School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska, United States of America.
PLoS One. 2011;6(7):e22721. doi: 10.1371/journal.pone.0022721. Epub 2011 Jul 29.
Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca(2+) signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca(2+) signals in ipRGCs independent of gap junction blockade.
METHODOLOGY/PRINCIPAL FINDINGS: To test the possibility that carbenoxolone directly inhibits light-evoked Ca(2+) responses in ipRGCs, the light-evoked rise in intracellular Ca(2+) (Ca(2+)) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in Ca(2+) in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.
CONCLUSIONS/SIGNIFICANCE: We demonstrate that the light-evoked rise in Ca(2+) in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in Ca(2+) in isolated ipRGCs is almost entirely due to Ca(2+) entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in Ca(2+) in ipRGCs by blocking L-type voltage-gated Ca(2+) channels. The ability of carbenoxolone to block evoked Ca(2+) responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in Ca(2+) is the output being measured.
表达光色素的视网膜神经节细胞是固有光敏的（ipRGCs）。这些神经节细胞光感受器通过谷氨酸能信号向参与各种功能的几个中枢靶标发送轴突。在视网膜内，ipRGCs 通过谷氨酸能信号向多巴胺能无长突细胞提供兴奋性驱动，并且 ipRGCs 通过缝隙连接与宽场 GABA 能无长突细胞偶联。然而，ipRGCs 通过缝隙连接与神经节细胞层中的其他视网膜神经元偶联的程度尚不清楚。广泛使用的缝隙连接抑制剂 carbenoxolone 大大减少了表现出非棒状、非锥体介导的光诱发 Ca(2+)信号的视网膜神经元的数量，表明在神经节细胞层中 ipRGCs 和非 ipRGCs 之间存在广泛的细胞间偶联。然而，carbenoxolone 可能独立于缝隙连接阻断而直接抑制 ipRGCs 中的光诱发 Ca(2+)信号。
方法/主要发现：为了测试 carbenoxolone 是否直接抑制 ipRGC 中的光诱发 Ca(2+)反应的可能性，使用 fura-2 成像在短期培养中离体保持的分离大鼠 ipRGC 中检查了光诱发的细胞内 Ca(2+)（Ca(2+)）升高。在分离的 ipRGC 中，浓度为 50 和 100 μM 的 carbenoxolone 完全消除了光诱发的 Ca(2+)升高。从 carbenoxolone 抑制中恢复是可变的。
结论/意义：我们证明，分离的哺乳动物神经节细胞光感受器中的光诱发 Ca(2+)升高被 carbenoxolone 抑制。由于分离的 ipRGC 中光诱发的 Ca(2+)增加几乎完全归因于通过 L 型电压门控钙通道的 Ca(2+)内流，并且 carbenoxolone 不在原位抑制 ipRGC 中的光诱发动作电位放电，因此 carbenoxolone 可能通过阻断 L 型电压门控 Ca(2+)通道来阻断 ipRGC 中的光诱发 Ca(2+)增加。在解释这种药理学试剂对视网膜或其他神经元回路的影响时，必须考虑到 carbenoxolone 阻断诱发的 Ca(2+)反应的能力，特别是如果测量的输出是 Ca(2+)的变化。
