Kirchgessner A L, Gershon M D, Liu K P, Tamir H
Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
J Neurosci. 1988 Oct;8(10):3879-90. doi: 10.1523/JNEUROSCI.08-10-03879.1988.
Previous studies have identified two neurectoderm-specific serotonin binding proteins (SBP), one with an apparent Mr of 45 kDa, and one of 56 kDa. The current experiments were undertaken to test the hypothesis that these proteins are specific components of serotonergic neurons. Since actin has been found to bind serotonin, the relationship of the 2 forms of SBP to actin was also investigated. Antisera against purified 45 and 56 kDa SBP were raised in rabbits and shown by analyses of immunoblots and differential absorption to be monospecific and not cross-reactive. Neither antiserum reacted with purified actin and none of 3 different anti-actin sera reacted with purified 45 or 56 kDa SBP. The antisera to 45 and 56 kDa were used for immunocytochemical localization of the proteins, which was compared to that of serotonin. SBP immunoreactivity was found in rat brain and spinal cord; however, no significant differences were observed in the pattern of distribution of 45 and 56 kDa SBP-immunoreactive structures. Immunostaining of neuronal perikarya by either SBP antiserum required pretreatment of animals with colchicine. The distribution of neurons and terminals labeled by each antiserum to SBP was similar to that of neurons and terminals labeled by anti-5-HT sera. SBP-immunoreactive neuronal perikarya were present in the nuclei raphe dorsalis, raphe centralis superior, raphe medianus, raphe magnus, raphe obscurus, raphe pallidus, dorsal to the medial lemniscus in the region of the B9 cell group, near the interpeduncular nucleus, in the area postrema, the pars compacta of the substantia nigra, the dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. SBP-immunoreactive fibers and terminals were present in many additional areas of the brain, as well as the spinal cord, where they paralleled those that were immunostained with antibodies to 5-HT. When double-immunostaining was used, serotonin and 45 and 56 kDa SBP immunoreactivities were found to be colocalized in both the brain and spinal cord. Cells and fibers found to be stained by one immunoreagent were also stained by the others; therefore, serotonergic neurons of the CNS probably contain both 45 and 56 kDa SBP. Moreover, it also seems likely that nonserotonergic neurons contain neither form of SBP. These data strongly suggest that SBP is an intrinsic and specific component of serotonergic neurons that can serve as a serotonergic marker.
以往的研究已鉴定出两种神经外胚层特异性血清素结合蛋白（SBP），一种表观分子量为45 kDa，另一种为56 kDa。开展当前实验以检验这些蛋白是血清素能神经元的特异性成分这一假说。由于已发现肌动蛋白可结合血清素，因此还研究了两种形式的SBP与肌动蛋白的关系。在兔体内制备了针对纯化的45 kDa和56 kDa SBP的抗血清，通过免疫印迹分析和差异吸收表明其具有单特异性且无交叉反应性。两种抗血清均不与纯化的肌动蛋白反应，三种不同的抗肌动蛋白血清也均不与纯化的45 kDa或56 kDa SBP反应。用针对45 kDa和56 kDa的抗血清进行蛋白的免疫细胞化学定位，并与血清素的定位进行比较。在大鼠脑和脊髓中发现了SBP免疫反应性；然而，在45 kDa和56 kDa SBP免疫反应性结构的分布模式上未观察到显著差异。用任何一种SBP抗血清对神经元胞体进行免疫染色都需要用秋水仙碱对动物进行预处理。每种抗SBP血清标记的神经元和终末的分布与抗5-羟色胺（5-HT）血清标记的神经元和终末的分布相似。SBP免疫反应性神经元胞体存在于中缝背核、中缝中央上核、中缝正中核、中缝大核、中缝隐核、中缝苍白核、B9细胞群区域内侧丘系背侧、脚间核附近、最后区、黑质致密部、下丘脑背内侧核和弓状核。SBP免疫反应性纤维和终末存在于脑的许多其他区域以及脊髓中，它们与用抗5-HT抗体免疫染色的区域平行。当进行双重免疫染色时，发现血清素以及45 kDa和56 kDa SBP的免疫反应性在脑和脊髓中均共定位。被一种免疫试剂染色的细胞和纤维也被其他试剂染色；因此，中枢神经系统的血清素能神经元可能同时含有45 kDa和56 kDa SBP。此外，非血清素能神经元似乎也不含这两种形式的SBP。这些数据强烈表明SBP是血清素能神经元的一种内在特异性成分，可作为血清素能标记物。
