In vivo patch-clamp analysis of the thermal response in VR-1 knockout mouse

最近，一个被命名为VR-1的辣椒素受体被克隆并进一步阐明了对有害热和质子的反应，并且被证明是TRP家族的一员。然而，该受体在体内的生理意义尚未阐明。为了阐明受体在体内的功能，我们需要一种在体膜片钳记录方法，使我们能够在生理条件下评估受体的功能。采用全细胞膜片钳技术记录了小鼠脊髓胶状质（SG）主要接受C类传入纤维的神经元。出乎意料的是，没有SG神经元测试响应有害的热，尽管从皮肤的C传入的denanderous终止。解剖学数据已经报道，深背角神经元的亚群将它们的树突延伸到SG并接收来自C传入纤维的输入。因此，我们接下来从深背角神经元进行记录并分析热反应。正如预期的那样，大约20%的神经元对有害的热刺激作出反应，增加了快速EPSC的弹幕。然而，在所有测试的神经元中没有观察到缓慢的突触电流，这表明热感觉是通过释放谷氨酸到深背角神经元的树突上来传递的。我们进一步证实了是否SG神经元接受C传入输入表达TRPV 1受体的辣椒素在记录过程中从SG神经元在脊髓切片。辣椒素的应用引起了mEPSC的弹幕，表明所有SG神经元接受C传入输入，赋予TRPV 1受体。这一明显的差异需要解决。

Recently a capsaicin receptor named VR-1 has been cloned and further clarified to respond to noxious heat and proton, and turned out to be one of the TRP family. However, the physiological significance of the receptor in in vivo has not been elucidated yet. To clarify the function of the receptor in in vivo condition, we need an in vivo patch-clamp recording method which enables us to evaluate receptor's function under the physiological conditions. Whole cell patch-clamp recordings were made from substantia gelatinosa (SG) neurons that received predominantly C afferent fibers in the mouse spinal cord in vivo. Unexpectedly, none of SG neurons tested responded to noxious heat, in spite of the densest termination of C afferents from the skin. Anatomical data has reported that a subpopulation of deep dorsal horn neurons extends their dendrites to the SG and receives inputs from C afferent fibers. Thus, we next made recordings from deep dorsal horn neurons and analyzed a heat response. As expected, about 20 % of neurons responded to noxious heat stimulation with increasing a barrage of fast EPSCs. However, no slow synaptic current was observed in all neurons tested, suggesting that the heat sensation is transmitted by a release of glutamate onto the dendrites of deep dorsal horn neurons. We further confirmed whether SG neurons received C afferent inputs expressing TRPV1 receptors by application of capsaicin during the recording from SG neurons in the spinal cord slices. Application of capsaicin elicited a barrage of mEPSCs, indicating that all SG neurons received C afferent inputs which were endowed the TRPV1 receptor. This clear discrepancy is required to be resolved.