Corbett Eric K A, Saha Sikha, Deuchars Jim, McWilliam Peter N, Batten Trevor F C
Institute for Cardiovascular Research, School of Medicine, Worsley Building, University of Leeds, LS2 9JT, Leeds, UK.
Auton Neurosci. 2003 May 30;105(2):105-17. doi: 10.1016/S1566-0702(03)00047-X.
The ionotropic glutamate receptor subunits expressed by vagal preganglionic neurones in the rat medulla oblongata were examined by using fluorescence immunolabelling combined with retrograde neuronal tracing. The general population of these neurones in the medulla was identified by intraperitoneal injections of Fluorogold and also with choline acetyltransferase antibodies. Cardiac projecting neurones were specifically identified by applying the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (DiI) to the heart or by injecting cholera toxin B-subunit into the pericardium. Both tracers labelled populations of neurones lying in the dorsal vagal nucleus, intermediate reticular formation and nucleus ambiguus, and when both tracers were applied simultaneously, approximately 50% of cells were dual-labelled. Control experiments established that the labelling was specific for neurones projecting to the heart. Most vagal preganglionic neurones, including those projecting to the heart, irrespective of their location in the medulla, had a similar profile of glutamate receptor immunoreactivity. Labelling of somata for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) subunit GluR1 was weak or absent, while labelling with antibodies directed to GluR2, a common sequence of GluR2 and GluR3, and GluR4 was moderate or intense. All neurones studied appeared to express the N-methyl-D-aspartate (NMDA) receptor subunit NR1, and while antibodies recognising the NR2A and NR2B splice variants gave strong labelling, immunoreactivity with a NR2B specific antibody was weaker. Weak to moderate labelling was seen in some neurones using antibodies to the kainate receptor subunits KA2 and GluR5-7. These results are consistent with neurophysiological data indicating the presence of AMPA, NMDA and kainate responses in cardiac vagal preganglionic neurones, and suggest that these neurones are similar to other vagal parasympathetic preganglionic neurones in expressing mainly AMPA receptor subunits GluR2-4.
运用荧光免疫标记结合逆行神经元追踪技术，对大鼠延髓中迷走神经节前神经元所表达的离子型谷氨酸受体亚基进行了检测。通过腹腔注射荧光金以及使用胆碱乙酰转移酶抗体，确定了延髓中这些神经元的总体分布。通过将荧光示踪剂1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青（DiI）应用于心脏，或向心包内注射霍乱毒素B亚基，特异性地识别了投射至心脏的神经元。两种示踪剂均标记了位于迷走神经背核、中间网状结构和疑核中的神经元群体，当同时应用这两种示踪剂时，约50%的细胞被双重标记。对照实验证实，这种标记对于投射至心脏的神经元具有特异性。大多数迷走神经节前神经元，包括那些投射至心脏的神经元，无论其在延髓中的位置如何，都具有相似的谷氨酸受体免疫反应性特征。α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）亚基GluR1的胞体标记较弱或不存在，而针对GluR2、GluR2和GluR3的共同序列以及GluR4的抗体标记则为中度或强烈。所有研究的神经元似乎都表达N-甲基-D-天冬氨酸（NMDA）受体亚基NR1，虽然识别NR2A和NR2B剪接变体的抗体产生了强烈标记，但与NR2B特异性抗体的免疫反应性较弱。使用针对海人藻酸受体亚基KA2和GluR5-7的抗体，在一些神经元中观察到了弱至中度的标记。这些结果与神经生理学数据一致，表明心脏迷走神经节前神经元中存在AMPA、NMDA和海人藻酸反应，并表明这些神经元在主要表达AMPA受体亚基GluR2-4方面与其他迷走神经副交感节前神经元相似。
