Viana F, Gibbs L, Berger A J
Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle 98195.
Neuroscience. 1990;38(3):829-41. doi: 10.1016/0306-4522(90)90075-f.
The use of in vitro preparations such as brain slices poses difficulties in determining the correct identity of cells under study. To circumvent this problem, we first used a fluorescence pre-labeling technique (rhodamine-dextran-lysine) to identify cranial motoneurons projecting to the tongue (hypoglossal motoneurons) in the guinea-pig. Following preparation of slices, cells were recorded intracellularly and their electrophysiological properties determined. The cells were then intracellularly stained with both a fluorescence label (Lucifer Yellow) and with the stable, non-fading label biocytin. Under fluorescent illumination, the great majority of recorded cells within the hypoglossal nucleus were double-labeled (rhodamine and Lucifer Yellow) suggesting that most are indeed motoneurons. Biocytin injected into the same motoneurons provided permanent and detailed images of their morphology. Intracellularly stained cells surrounding the hypoglossal nucleus were not labeled with rhodamine and had distinct electro-physiological properties. The use of the retrogradely transported marker rhodamine-dextran-lysine allows the unambiguous identification of motoneurons in a brainstem slice. The combined intracellular injection of Lucifer Yellow and biocytin provides a simple means of melding the advantages of a fluorescent label (compatible with other fluorescence labels and with immunocytochemistry) with the benefits of a stable, non-fading, electron-dense marker. Application of this technique should prove useful in establishing morphological and functional correlates in other areas of the CNS.
使用诸如脑片之类的体外制剂在确定所研究细胞的正确身份方面存在困难。为了规避这个问题，我们首先使用荧光预标记技术（罗丹明 - 葡聚糖 - 赖氨酸）来识别豚鼠中投射到舌头的颅运动神经元（舌下运动神经元）。制备切片后，对细胞进行细胞内记录并确定其电生理特性。然后用荧光标记物（路西法黄）和稳定的、不褪色的标记物生物素对细胞进行细胞内染色。在荧光照明下，舌下神经核内绝大多数记录的细胞被双重标记（罗丹明和路西法黄），这表明大多数确实是运动神经元。注入同一运动神经元的生物素提供了它们形态的永久且详细的图像。舌下神经核周围细胞内染色的细胞未被罗丹明标记，并且具有独特的电生理特性。逆行运输标记物罗丹明 - 葡聚糖 - 赖氨酸的使用使得能够在脑干切片中明确识别运动神经元。路西法黄和生物素的联合细胞内注射提供了一种简单的方法，将荧光标记物（与其他荧光标记物和免疫细胞化学兼容）的优点与稳定的、不褪色的、电子致密标记物的优点相结合。这项技术的应用在建立中枢神经系统其他区域的形态和功能相关性方面应该会被证明是有用的。
