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

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。

關鍵字

腦幹； A7核區；食慾蛋白；全細胞電生理記錄； TRPV1通道；痛覺傳導

並列摘要

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.

並列關鍵字

brainstem ； A7 cell group ； orexin ； whole-cell recording ； TRPV1 channels ； pain transmission

參考文獻

Bajic, D., and Proudfit, H.K. (1999). Projections of neurons in the periaqueductal gray to pontine and medullary catecholamine cell groups involved in the modulation of nociception. The Journal of comparative neurology 405, 359-379.

Bajic, D., Van Bockstaele, E.J., and Proudfit, H.K. (2001). Ultrastructural analysis of ventrolateral periaqueductal gray projections to the A7 catecholamine cell group. Neuroscience 104, 181-197.

Benham, C.D., Gunthorpe, M.J., and Davis, J.B. (2003). TRPV channels as temperature sensors. Cell Calcium 33, 479-487.

Berg, A.P., Sen, N., and Bayliss, D.A. (2007). TrpC3/C7 and Slo2.1 are molecular targets for metabotropic glutamate receptor signaling in rat striatal cholinergic interneurons. J Neurosci 27, 8845-8856.

Bernardis, L.L., and Bellinger, L.L. (1993). The lateral hypothalamic area revisited: neuroanatomy, body weight regulation, neuroendocrinology and metabolism. Neuroscience and biobehavioral reviews 17, 141-193.

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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

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