DISSOCIATED HIGH-PURITY DOPAMINERGIC NEURON CULTURES FROM THE SUBSTANTIA-NIGRA AND THE VENTRAL TEGMENTAL AREA OF THE POSTNATAL RAT
DISSOCIATED HIGH-PURITY DOPAMINERGIC NEURON CULTURES FROM THE SUBSTANTIA-NIGRA AND THE VENTRAL TEGMENTAL AREA OF THE POSTNATAL RAT
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DOI：
10.1016/0306-4522(92)90101-7
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发表时间：
1992-07-01
期刊：
影响因子：
3.3
通讯作者：
NAKAJIMA, Y
中科院分区：
文献类型：
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作者：
MASUKO, S;NAKAJIMA, S;NAKAJIMA, Y
我们从出生后(两到三天)大鼠的黑质和腹侧被盖区获得了分离的原代神经元培养。制作脑片后，分别解剖黑质和腹侧被盖区。将取出的脑组织碎片分离并培养在神经胶质饲养层上。对生长2~3周的培养细胞进行酪氨酸羟化酶和GABA免疫细胞化学双重标记，黑质培养物中有42%的多巴胺能神经元和39%的GABA能神经元，而腹侧被盖区培养物中有65%的多巴胺能神经元和21%的GABA能神经元。多巴胺能神经元以粗直的初级突起为特征，分为多个分支。静脉曲张主要见于突起的远端。与之相反，GABA能神经元具有高度分枝的粗细初级突起，树枝密集，有大量的突起。在来自黑质的多巴胺能神经元和来自腹侧被盖区的多巴胺能神经元中，约70%的多巴胺能神经元和约35%的被盖区的多巴胺能神经元共存。每次生理实验后，免疫细胞化学标记证实该细胞为多巴胺能细胞。单动作电位的特性与已报道的脑片多巴胺能神经元相似，动作电位高度为92 mV，时程为1.6ms。神经元表现为高静息电位，无动作电位自发放电。恒流去极化引起了一连串的动作电位。在大多数细胞中，火车在几秒钟内停止发射，而在一些细胞中，它持续了无限期。当细胞超极化时，电压响应开始缓慢下降(SAG)，这表明存在超极化激活电流(时间依赖的内向整流)。这些结果表明，使用我们的培养方法，可以获得分离的新生大鼠黑质和腹侧被盖区的培养。由于它们富含功能性多巴胺能神经元，这些培养物将是研究多巴胺能神经元各种特性的有用工具。
We have developed dissociated primary neuronal cultures obtained from the substantia nigra and from the ventral tegmental area of postnatal rats (two to three days old). After making brain slices, the regions of the substantia nigra and the ventral tegmental area were separately dissected. The removed fragments of brain tissue were dissociated and cultured on a glial feeder layer. Double immunocytochemical labeling for tyrosine hydroxylase and GABA on cultures grown for two to three weeks showed the presence of 42% dopaminergic and 39% GABAergic neurons in substantia nigra cultures, whereas in ventral tegmental area cultures there were 65% dopaminergic and 21% GABAergic neurons. The dopaminergic neurons were characterized by thick and straight primary processes dividing into several branches. Varicosities were found mainly on distal parts of the processes. In contrast, GABAergic neurons possessed highly branched thick and thin primary processes with intensive arborization and numerous varicosities. Co-existence of dopamine and cholecystokinin was found in about 70% of dopaminergic neurons from the substantia nigra and in about 35% of dopaminergic neurons from the ventral tegmental area.Physiological properties of these cultured dopaminergic neurons were investigated with the whole-cell version of the patch-clamp method. After each physiological experiment, immunocytochemical labeling confirmed that the cell was dopaminergic. Properties of single action potentials, with an action potential height of 92 mV and duration of 1.6 ms, were similar to those reported for dopaminergic neurons in brain slices. The neurons showed a high resting potential, and no spontaneous firing of action potentials. Constant current depolarizations elicited trains of action potentials. In the majority of cells, the train stopped firing within a few seconds, while in some cells it lasted indefinitely. When the cell was hyperpolarized, the voltage response started to decline slowly (sag), indicating the presence of hyper-polarization-activated currents (time-dependent inward rectification).These results show that by using our culture method it is possible to obtain separate dissociated cultures of the substantia nigra and the ventral tegmental area from newborn rats. Because they are rich in functional dopaminergic neurons, these cultures will be a useful tool for studying various properties of dopaminergic neurons.