- 學位論文
中腦環導水管灰質中催產素系統在小鼠緊張導致止痛之角色
The role of the oxytocin system in the periaqueductal gray in stress-induced analgesia in mice
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摘要
催產素（oxytocin）為腦下垂體釋放出的荷爾蒙之一，會在分娩和哺乳期間大量釋放。同時，催產素在中樞系統中也具有神經傳導物質的功能，可以透過活化催產素受器（oxytocin receptor, OTR），一種屬於Gq蛋白偶合受器家族的受器，進行神經元之間的訊息傳遞。Oxytocin具有調節社交行為和非社交行為的作用；其中非社交行為包括食慾、痛覺、壓力所誘導之成癮藥物的復發行為和緊張所致的止痛反應（stress-induced analgesia, SIA）。SIA是當人類或動物緊張時痛覺感受程度降低的一種現象。藥物產生的止痛作用或是緊張所致的止痛反應皆由負責下行性疼痛抑制訊息的中腦環導水管灰質（periaqueductal gray, PAG）腦區所調控，抑制中腦環導水管灰質神經細胞的GABA transmission可以活化下行性疼痛抑制路徑達到止痛效果。有研究指出壓力下或是緊張時，下視丘的oxytocin神經細胞會釋放oxytocin到大腦和脊髓中。目前已知，脊髓中的oxytocin具有止痛作用也貢獻於SIA，而大腦中的oxytocin還未被發現是否有貢獻於SIA中。催產素和血管加壓素，另一種由腦下垂體釋放的神經胜肽/荷爾蒙，會與OTR和血管加壓素受器（vasopressin receptor）結合。過去有電生理的研究顯示，在下視丘的神經細胞發現oxytocin 可活化突觸後的OTR，藉此活化phospholipase C （PLC）的訊息傳遞途徑，繼而促進內生性大麻酯分子（endocannabinoids）的生成。這些內生性大麻酯分子會逆行活化位於突觸前的CB1R，進而抑制GABA釋放。在大鼠的研究也發現將oxytocin直接注入腦室或是不同的腦區會產生止痛作用，可是其作用機制目前還是未知。 因此，我們在這篇論文中使用藥理學和行為的方式驗證（1）緊張時中腦環導水管灰質腹外側區（ventrolateral periaqueductal gray, vlPAG）oxytocin系統是否有參與SIA、（2）活化vlPAG中的OTR是否可以產生止痛作用、（3）活化OTR產生的止痛作用是否經由內生性大麻酯系統所媒介、（4）活化OTR產生的止痛作用是否為鴉片系統依賴性的反應。 我們發現給予小鼠束縛壓力後其縮腳潛伏期（paw withdrawal latency）有明顯增長，確認了緊張時會產生止痛反應。在實驗中的緊張所致止痛作用可以藉由先在腹腔或是vlPAG投與L-368,899（OTR拮抗劑）抑制掉，而其使用劑量並不影響小鼠的活動能力。第二，經束縛壓力處理過的小鼠相較於控制組，其vlPAG均質液中oxytocin胜肽量有明顯增加。由免疫螢光染色結果發現小鼠下視丘oxytocin神經細胞經由束縛壓力處理後有被激活的趨勢。以上的結果顯示緊張時有成功誘導止痛反應，這止痛作用可能來自於緊張時釋放到vlPAG中的oxytocin活化OTR的結果。第三，我們在vlPAG內注射不同劑量的oxytocin（0.01-1 nmol；i.pag.）。在只有0.1 nmol oxytocin劑量下產生具有止痛效果的趨勢，然而在1 nmol劑量則出現pronociception。第四，在vlPAG內注射不同劑量的TGOT（OTR活化劑）皆具有止痛效果，而其縮腳潛伏期呈現鐘形劑量反應曲線。0.03 nmol TGOT劑量下產生最顯著止痛反應而小鼠的活動能力也不受影響，且0.03 nmol TGOT（i.pag.）下產生的止痛可以被經由vlPAG注射L-368,899所抑制。第五，由於vlPAG注射TGOT產生的止痛反應能被AM251（CB1受器拮抗劑；i.pag.）和naloxone（鴉片類受器拮抗劑；i.pag.）所阻斷，指出CB1R和opioid receptors皆參與在TGOT的止痛作用中。 本篇論文的實驗結果可以推測出活化vlPAG中的OTR可以產生顯著的止痛作用，所產生的止痛作用主要由內生性大麻酯與部分鴉片類所媒介。活化OTR產生止痛反應的途徑可能有貢獻於束縛壓力誘導的緊張所致止痛作用。
並列摘要
Oxytocin is a well-known pituitary hormone involved in parturition, lactation and maternal care. It also acts as a neurotransmitter through the oxytocin receptor (OTR), a GqPCR family, playing a pivotal role in both social bonding and non-social behaviours. The latter encompasses feeding, pain perception and several stress-induced responses, such as stress-induced drug reinstatement and stress-induced analgesia (SIA). SIA is a phenomenon whereby the pain sensation of the subject is suppressed upon exposure to stressful stimuli. Either drug-induced analgesic effect or SIA is mediated by the descending pain inhibitory pathway where the midbrain periaqueductal gray (PAG) is the major integrating hub at the supraspinal level, inhibition of the GABA transmission in PAG leads to the activation of descending pain inhibitory pathway. It has been shown that stress can activate the hypothalamic