Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina
Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina
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完整视网膜中 GFP 表达细胞群的电生理学特征
DOI：
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Karin Dedek
中科院分区：
文献类型：
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作者：
M. Pottek;Gabriel C. Knop;R. Weiler;Karin Dedek
研究完整组织中特定神经元的生理特性和突触连接对于那些缺乏明显形态特征或显示出低群体密度的细胞来说是一个挑战。这尤其适用于视网膜无长突细胞，这是一种异常多样的中间神经元，在哺乳动物中包括大约30种亚型1。虽然是通过塑造视网膜输出2的视觉处理的关键部分，但到目前为止，这些亚型中的大多数尚未在功能背景下进行研究，因为用记录电极遇到这些细胞是罕见的事件。最近，大量的转基因小鼠品系是可用的，其在膜受体或酶的启动子的控制下表达荧光标记物如绿色荧光蛋白（GFP），所述膜受体或酶仅对给定组织中的神经元的子集具有特异性3，4。因此，这些预先标记的细胞可以在显微镜控制下直接进行微电极靶向，从而可以原位系统地研究其生理特性。然而，荧光标记物的激发伴随着活组织的光毒性风险。在视网膜中，这种方法还受到以下问题的阻碍：激发光引起感光器的适当刺激，从而造成色素漂白并将视网膜回路转移到光适应条件中。这些缺点通过使用由锁模激光器在飞秒范围的短脉冲中递送的红外激发来克服。双光子激发为荧光团激发提供了足够的能量，同时将激发限制在小的组织体积，从而最大限度地减少光损伤的危害5。此外，它使视网膜对视觉刺激有反应，因为红外光（>850 nm）仅被色素吸收很少。在这篇文章中，我们证明了使用转基因小鼠视网膜获得电生理原位记录GFP表达细胞，视觉上有针对性的双光子激发。视网膜准备并保持在黑暗中，并可以受到通过显微镜聚光镜投射的光学刺激（图1）。光响应的膜片钳记录可以与染料填充相结合，以揭示形态学并检查间隙连接介导的染料与相邻细胞的偶联，从而可以在不同的实验水平上研究靶细胞。
Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event. Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments6. In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.
DOI：
10.1073/pnas.96.12.7035
发表时间：
1999-06-08
影响因子：
11.1
作者：
Denk, W;Detwiler, PB
通讯作者：
Detwiler, PB