食慾蛋白-A對大鼠A7核區正腎上腺素神經元細胞膜興奮性之影響

Abstract

食慾蛋白(Orexin)為大鼠下視丘一群神經元所分泌的多肽神經傳導物質，它的神經末稍遍布全腦，調控了許多生理上功能，包括痛覺的傳遞。而許多研究指出，A7兒茶酚胺細胞群投射軸突至脊髓背角處，並可產生止痛的效果。為了解食慾蛋白是否在腦幹中調控A7兒茶酚胺神經細胞群的活動，本實驗進行了型態上與電生理上的研究。染色結果顯示，A7核區中有很豐富的食慾蛋白神經末稍分布。從腦薄片上的全細胞電生理紀錄則可發現，給予食慾蛋白後，這些神經元的自發性動作電位頻率有顯著的提升，而如果將膜電位箝制在-70mV，則可觀察到食慾蛋白-A引發了一往細胞內流的電流。此種興奮性的效果會被食慾蛋白受體1的阻斷劑—SB-334867所抑制。除此之外，該電流的反轉電位接近-34mV，且若將細胞外大部分鈉離子取代為NMDG，便偵測不到此食慾蛋白引起的電流，暗示著食慾蛋白可能開啟了一種無選擇性的陽離子通道，如Transient Receptor Potential(TRP)通道。事實上，這股電流在一些非專一性TRP通道的抑制劑(如2-APB, SKF-96365, Ruthenium red)下均有效被抑制；另外，capsazepine(一種專一性的TRPV1通道的拮抗劑)也能顯著減少此食慾蛋白引發的電流。免疫染色結果指出在這些神經元上的確有TRPV1通道的分布，而且此通道的活化劑Capsaicin也能夠引發內流電流，其反轉電位亦接近於-34mV，種種電生理特性皆與食慾蛋白-A引起之反應相符。本研究顯示，食慾蛋白-A會作用在A7兒茶酚胺細胞群的食慾蛋白受體1上，並可能打開了TRPV1通道，進而提升這些神經元的膜興奮性。

Orexin is a neuropeptide that involves in multiple brain functions, including pain modulation. Therefore we investigated the effect of orexin-A on noradrenergic (NAergic) neurons of A7 catecholamine cell group, which projects NAergic fibers to the dorsal spinal cord and plays a role in spinal antinociception. From whole cell recordings of NAergic neurons in the rat brain slices, bath application of orexin-A increases spontaneous firing rate under current clamp mode and activates an inward current(IOXA) under voltage clamp mode with holding voltage -70mV. SB-334867, an orexin receptor 1 antagonist, blocked this response. In addition, IOXA reversed at -33.9mV and was almost eliminated when extracellular Na+ was substituted by N-methyl-D-glucamine(NMDG), suggesting IOXA was mediated by a nonselective cation conductance, presumably by the transient receptor potential(TRP) channels. Indeed, IOXA was significantly blocked by several antagonists of TRP channels, such as 2-APB, SKF-96365, ruthenium red, and a specific TRPV1 antagonist capsazepine. We then tested whether TRPV1 expresses on A7 NAergic neurons. Application of specific TRPV1 agonist capsaicin produced an inward current, which reversed at -33.4mV and was sensitive to both NMDG and capsazepine. Rich expression of TRPV1 proteins was also found on these NAergic neurons using immunohistochemistry techniques. Taken together, we conclude that orexin-A acting on OXR1 excites NAergic neurons in A7 catecholamine cell group through opening TRPV1 channels. Other TRP-like channels may also mediate the response.