Marcel D, Pollard H, Verroust P, Schwartz J C, Beaudet A
Montreal Neurological Institute, Montreal, Quebec, Canada.
J Neurosci. 1990 Aug;10(8):2804-17. doi: 10.1523/JNEUROSCI.10-08-02804.1990.
The fine structural distribution of the enzyme-neutral endopeptidase EC 3.4.24.11 (enkephalinase) was examined by immunoradioautography (using an iodinated monoclonal antibody) and peroxidase immunocytochemistry (using the same probe in nonradioactive form) in the neostriatum of the rat. At the light microscopic level, both techniques revealed a heterogeneous distribution of immunoreactive enkephalinase in the caudoputamen, characterized by the presence of patches of intense immunolabeling prominent against a relatively strong immunoreactive matrix, a pattern reminiscent of mu opioid receptors radioautographically labeled in the same region. Pilot experiments indicated that fixation of the brain with a mixture of 4% paraformaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid did not modify the distribution and only slightly reduced the intensity of striatal enkephalinase antigenicity, provided that the post-fixation period did not exceed 1 hr. In the neostriatum of animals fixed according to this protocol, enkephalinase immunoreactivity was found by electron microscopic immunoradioautography to be exclusively confined to neuronal and glial membrane interfaces. Immunoperoxidase cytochemistry confirmed the association of immunoreactive enkephalinase with the plasma membrane of neurons and, to a lesser extent, of astrocytes and oligodendrocytes. Both immunoradioautographic and immunoperoxidase techniques revealed a predominant association of the enzyme with neuronal perikarya and dendrites. The morphological features of the labeled perikarya, together with the presence of immunoreactive dendritic spines, suggested that some of these neurons corresponded to striatofugal medium spiny neurons. Immunoreactive enkephalinase was also detected at the level of myelinated and unmyelinated axons and axon terminals. These axons could potentially have originated from intrinsic striatal neurons or from the substantia nigra. Statistical analysis of silver grain distribution in electron microscopic immunoradioautographs indicated that immunoreactive enkephalinase was not preferentially concentrated at the level of specific membrane interfaces, but rather, was more or less uniformly distributed on the surface of neurons and/or glial cells. A similarly diffuse localization of the enzyme was apparent in peroxidase-reacted material, though the latter technique also revealed a microheterogeneity in the deposition of the reaction product along the labeled membranes. Finally, quantitative analysis of immunoradioautographs clearly indicated an absence of enkephalinase enrichment at the level of synaptic junctions. The similarity between the light and electron microscopic distribution of enkephalinase observed in the present study, and that previously reported for mu opioid receptors, lends support to the concept that this ectoenzyme may be involved in the inactivation of endogenous opioids in the mammalian neostriatum.
采用免疫放射自显影术（使用碘化单克隆抗体）和过氧化物酶免疫细胞化学方法（使用相同的非放射性形式探针），对大鼠新纹状体中酶中性内肽酶EC 3.4.24.11（脑啡肽酶）的精细结构分布进行了研究。在光学显微镜水平上，两种技术均显示脑啡肽酶免疫反应性在尾壳核中呈异质性分布，其特征是在相对较强的免疫反应性基质中存在密集免疫标记的斑块，这种模式让人联想到在同一区域放射自显影标记的μ阿片受体。初步实验表明，用4%多聚甲醛、0.05%戊二醛和0.2%苦味酸的混合物固定大脑不会改变分布，并且只要固定后时间不超过1小时，只会略微降低纹状体脑啡肽酶抗原性的强度。在按照该方案固定的动物新纹状体中，通过电子显微镜免疫放射自显影发现脑啡肽酶免疫反应性仅局限于神经元和神经胶质膜界面。免疫过氧化物酶细胞化学证实免疫反应性脑啡肽酶与神经元的质膜相关，在较小程度上也与星形胶质细胞和少突胶质细胞的质膜相关。免疫放射自显影术和免疫过氧化物酶技术均显示该酶主要与神经元胞体和树突相关。标记胞体的形态特征以及免疫反应性树突棘的存在表明，其中一些神经元对应于纹状体传出中型多棘神经元。在有髓和无髓轴突以及轴突终末水平也检测到了免疫反应性脑啡肽酶。这些轴突可能源自纹状体内在神经元或黑质。对电子显微镜免疫放射自显影片中银粒分布的统计分析表明，免疫反应性脑啡肽酶并非优先集中在特定膜界面水平，而是或多或少均匀地分布在神经元和/或神经胶质细胞表面。在过氧化物酶反应物质中，该酶也有类似的弥散定位，尽管后一种技术还显示沿着标记膜反应产物的沉积存在微异质性。最后，对免疫放射自显影片的定量分析清楚地表明在突触连接水平不存在脑啡肽酶富集。本研究中观察到的脑啡肽酶在光学和电子显微镜水平的分布与先前报道的μ阿片受体分布相似，这支持了这种外切酶可能参与哺乳动物新纹状体内源性阿片类物质失活的概念。
