生物素胺（神经生物素^（TM））细胞内染色法与辣根过氧化物酶逆行标记法的结合──研究神经元通路的一个可靠方法

INTRACELLULAR BIOTINAMIDE (NEUROBIOTIN￣(TM))STAINING COMBINED WITH RETROGRADE HORSERADISH PEROXIDASE LABELING ── A RELIABLE MEANS TO DETECT NEURONAL PATHWAYS

本文阐明了利用细胞内神经生物素（Ｎｅｕｒｏｂｉｏｔｉｎ）染色与ＨＲＰ逆行追踪相结合的方法，研究大鼠三叉神经本体感觉的传导通路．首先将ＨＲＰ分别注射于或微电泳亚对侧丘脑腹后内侧核和后核群，同侧颈髓Ｃ２～Ｃ４平面；同侧闭口肌或同侧三叉神经运动核。２４～４８小时后，用电生理的方法鉴定１～５根闭口肌肌梭传入轴突，分别于细胞内注射神经生物素。ＨＲＰ用ＴＭＢ成色并以钨酸钠作为稳定剂，神经生物素用免疫组化的方法显示．待ＨＲＰ和神经生物素呈色后光镜下发现；在三叉神经运动核，三叉神经感觉核簇和邻近网状结构的ＨＲＰ逆行标记细胞被神经生物素细胞内染色的肌梭传入终扣紧密相贴（Ａｐｐｏｓｉｔｉｏｎ）．这些紧密相贴的结构在电镜下证实为细胞内染色的终扣和ＨＲＰ逆行标记细胞的胞体和的突之间的突触联系．因此，这些ＨＲＰ标记的神经元分别中继来自闭口肌肌梭的三叉神经本体感觉信息至丘脑、颈髓和三叉神经运动核等，分别形成三叉－丘脑和三叉－颈髓的本体感觉传导通路以及与三叉神经运动核之间的直接联系．细胞内神经生物素注射与ＨＲＰ逆行追踪相结合的方法提供了一种追踪中枢神经系统神经环路和神经通路的可靠手段．本方法优越于亲神经病毒追除、原癌基因表达以及神经元溃?

In this paper we describe a method for combining intracellular biotinamide hydrochloride (NeurobiotinTM) labeling with retrograde horseradish peroxidase (HRP) labeling which was developed to study the trigeminal proprioceptive pathways in the rat. Initially HRP was injected or iontophoresed into either the: ventroposteriomedial nucleus and posterior group of the contralateral thalamus ; ipsilateral cervical spinal cord (C2～C4) ; ipsilateral jaw-closing muscles; or ipsilateral trigeminal motor nucleus (Vmo). After 24～48 hours, 1～5 jaw-musclespindle afferent axons were physiologically identified, and intracellularly injected with Neurobiotin. HRP was processed using tetramethylbenzidine as the chromogen and sodium tungstate as the stabilizer while Neurobiotin wasprocessed immunohistochemically. Following these, we observed neurons retrogradely labeled with HRP in thetrigeminal sensory nuclei, Vmo and adjacent reticular formation which were closely apposed by jaw-muscle spindleafferent boutons intracellularly stained with Neurobiotin. Using electron microscopic techniques, these appositionswere shown to contain synapses between intracellularly-stained boutons and the cell bodies and dendrites of theneurons retrogradely labeled with HRP. These HRP-labeled neurons are therefore most likely to relay trigeminalproprioceptive and/or kinesthetic information from jaw-muscle spindle afferents to the thalamus, cervical spinal cord and trigeminal motor nucleus. This method combining intracellular Neurobiotin injection with retrograde HRPlabeling provides a reliable means to trace neuronal circuits and pathways in the central nervous system. Thismethod has numerous advantages over transport via neurotropic viruses ,oncogene expression(c-fos, etc.) and the combinaton of neuronal degeneration with HRP which include its high specificity efficiency, combination of physiology and morphology,and morphology, and adaptation for light and electron microscopy. This method can also be combined withanti-neurotransmitter immunocytochemistry to discern chemical properties and integrative neuronal relationships.