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

最近，一个辣椒素受体VR-1被克隆并被进一步阐明，它可以响应有毒的热量和质子，并被证明是Trp家族的一员。然而，该受体在体内的生理意义尚未阐明。为了阐明受体在活体条件下的功能，我们需要一种在体膜片钳记录方法来评估受体在生理条件下的功能。全细胞膜片钳记录来自胶状质(SG)神经元，在活体小鼠脊髓中主要接受C传入纤维。出乎意料的是，尽管皮肤上C传入神经元的密度最高，但被测试的SG神经元中没有一个对有毒的高温做出反应。解剖学数据已报道，深背角神经元的一个亚群将它们的树突延伸到SG，并接受来自C传入纤维的输入。因此，我们接下来记录了深层背角神经元，并分析了热反应。正如预期的那样，大约20%的神经元对伤害性热刺激的反应是通过增加一连串的快速EPSCs。然而，在所有被测试的神经元中都没有观察到缓慢的突触电流，这表明热感觉是通过谷氨酸释放到深背角神经元的树突上来传递的。我们在脊髓脑片的SG神经元记录过程中应用辣椒素进一步证实了SG神经元是否接受了表达TRPV1受体的C传入输入。应用辣椒素可引起一连串的mEPSCs，表明所有的SG神经元都接受了被赋予TRPV1受体的C传入输入。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.