oxytocin neurons and release oxytocin into the brain and the spinal cord. Oxytocin has been reported to be antinociceptive at the spinal level, contributing SIA. However, it remains unclear whether oxytocin can at the supraspinal level to mediate SIA. Oxytocin and vasopressin, another pituitary neuropeptide/hormone, have cross interactions with OTRs and vasopressin receptors. An electrophysiological study has showed that in hypothalamic neurons, activating postsynaptic OTRs can inhibit GABA release via generating endocannabinoid that engages retrogradely at presynaptic cannabinoid 1 (CB1) receptors. In rats, there were some studies showing that oxytocin was antinociceptive when given at the supraspinal level while the underlying mechanism(s) remain confusing. Collectively, in this study we elucidated (i) the contribution of the oxytocin system in SIA, (ii) the anti-nociceptive effect of OTR activation in the mouse ventrolateral-PAG (vlPAG), (iii) whether the OTR-induced antinociception is an endocannabinoid-dependent effect, and (iv) whether the OTR-induced antinociception interacts with the opioid system, using pharmacological and behavioural approaches. We found that mice receiving a 30-minute restraint stress showed significantly longer hot-plate latency and higher oxytocin levels in the vlPAG than unrestrained mice. This SIA was prevented by either intraperitoneal (i.p) or intra-periaqueductal gray (i.pag.) microinjection of L-368,899 at doses that did not affect the spontaneous locomotor function. These results suggest that SIA is successfully established and this SIA is possibly due to elevated oxytocin acting at OTRs in the vlPAG. Next, we performed i.pag. microinjection of a broad range (0.001-1nmol) of synthetic oxytocin in mice. Unexpectedly, oxytocin (i.pag.) only at 0.1 nmol produced a marginal antinociception while at 1 nmol displayed pronociceptive effect. We, therefore, further examined effects of TGOT, an OTR-selective agonist, on the mouse hot-plate test. TGOT was antinociceptive at all tested doses, while produced a bell-shape dose-response curve in increasing the hot-plate latency, with the maximal antinociceptive effect at 0.03 nmol (i,pag.). This antinociceptive effect was blocked by i.pag. pretreatment with that antagonist of OTRs (L-368,899), CB1Rs (AM251) and non-selective opioid receptors (naloxone)., suggesting the OTR, CB1R and opioid receptors in the vlPAG are involved in the antinociceptive effect of TGOT. These results suggest that activating the OTRs in the vlPAG induces a significant antinociceptive effect, which is mainly mediated by endocannabinoids and also partially opioid-dependent. This antinociceptive effect may contribute to SIA induced by restraint stress.
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