Intravenous injection of Evans Blue labels magnocellular neuroendocrine cells of the rat supraoptic nucleus in situ and after dissociation.
Intravenous injection of Evans Blue labels magnocellular neuroendocrine cells of the rat supraoptic nucleus in situ and after dissociation.
复制标题
静脉注射伊文思蓝原位和解离后标记大鼠视上核的大细胞神经内分泌细胞。
DOI：
10.1016/0306-4522(92)90498-q
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发表时间：
1992
期刊：
影响因子：
3.3
通讯作者：
Cobbett,P
中科院分区：
文献类型：
--
作者：
Weiss,ML;Cobbett,P
先前的研究已经证明，静脉注射神经元示踪剂，如辣根过氧化物酶或Fast Blue，可以逆行标记位于血脑屏障外的大脑区域的神经元，如下丘脑的大细胞神经内分泌神经元。本研究显示，静脉注射荧光逆行示踪剂Evans Blue 24小时后，相同数量的大细胞神经内分泌神经元在室旁、视上和副大细胞核中被标记。室旁核的细胞旁神经内分泌细胞也被标记。大多数Evans blue标记的视上核大细胞神经内分泌细胞可以用免疫细胞化学染色检测神经physins，这表明这些神经元在接受荧光染料后继续产生肽激素。Evans Blue逆行标记的视上细胞超微结构观察显示95%的神经元健康。超微结构未见变性、过度刺激或轴浆血流中断的证据。用埃文斯蓝标记神经内分泌细胞并没有改变大细胞的大小、动物的体液平衡或摄食行为。在24小时前注射Evans Blue的动物的视上核进行酶/机械解离后，观察到通常含有荧光物质的相亮神经元，从而确定这些神经元为神经内分泌。经鉴定的神经内分泌细胞的记录显示，这些神经元产生自发或电流触发的超调动作电位，并伴有后超极化，静息膜电位为负。在去极化电流注入引起的动作电位爆发过程中，观察到动作电位展宽是大细胞神经元的一个特征。综上所述，这项工作表明Evans Blue在使用的剂量下是无毒的，并且它提供了一种方法来鉴定由成人下丘脑制成的原代细胞培养中的单个神经内分泌细胞，用于电压钳记录。
Previous work has demonstrated that intravenous injection of neuronal tracers, e.g. horseradish peroxidase or Fast Blue, can retrogradely label neurons in brain areas that project outside the blood-brain barrier, e.g. magnocellular neuroendocrine neurons of the hypothalamus. Here we have shown that 24 h after intravenous injection of the fluorescent retrograde tracer Evans Blue, the same population of magnocellular neuroendocrine neurons is labeled in the paraventricular, supraoptic and accessory magnocellular nuclei. Parvicellular neuroendocrine cells in the paraventricular nuclei are also labeled. Most Evans Blue-labeled magnocellular neuroendocrine cells in the supraoptic nucleus could be stained immunocytochemically for neurophysins, suggesting that these neurons continue to produce their peptide hormones after taking up the fluorescent dye. Ultrastructural observation of supraoptic cells retrogradely labeled with Evans Blue shows that 95% of the neurons appeared healthy. There was no ultrastructural evidence of degeneration, hyperstimulation, or interruption of the axoplasmic flow. Labeling the neuroendocrine cells with Evans Blue did not alter the size of magnocellular cells, the animal's fluid balance or ingestive behavior. Following enzymatic/mechanical dissociation of the supraoptic nucleus from animals that had been injected with Evans Blue 24 h previously, phase-bright neurons that often contained fluorescent material were observed, thus identifying these neurons as neuroendocrine. Recording from identified neuroendocrine cells showed that these neurons generated spontaneous or currentevoked overshooting action potentials with an afterhyperpolarization and had negative resting membrane potentials. Action potential broadening, a feature of magnocellular neurons, was observed during bursts of action potentials elicited by depolarizing current injection.Taken together, this work would suggest that Evans Blue is non-toxic at the doses used and that it provides a method to identify single neuroendocrine cells in primary cell cultures made from adult hypothalamus for voltage-clamp recordings.