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Abstract：
Previous behavioral studies have revealed that CaV3.2 T-type calcium channels support peripheral nociceptive transmission and electrophysiological studies have established the presence of T-currents in putative nociceptive sensory neurons of dorsal root ganglion (DRG). To date, however, the localization pattern of this key nociceptive channel in the soma and peripheral axons of these cells has not been demonstrated due to lack of isoform-selective anti-CaV3.2 antibodies. In the present study a new polyclonal CaV3.2 antibody is used to localize CaV3.2 expression in rodent DRG neurons using different staining techniques including confocal and electron microscopy (EM). Confocal microscopy of both acutely dissociated cells and short-term cultures demonstrated strong immunofluorescence of anti-CaV3.2 antibody that was largely confined to smaller diameter DRG neurons where it co-localized with established immuno-markers of unmyelinated nociceptors, such as, CGRP, IB4 and peripherin. In contrast, a smaller proportion of these CaV3.2-labeled DRG cells also co-expressed neurofilament 200 (NF200), a marker of myelinated sensory neurons. In the rat sciatic nerve preparation, confocal microscopy demonstrated anti-CaV3.2 immunofluorescence which was co-localized with both peripherin and NF200. Further, EM revealed immuno-gold labeling of CaV3.2 preferentially in association with unmyelinated sensory fibers from mouse sciatic nerve. Finally, we demonstrated the expression of CaV3.2 channels in peripheral nerve endings of mouse hindpaw skin as shown by co-localization with Mrgpd-GFP-positive fibers. The CaV3.2 expression within the soma and peripheral axons of nociceptive sensory neurons further demonstrates the importance of this channel in peripheral pain transmission.
摘要：
先前的行为研究表明，CaV3.2T型钙通道支持外周伤害性传递，电生理学研究已确定在背根神经节（DRG）的假定伤害性感觉神经元中存在T电流。迄今为止,然而,由于缺乏同工型选择性抗CaV3.2抗体,该关键伤害性通道在这些细胞的体细胞和外周轴突中的定位模式尚未得到证实.在本研究中，一种新的多克隆CaV3.2抗体用于使用不同的染色技术（包括共聚焦和电子显微镜（EM））定位啮齿动物DRG神经元中的CaV3.2表达。急性解离细胞和短期培养物的共聚焦显微镜显示抗CaV3.2抗体的强免疫荧光，该抗体主要局限于较小直径的DRG神经元，在那里它与已建立的无髓伤害感受器的免疫标记物共定位。例如,CGRP,IB4和外周蛋白。相比之下,较小比例的这些CaV3.2标记的DRG细胞也共表达神经丝200（NF200），有髓鞘的感觉神经元的标记。在大鼠坐骨神经准备中，共聚焦显微镜显示抗CaV3.2免疫荧光与外周蛋白和NF200共定位.Further,EM显示CaV3.2的免疫金标记优先与小鼠坐骨神经的无髓鞘感觉纤维相关。最后,通过与Mrgpd-GFP阳性纤维的共定位，我们证明了CaV3.2通道在小鼠后爪皮肤周围神经末梢的表达。伤害性感觉神经元的体细胞和外周轴突内的CaV3.2表达进一步证明了该通道在外周疼痛传递中的重要性。
