食慾蛋白神經軸突末端在A7核區的超微結構

Abstract

食慾蛋白(orexin)為下視丘分泌的神經蛋白，它參與調節許多的腦部功能，其中包括痛覺的抑制。 有研究指出，A7的兒茶酚胺神經纖維投射到脊髓背角處並且調節脊髓的止痛機制，而食慾蛋白A對於A7的兒茶酚胺神經有興奮的效果。然而，與A7的兒茶酚胺神經細胞相關的食慾蛋白A神經末梢之型態沒有詳細的被描述。因此，本實驗使用小鼠抗酪氨酸羥化酶作為一級抗體標定A7的兒茶酚胺神經細胞，二級抗體使用結合直徑一奈米金粒子的抗小鼠免疫球蛋白，再用銀粒子增加金粒子的直徑，使標定結果可以直接在穿透式電子顯微鏡下被觀察。為了分辨與A7兒茶酚胺神經細胞不同，一級抗體使用兔抗食慾蛋白A來標定有食慾蛋白A的神經纖維。即可在光學顯微鏡下觀察到大量黑色的食慾蛋白A神經纖維在位於三叉神經核區兩百微米前方的A7核區，並且和由酪氨酸羥化酶標定A7兒茶酚胺神經細胞的細胞本體或樹突形成直接的連繫。在電子顯微鏡下，這些接觸是有功能的突觸，在特定區域有突觸前和突觸後細胞膜以及清晰可見的突觸間隙，有增厚的突觸後膜，還有突觸小泡聚集在突觸前側。這些結果顯示食慾蛋白A可經由調控下行的正腎上腺素系統來止痛。

Orexin is a hypothalamic neuropeptide that is involved in many brain functions, including neuropathic pain modulation. Our previous results have shown that orexin-A has excitatory effect on noradrenergic (NAergic) neurons of A7 catecholamine cell groups, which projects NAergic fibers to the dorsal spinal cord and plays a role in spinal antinociception. However morphological detail of that orexin-A terminals associated with NAergic A7 neurons has not been described. Here, we use mouse anti-tyrosine hydroxylase (TH) to identify NAergic neurons in A7 area, and emerge by 1nm gold conjugated anti-mouse IgG, and then intensify by silver enhancement. To distinguish orexin- immunoreactive (ir) fibers from NAergic neurons, we use rabbit anti-orexin-A to identify orexin containing fibers. At light microscopic level, there were many of orexin-ir buttons which were stained in black and found to make physical contacts on the dendrites and soma of A7 NAergic neurons, identified as their immunostaining profile of showing TH-ir and location of ~ 200 micrometers rostral to the trigeminal motor nucleus. Under electron microscopic observation, these contacts were confirmed to be functional synaptic connections as a restricted zone of parallel pre- and postsynaptic membrane specializations with visible synaptic cleft, and/or associated postsynaptic thickening, and the accumulation of synaptic vesicles in the presynaptic profile were identified. These results suggest that orexin-A might exert analgesia through modulation of descending NAergic system.