Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, California 90095, USA.
J Comp Neurol. 2010 May 15;518(10):1647-69. doi: 10.1002/cne.22294.
Gamma-aminobutyric acid (GABA) is likely expressed in horizontal cells of all species, although conflicting physiological findings have led to considerable controversy regarding its role as a transmitter in the outer retina. This study has evaluated key components of the GABA system in the outer retina of guinea pig, an emerging retinal model system. The presence of GABA, its rate-limiting synthetic enzyme glutamic acid decarboxylase (GAD(65) and GAD(67) isoforms), the plasma membrane GABA transporters (GAT-1 and GAT-3), and the vesicular GABA transporter (VGAT) was evaluated by using immunohistochemistry with well-characterized antibodies. The presence of GAD(65) mRNA was also evaluated by using laser capture microdissection and reverse transcriptase-polymerase chain reaction. Specific GABA, GAD(65), and VGAT immunostaining was localized to horizontal cell bodies, as well as to their processes and tips in the outer plexiform layer. Furthermore, immunostaining of retinal whole mounts and acutely dissociated retinas showed GAD(65) and VGAT immunoreactivity in both A-type and B-type horizontal cells. However, these cells did not contain GAD(67), GAT-1, or GAT-3 immunoreactivity. GAD(65) mRNA was detected in horizontal cells, and sequencing of the amplified GAD(65) fragment showed approximately 85% identity with other mammalian GAD(65) mRNAs. These studies demonstrate the presence of GABA, GAD(65), and VGAT in horizontal cells of the guinea pig retina, and support the idea that GABA is synthesized from GAD(65), taken up into synaptic vesicles by VGAT, and likely released by a vesicular mechanism from horizontal cells.
γ-氨基丁酸（GABA）可能在所有物种的水平细胞中表达，尽管相互矛盾的生理发现导致了关于其在外网状层作为递质的作用的相当大的争议。本研究评估了豚鼠外网状层 GABA 系统的关键成分，豚鼠是一种新兴的视网膜模型系统。使用免疫组织化学和特征明确的抗体评估了 GABA、其限速合成酶谷氨酸脱羧酶（GAD(65)和 GAD(67)同工型）、质膜 GABA 转运体（GAT-1 和 GAT-3）和囊泡 GABA 转运体（VGAT）的存在。还使用激光捕获显微解剖和逆转录聚合酶链反应评估了 GAD(65)mRNA 的存在。特异性 GABA、GAD(65)和 VGAT 免疫染色定位于水平细胞体，以及外丛状层中的其突起和顶端。此外，对整个视网膜和急性分离的视网膜进行免疫染色显示 A 型和 B 型水平细胞中均存在 GAD(65)和 VGAT 免疫反应性。然而，这些细胞不含有 GAD(67)、GAT-1 或 GAT-3 免疫反应性。在水平细胞中检测到 GAD(65)mRNA，并且扩增的 GAD(65)片段的测序显示与其他哺乳动物 GAD(65)mRNA 约 85%的同一性。这些研究表明 GABA、GAD(65)和 VGAT 存在于豚鼠视网膜的水平细胞中，并支持 GABA 由 GAD(65)合成、通过 VGAT 摄取到突触小泡中、并可能通过水平细胞的囊泡机制释放的观点。
