Clarke N P, Bevan M D, Cozzari C, Hartman B K, Bolam J P
MRC Anatomical Neuropharmacology Unit, Department of Pharmacology, Oxford, U.K.
Neuroscience. 1997 Nov;81(2):371-85. doi: 10.1016/s0306-4522(97)00247-9.
Several lines of evidence suggest that the cholinergic neurons of the mesopontine tegmentum contain elevated levels of glutamate and are the source of cholinergic terminals in the subthalamic nucleus and entopeduncular nucleus. The object of this study was to test whether cholinergic terminals in the entopeduncular nucleus and subthalamic nucleus, also express relatively high levels of glutamate. To address this, double immunocytochemistry was performed at the electron microscopic level. Perfuse-fixed sections of rat brain were immunolabelled to reveal choline acetyltransferase by the pre-embedding avidin-biotin-peroxidase method. Serial ultrathin sections of cholinergic terminals in both the entoped uncular nucleus and subthalamic nucleus were then subjected to post-embedding immunocytochemistry to reveal glutamate and GABA. Quantification of the immunogold labelling showed that choline acetyltransferase-immunopositive terminals and boutons in both regions were significantly enriched in glutamate immunoreactivity and had significantly lower levels of GABA immunoreactivity in comparison to identified GABAergic terminals. Furthermore, the presumed transmitter pool of glutamate i.e. that associated with synaptic vesicles, was significantly greater in the choline acetyltransferase-positive terminals than identified GABA terminals, albeit significantly lower than in established glutamatergic terminals. In the entopeduncular nucleus, a small proportion of cholinergic terminals displayed high levels of GABA immunoreactivity. Taken together with other immunocytochemical and tracing data, the elevated levels of glutamate in cholinergic terminals in the entopeduncular nucleus and subthalamic nucleus, is further evidence adding weight to the suggestion that acetylcholine and glutamate may be co-localized in both the perikarya and terminals of at least a proportion of neurons of the mesopontine tegmentum.
多项证据表明，中脑桥脑被盖区的胆碱能神经元谷氨酸水平升高，是丘脑底核和脚内核中胆碱能终末的来源。本研究的目的是测试脚内核和丘脑底核中的胆碱能终末是否也表达相对高水平的谷氨酸。为解决这一问题，在电子显微镜水平上进行了双重免疫细胞化学实验。通过包埋前抗生物素蛋白-生物素-过氧化物酶法对大鼠脑的灌注固定切片进行免疫标记以显示胆碱乙酰转移酶。然后，对脚内核和丘脑底核中胆碱能终末的连续超薄切片进行包埋后免疫细胞化学，以显示谷氨酸和γ-氨基丁酸（GABA）。免疫金标记的定量分析表明，与已确定的GABA能终末相比，这两个区域中胆碱乙酰转移酶免疫阳性的终末和终扣的谷氨酸免疫反应性显著富集，而GABA免疫反应性水平显著较低。此外，胆碱乙酰转移酶阳性终末中假定的谷氨酸递质池，即与突触小泡相关的部分，虽然明显低于已确定的谷氨酸能终末，但显著大于已确定的GABA终末。在脚内核中，一小部分胆碱能终末显示出高水平的GABA免疫反应性。结合其他免疫细胞化学和追踪数据，脚内核和丘脑底核中胆碱能终末的谷氨酸水平升高，进一步证明了至少一部分中脑桥脑被盖区神经元的胞体和终末中乙酰胆碱和谷氨酸可能共定位的观点。
