Biocytin在在体脊髓细胞内标记中的应用

细胞内记录脊髓背角神经元之后，将玻璃微电极中的ｂｉｏｃｙｔｉｎ用去极化方波电流导入。切片经ＡＢＣ法孵育，ＤＡＢ呈色。标记细胞呈Ｇｏｌｇｉ样，胞体直径从数μｍ到数＋μｍ不等，形态各异；树突树完整，光滑或有棘；轴空有时可以显示，如出现串珠样轴突终末及侧枝则提示细胞标记完整。上述结果表明，在在体细胞内记录与细胞内标记相结合中，ｂｉｏｃｙｔｉｎ能够完整地显示所记录细胞的形态。特别是在用于标记中、小细胞有其独到之处。

APPLICATION OF BIOCYTIN IN INTRACELLULAR STAINING OF CELLS IN THE SPINAL CORD DORSAL HORN OF CATS IN VIVO

It is generally agreed that the superficial dorsal horn of the spinal cord,including Rexed Ⅰand Ⅱ, is an integral component of the central representation of pain and temperature sensation. It is also clear that the vast majority of cells in the Rexed Ⅱor marginal zone,if not all,project to the supraspinal structures. like the thalamus, lateral parabrachial nucleus and other brainstem nuclei, while almost all cells in the Rexed Ⅱor substantia gelatinosa are interneurons or propriospinai neurons with few exception,suggesting an existence of functional differentiation between the Rexed Ⅰand Ⅱcells. However, physiological studies combining intracellular recording and injection of horseradish peroxidase (HRP) did not reveal such differentiation. One may argue whether there is or is not a sampling problem due to limitation of methedology. Therefore, the present experiments were designed to explore any existing possible correlation between cellular morphology and physiology in the Rexed Ⅰlayer using new intracellular tracer,biocytin. Using standard intracellular recording and staining techniques in the present study. cells in the superficial dorsal horn of the spinal cord at the lumbar enlargement were recorde and stained with glass microelectrcodes containing biorytin in barbiturate anesthetized cats. Serial 50 μm horizontal sections were proressed using the ABC method. It was found that the vast majority ofphysiologically identified Rexed Ⅰcells could be classified into three types on the basis of their morphology.i.e.pyramidal.fusiform and multipolar cells. pyramidal cells had three major dendrites extending horizontally from the base and vertically from the apex of the cell body. Fusiform cells had two major longitudinal dendrites. Multipolar cells gave off four ur more radiating dendrites. The dendritic trees of pyramidal and fusiform cells were longitudinally oriented. extending more than 1 or 2 mm in the longitudinal axis of the spinal cord, while only extending less than 400 and 100 μm in the mediolateral and dorsoventral directions. respectively. The dendritic trees of multipolar cells also extended for more than 1 or 2 mm in the longitudinal direction. While more than 600 and 150 μm in the mediolateral and dorsoventral directions. respectively. The dendrites were spiny or smooth.The soma size ranged from several to tens of μm. The axons of cells in the Rexed Ⅰlayer were rarely visualized. The axons and their collaterals of Rexed Ⅱor Ⅴcells. however. could be normally identified. Physiologically, Rexed Ⅰcells could be also classified into three groups according to their responses to natural peripheral stimuli.i.e.ncaiceptive specific cells responded to heat and pinch stimuli;cold specific cells responded only to cold stimulus. and multireceptive cells could be activateted by noxious heat.pinch and cold stimuli. Biocytin labeling showed that cold and multireceptive cells were pyramidal and multipolar in shape. Respec tively. while the nociceptive cells fell into fusiform and multipolar types.The present results indicate that like HRP. as an intracellular tracer. biocytin produces a Golgi-like image. In additional to medium and large neurons. biocytin also labels some small cells in the Rexed Ⅰlayer, which could not be stained by HRP, probably because of its very large molecular size. Some correlation between morphology and physiology of cells in the Rexed Ⅰlayer were also also obtained. Thus. the present study favors the application of biocytin as an intracellular tracer in in vivo preparations.