with a double-labeling procedure that entailed the intracellular deposition of a fluorescent dye (Lucifer yellow or ethidium bromide), followed by processing for tyrosine hydroxylase immunocytochemistry. DA neurons identified in this manner were observed to have resting membrane potentials between -50 and -75 mV, input resistances of 50-360 M omega, and membrane time constants of 4.1-14.1 msec. Forty-seven percent of these cells displayed spontaneous activity that was irregular in nature and often contained bursts (burst length was between two and six action potentials). The DA neurons displayed a variety of ionic conductances, including (1) a Na+ conductance (gNa) that underlies the action potential, (2) Ca2+ conductances (gCa) that mediate the nonsomatic low- and high-threshold spikes observed, and (3) at least three K+ conductances (gK). Voltage-clamp analysis revealed several distinct transmembrane ionic currents, including (1) a large, rapidly inactivating tetrodotoxin-sensitive inward Na+ current (INa), (2) a 4-aminopyridine-sensitive, transient early outward K+ current that required a conditioning hyperpolarization of the membrane to be activated by a subsequent depolarization (A-current, IA), (3) a slowly developing inward current that was seen only after a conditioning hyperpolarization of the membrane and that was dependent on the presence of external Ca2+ ions (ICa), and (4) a late-onset, noninactivating K+ current. Between 25% and 54% of the late-onset K+ current was Ca(2+)-dependent and was not affected by tetraethylammonium ions. This current was termed IAHP. The remaining current was not sensitive to changes in the extracellular Ca2+ concentration but was blocked by external tetraethylammonium. This current was termed IK. The direct pressure application of DA (1-200 microM) onto the soma dose-dependently hyperpolarized these neurons; this effect was potentiated by the presence of the catecholamine reuptake blocker cocaine hydrochloride (10-200 microM). Under voltage-clamp conditions, DA was observed to increase IK significantly and had little effect on IAHP.(ABSTRACT TRUNCATED AT 400 WORDS)
利用组织化学和电生理技术，对源自胚胎小鼠中脑（E13天）的原代解离细胞培养物中含多巴胺（DA）的神经元进行了研究。DA神经元呈现出两种不同的形态，即梭形和多极形，倾向于成群分布，并将树突组织成共同的束状结构。虽然这些神经元表达细胞表面标志物乙酰胆碱酯酶，但由于在非多巴胺能细胞中也观察到这种酶的存在，因此不能用该酶的存在来明确鉴定DA神经元。因此，根据其独特的形态将神经元鉴定为DA神经元，并且通过双标记程序验证了这种鉴定，该程序包括在细胞内注入荧光染料（路西法黄或溴化乙锭），然后进行酪氨酸羟化酶免疫细胞化学处理。以这种方式鉴定的DA神经元的静息膜电位在-50至-75 mV之间，输入电阻为50 - 360 MΩ，膜时间常数为4.1 - 14.1毫秒。这些细胞中有47%表现出自发性活动，其性质不规则，且常包含爆发性活动（爆发长度在2至6个动作电位之间）。DA神经元表现出多种离子电导，包括：（1）构成动作电位基础的Na⁺电导（gNa）；（2）介导所观察到的非躯体低阈值和高阈值尖峰的Ca²⁺电导（gCa）；（3）至少三种K⁺电导（gK）。电压钳分析揭示了几种不同的跨膜离子电流，包括：（1）一种大的、快速失活的对河豚毒素敏感的内向Na⁺电流（INa）；（2）一种对4 - 氨基吡啶敏感的、瞬时早期外向K⁺电流，该电流需要膜的预处理超极化才能被随后的去极化激活（A电流，IA）；（3）一种仅在膜的预处理超极化后出现且依赖于外部Ca²⁺离子存在的缓慢发展的内向电流（ICa）；（4）一种延迟出现的、非失活的K⁺电流。迟发性K⁺电流的25%至54%是Ca²⁺依赖性的，且不受四乙铵离子的影响。这种电流被称为IAHP。其余电流对细胞外Ca²⁺浓度的变化不敏感，但被外部四乙铵阻断。这种电流被称为IK。将DA（1 - 200 μM）直接压力施加到胞体上会使这些神经元剂量依赖性地超极化；儿茶酚胺再摄取阻滞剂盐酸可卡因（10 - 200 μM）的存在会增强这种效应。在电压钳条件下，观察到DA显著增加IK，而对IAHP影响很小。（摘要截断于400字）
