Drake C T, Patterson T A, Simmons M L, Chavkin C, Milner T A
Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021, USA.
J Comp Neurol. 1996 Jul 1;370(3):377-95. doi: 10.1002/(SICI)1096-9861(19960701)370:3<377::AID-CNE8>3.0.CO;2-1.
Physiological and pharmacological studies have suggested that kappa opioid receptors (KORs) may be located presynaptically in the guinea pig hippocampal formation. In the present study, KOR-like immunoreactivity (-LI) was examined by using a rabbit antibody raised against a synthetic peptide from the carboxyl terminus of a cloned rat kappa receptor (KT). The specificity of affinity-purified KT antibody was confirmed by Western blotting, enzyme-linked immunosorbent assay, immunolabeling of KORs expressed in Xenopus oocytes, and immunocytochemical preadsorption controls. Specificity also was demonstrated by the light microscopic distribution of KT-LI in sections through the forebrain and the pons, which was largely consistent with the distribution of KORs previously reported, and resembled that of immunoreactivity for dynorphin B, an endogenous ligand for KORs. Detailed analysis of the hippocampal formation revealed that KT-LI was located predominantly in thin processes in the granule cell and inner molecular layers of the dentate gyrus. A few KT-labeled processes were also present in stratum lacunosum-moleculare of the CA1 region and all layers of the CA3 region of the hippocampus. By electron microscopy, KT-LI was restricted to unmyelinated axons and axon terminals, and was associated with plasma membranes, large dense-core vesicles, and cytoplasmic surfaces of small vesicles. In the dentate gyrus, immunolabeled terminals formed asymmetric synapses with granule cell perikarya and large unlabeled dendrites. In the CA3 region of hippocampus, KT-LI was present in small unmyelinated axons. The results of this study 1) demonstrate the specificity of the KT antibody, 2) show that the distribution of KT labeling corresponds well with previous KOR and dynorphin localization in many regions, and 3) provide ultrastructural evidence that KORs are located presynaptically in the guinea pig hippocampal formation.
生理学和药理学研究表明，κ阿片受体（KORs）可能位于豚鼠海马结构的突触前。在本研究中，使用针对克隆大鼠κ受体（KT）羧基末端合成肽产生的兔抗体检测了KOR样免疫反应性（-LI）。通过蛋白质印迹、酶联免疫吸附测定、非洲爪蟾卵母细胞中表达的KORs的免疫标记以及免疫细胞化学预吸附对照，证实了亲和纯化的KT抗体的特异性。KT-LI在前脑和脑桥切片中的光学显微镜分布也证明了其特异性，这在很大程度上与先前报道的KORs分布一致，并且类似于KORs的内源性配体强啡肽B的免疫反应性分布。对海马结构的详细分析表明，KT-LI主要位于齿状回颗粒细胞层和内分子层的细纤维中。海马CA1区的腔隙分子层和CA3区的所有层中也有一些KT标记的纤维。通过电子显微镜观察，KT-LI仅限于无髓轴突和轴突终末，并与质膜、大型致密核心囊泡和小囊泡的胞质面相关。在齿状回中，免疫标记的终末与颗粒细胞胞体和大型未标记的树突形成不对称突触。在海马CA3区，KT-LI存在于小型无髓轴突中。本研究结果1）证明了KT抗体的特异性，2）表明KT标记的分布与许多区域先前的KOR和强啡肽定位非常吻合，3）提供了超微结构证据，表明KORs位于豚鼠海马结构的突触前。
