Magnusson K R, Madl J E, Clements J R, Wu J Y, Larson A A, Beitz A J
Department of Veterinary Biology, College of Veterinary Medicine, University of Minnesota, St. Paul 55108.
J Neurosci. 1988 Dec;8(12):4551-64. doi: 10.1523/JNEUROSCI.08-12-04551.1988.
Two monoclonal antibodies against fixative-modified taurine, Tau1 and Tau2, were produced, characterized, and used in the present study to analyze the distribution of taurine in the cerebellum of the rat. In addition, immunohistochemical colocalization experiments were performed to determine whether cerebellar neurons contain both taurine and its synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSADC). In ELISAs, both Tau1 and Tau2 displayed high affinities for taurine conjugated to various carrier proteins and possessed some cross-reactivity for other amino acids which are present in lower concentrations in the brain than taurine. Tau2 was found to recognize only taurine and hypotaurine when paraformaldehyde was used to fix the amino acids to carrier proteins. With the use of glutaraldehyde fixation, Tau1 cross-reacted with conjugates of beta-alanine and hypotaurine and Tau2 cross-reacted strongly with conjugates of cysteic acid and hypotaurine and weakly with cysteine sulfinic acid. Despite different cross-reactivities, Tau1 and Tau2 exhibited almost identical patterns of neuronal staining in bands of Purkinje cells in the cerebellum. Staining of Purkinje cell dendrites was more prominent than staining of the soma. Light immunoreactivity was present in Golgi, stellate, and basket cells. A scattered population of granule cells displayed taurine-like immunoreactivity at the electron microscopic level. Immunostaining was identified in some terminals in the Purkinje cell layer and in a limited number of mossy fibers. Tau2-like immunoreactivity was colocalized with CSADC-like immunoreactivity in the cerebellar neurons described above. These immunoreactive cells may represent a subpopulation of neurons that contain a higher concentration of taurine than neighboring cells due to their ability to synthesize taurine. The intense immunoreactive staining of Purkinje cell dendrites provides support for the hypothesis that calcium-dependent release of taurine in the cerebellum may originate primarily from dendritic rather than synaptic processes and suggests a neuromodulator role for taurine in the cerebellum.
制备、鉴定了两种抗固定剂修饰牛磺酸的单克隆抗体Tau1和Tau2，并在本研究中用于分析大鼠小脑内牛磺酸的分布。此外，进行了免疫组织化学共定位实验，以确定小脑神经元是否同时含有牛磺酸及其合成酶半胱氨酸亚磺酸脱羧酶（CSADC）。在酶联免疫吸附测定（ELISA）中，Tau1和Tau2对与各种载体蛋白结合的牛磺酸均表现出高亲和力，并且对脑内浓度低于牛磺酸的其他氨基酸具有一定交叉反应性。当使用多聚甲醛将氨基酸固定到载体蛋白上时，发现Tau2仅识别牛磺酸和亚牛磺酸。使用戊二醛固定时，Tau1与β-丙氨酸和亚牛磺酸的结合物发生交叉反应，Tau2与半胱磺酸和亚牛磺酸的结合物强烈交叉反应，与半胱氨酸亚磺酸的结合物弱交叉反应。尽管存在不同的交叉反应性，但Tau1和Tau2在小脑浦肯野细胞带中表现出几乎相同的神经元染色模式。浦肯野细胞树突的染色比胞体的染色更明显。高尔基体、星状细胞和篮状细胞中有轻度免疫反应性。在电子显微镜水平上，散在的颗粒细胞群体显示出牛磺酸样免疫反应性。在浦肯野细胞层的一些终末和有限数量的苔藓纤维中鉴定出免疫染色。上述小脑神经元中，Tau2样免疫反应性与CSADC样免疫反应性共定位。这些免疫反应性细胞可能代表了一类神经元亚群，由于它们具有合成牛磺酸的能力，其所含牛磺酸浓度高于相邻细胞。浦肯野细胞树突强烈的免疫反应性染色支持了以下假说：小脑中钙依赖性牛磺酸释放可能主要源于树突而非突触过程，并提示牛磺酸在小脑中具有神经调节作用。
