Functional analysis of purinergic receptors using IMCT technology in transgenic mouse retina

应用免疫组织化学和膜片钳技术研究了P2X嘌呤受体家族在小鼠视网膜胆碱能无长突细胞中的分布。P2X2-嘌呤受体的免疫反应选择性地分布在OFF型胆碱能无长突细胞中，而不在ON型胆碱能无长突细胞中。当IMCT技术从视网膜回路中选择性地切除胆碱能无长突细胞时，OFF型胆碱能无长突细胞中P2X2-嘌呤受体的免疫反应性消失。这些免疫组织化学研究表明，P2X2-嘌呤受体选择性地表达在OFF型胆碱能无长突细胞上。在转基因小鼠视网膜切片制备中，通过绿色荧光蛋白信号鉴定胆碱能无长突细胞。在膜片钳记录下，OFF型胆碱能无长突细胞对ATP的反应大于ON型胆碱能无长突细胞。在非胆碱能无长突细胞中未观察到对ATP的反应。OFF型胆碱能无长突细胞对ATP的反应由P2X2-嘌呤受体介导。电生理数据也支持P2X2-嘌呤受体选择性表达于OFF型胆碱能无长突细胞。ATP还增加了GABA能的输入到ON型和OFF型胆碱能无长突细胞的树突。GABA能输入的增加由不同类型的P2X-嘌呤受体介导。

We investigated the distribution of P2X-purinoceptor families in the cholinergic amacrine cells of the mouse retina by the immunohistochemical technique and the patch clamp technique. The immunoreactivity for P2X2-purinoceptors selectively distributed in the OFF-type cholinergic amacrine cells but not in the ON-type cholinergic amacrine cells. The immunoreactivity for P2X2-purinoceptors in the OFF-type cholinergic amacrine cells disappeared when IMCT technology was introduced to ablate the cholinergic amacrine cells selectively from the retinal circuit. These immunohistochemical studies indicate that P2X2-purinoceptors are selectively expressed on the OFF-type cholinergic amacrine cells. In the slice preparation of the transgenic mouse retina, the cholinergic amacrine cells were identified by the signals of green fluorescent protein. Under the patch clamp recordings, responses to ATP in the OFF-type cholinergic amacrine cells were greater than those in the ON-type cholinergic amacrine cells. No responses to ATP were observed in the non-cholinergic amacrine cells. The responses of ATP in the OFF-type cholinergic amacrine cells were mediated by the P2X2-purinoceptors. The electrophysiological data also support that P2X2-purinoceptors are selectively expressed in the OFF-type cholinergic amacrine cells. ATP also increased the GABAergic inputs to the dendrites of both the ON-type and the OFF-type cholinergic amacrine cells. The increase in the GABAergic inputs were mediated by the different types of P2X-purinoceptors.