Orexin A及[Ala11, D-Leu15] orexin-B在大鼠環導水管灰質切片作用之電生理研究

Abstract

Orexins，又稱做hypocretins，是在1998年所發現的神經胜肽，有兩個成員，orexin A( hypocretin 1 )及orexin B( hypocretin 2 )。Orexin A是由33個胺基酸所組成，並且有兩個雙硫鍵，而orexin B則是28個胺基酸所組成；orexin A及orexin B的胺基酸序列的同源相似性為46%。 Orexin受體屬於G蛋白偶合受體( G-protein coupled receptor )，有兩種亞型，分別是OX1R及OX2R；OX1R對於orexin A的親和力比orexin B高，但是OX2R對於兩者的親和力則相似。由於含有orexins的神經元主要位於下視丘，並且軸突會延伸至許多的腦區，例如：midbrain、cerebral cortex、hippocampus、septum、brain stem、pituitary及cerebellum等，因此推測orexins可能與許多生理作用有關，包括痛覺調控、覺醒、進食、內分泌、促進活動力及活化自主神經功能等。 由於2001年在動物的疼痛模式上觀測到，在老鼠腦室給予orexins會產生止痛的效果，而且在疼痛傳導路徑上，PAG位於上傳疼痛產生路徑與下傳疼痛抑制傳導路徑的樞紐，再加上orexins及其受體皆有分佈於PAG，因此，本實驗用盲補綴全細胞記錄的電生理方法觀測orexins在PAG所引發的影響，以研究orexins在vlPAG對神經細胞引起的作用，以解釋orexins在腦部( supraspinal )產生止痛的機制。 本實驗結果發現，在中腦的vlPAG給予orexin A 10~300 nM，發現會有去極化現象產生，並且會依劑量增加導致去極化現象增強的情況發生，而orexin A的EC50為12.3 ± 0.3 nM；當orexin A產生去極化現象後，再同時給予SB-334867 3 micromolar ( 為OX1R拮抗劑 )，會有去極化現象被反轉的現象，可推知orexin A所引發的去極化現象是經由OX1R而來，但由於去極化現象無法完全被SB-334867所抑制，推測無法完全抑制的部份可能是經由OX2R而來；當給予OX2R致效劑[Ala11, D-Leu15] orexin-B 100~300 nM，會使vlPAG神經細胞產生去極化的反應，而此結果不能被SB-334867對抗，顯示活化vlPAG的OX2R亦產生去極化反應，此結果更支持orexin A所產生無法被SB-334867所抑制的去極化反應是經由OX2R而來。 由於在2003年有報導指出orexin A 會抑制GIRK channel的電流，因此為了觀察orexin A是否影響vlPAG上的GIRK channel，因此使用hyperpolarization ramp誘發vlPAG細胞的GIRK channel開啟，再加入orexin A100 nM，發現對於GIRK channel的電流沒有影響，之後再加入BaCl2，可以明顯看見hyperpolarization ramp所引起的電流有減小的情況，分析BaCl2抑制的電流，確實是GIRK 電流；因此推測可能是因GIRK channel在此狀況下的基本活性極低，所以先給予N/OFQ 300 nM誘發GIRK電流後，再給予orexin A 100 nM，結果發現 orexin A對於經由N/OFQ所誘發的GIRK電流並無影響，分析加入N/OFQ後所誘導細胞膜電流，確實是GIRK電流，因此可以確定Orexin A所引起的細胞膜電位改變並非經由GIRK channel；之後以一連串不同的voltage steps觀測orexin A在vlPAG所影響的離子通道，但由於分析過後所得的I-V relationship無一定規則，而無法得知是影響何種離子通道。 本論文結論在vlPAG神經細胞：(1) Orexin A所產生去極化反應，大多是經由OX1R而來；(2) [Ala11, D-Leu15] orexin-B所產生的去極化反應無法被SB-334867反轉，推測是由OX2R而來；(3) Orexin A所引起的細胞膜電位改變並非經由GIRK channel。

Orexins, also known as hypocretins, are a family of hypothalamic neuropeptides derived from prepro-orexin which include: orexin A (hypocretin 1) and orexin B (hypocretin 2). Orexin A and orexin B each consist of 33 and 28 amino acids, respectively, and share 46% sequence homology. Two receptors, OX1R and OX2R, were identified from G-protein coupled orphan receptors. OX1R has a higher affinity for orexin A, while OX2R displays equal affinity to both. Orexin-containing neurons are localized in the hypothalamus and project widely to numerous brain regions, including the periaqueductal gray (PAG). Orexins have been implicated in the several brain functions, including arousal, feeding, pain regulation, hyperlocomotion and autonomic stimulation. Previous reports have implicated that orexins have an antinociceptive effect in animal pain models, suggesting that orexins are involved in pain regulation. Furthermore, the PAG has been defined as a brain region with a critical role in the pain pathway. With regard to these findings, we investigated the effects of orexins in rat midbrain ventrolateral PAG (vlPAG) neurons using blind patch whole cell clamp recording. vlPAG neurons treated with orexin A (10~300 nM) were observed to dose-dependently induce membrane potential depolarization. The EC50 of orexin A was 12.3 ± 0.3 nM. Spontaneous firing activity was increased in 3/75 neurons after application of orexin A and just 45% of the recorded neurons were affected by orexin A. Orexin A-induced depolarization in vlPAG neurons was incompletely inhibited by SB-334867 (3 micromolar), a selective OX1R antagonist. Membrane potential depolarization was also induced when neurons were treated with [Ala11, D-Leu15] orexin-B (100~300 nM), an OX2R agonist. Yet this effect could not be reversed by SB-334867 (3 micromolar). The application of 300 nM [Ala11, D-Leu15] orexin-B was able to induce greater increments of membrane potential changes than the 100 nM dosage. According to a 2003 study, orexin A inhibited GIRK-mediated currents. Here, we used hyperpolarization ramps to research whether the effects of orexin A were mediated through GIRK channels in vlPAG neurons. We observed that orexin A (100 nM) did not change hyperpolarization ramp-induced currents, but the current was inhibited after treatment with 0.3 mM BaCl2. The BaCl2-inhibited currents were characterized with inward rectification and the reversal potential was near the equilibrium potential of K+ ions according to the Nernst equation. In another experiment, vlPAG neurons were first treated with N/OFQ (300 nM) to induce a GIRK-mediated current, and then treated with orexin A (100 nM). We did not observe any changes in the N/OFQ-induced current which was also a GIRK-mediated current. Furthermore, we observed the I-V relationships of orexin A-induced currents, but it was not constant. In conclusion, orexin A could induce membrane potential depolarization in a dose-dependant manner, and this effect could almost be reversed using SB-334867, a selective OX1R antagonist. Compared with orexin-A, 300 nM of [Ala11, D-Leu15] Orexin-B was more effective in altering the neuron membrane potential compared to 100 nM [Ala11, D-Leu15] orexin-B. We found that [Ala11, D-Leu15] orexin-B-induced depolarization was not affected when neurons were co-treated with [Ala11, D-Leu15] orexin-B and SB-334867. In vlPAG neurons, orexin A-induced depolarization did not seem to be via GIRK channels. In addition, I-V relationships created from orexin A-induced currents were found to be inconsistent. Thus, the mechanisms behind orexin A-induced effects in vlPAG neurons are still unclear.