苦藍盤萃取物KLP-1調控小腦切片中α6GABAAR媒介之持續抑制神經活性與修復K它命、MK-801和甲基安非他命引發前脈衝抑制缺損之作用

KLP-1 modulates α6-containing GABAAR-mediated tonic inhibition in cerebellar slices and reinstates ketamine-, MK-801- and methamphetamine-induced disruption of prepulse inhibition

周瑞峰(Jui-Feng Chou)

指導教授 ： 邱麗珠

國立臺灣大學/醫學院/藥理學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10285

摘要

一則臨床報告發現，一位患有妥瑞症（Tourette syndrome）小孩病患，其在接受許多臨床抗抽動藥物後，不見其療效。 而在服用苦藍盤汁液後，症狀獲得有效緩解。 而從苦藍盤中所得到的有效成分KLP-1，已在本實驗室證實其會抑制甲基安非他命所造成的活動力增加，且會修復其造成前脈衝抑制不良的現象。
相對於benzodiazepine，KLP-1已被證實可正向調節蛙卵上所表現的GABAA 接受器，也包含α6GABAA 接受器 (α6GABAAR)。 而α6GABAAR只存在於小腦內的顆粒細胞上，且其主導持續性抑制的功能，來控制小腦神經迴路的訊息傳遞。
第一部分的動物行為模式實驗，進一步探討KLP-1對前脈衝抑制的影響。 使用甲基安非他命(2 mg/kg)，K它命(30 mg/kg)或MK-801(0.3 mg/kg)來造成前脈衝抑制不良的現象，再給予KLP-1，來觀察其效果。 結果發現，不論是腹腔注射或小腦埋管給藥，皆會修復甲基安非他命，K它命或MK-801，所造成前脈衝抑制不良的現象。 且小腦內注射α6GABAA 接受器的活化劑，Ro-154513可修復K它命所造成前脈衝抑制不良的現象。 另外，相較於KLP-1與Ro-154513，diazepam 無法作用於α6GABAAR。 小腦內注射diazepam後，並無法修復K它命所造成前脈衝抑制不良的現象。 另外也使用對於α6GABAA 接受器有高選擇性的阻斷劑，furosemide可有效的抑制KLP-1與Ro-154513修復K它命所造成前脈衝抑制不良的效果。 因此可知道的是，KLP-1可修復前脈衝抑制不良的效果，有可能是透過活化小腦顆粒細胞上的α6GABAAR。
第二部份，我們利用visual patch clamp whole-cell recording電生理技術，來記錄顆粒細胞上的持續抑制性電流。 一開始藉由單獨bicuculline 10 μM會造成holding current往上移動(outward shift)來證實持續抑制性電流的存在，而之後發現furosemide 100 μM也會抑制此持續抑制性電流，表示其α6GABAAR所主導之現象。 接著給予不同濃度的KLP-1，發現holding current並沒有明顯的改變，即使改變加藥順序(先給bicuculline再給KLP-1 100 μM)，也無法觀察到KLP-1的效果。 接著利用給予可選擇性活化α6GABAAR的低濃度THIP或抑制GABA回收的NO-711來增強持續抑制性電流的表現，而在給予KLP-1 100 μM後，仍然無法觀察到顯著的效果。
因此，對於KLP-1會修復甲基安非他命，K它命或MK-801來造成前脈衝抑制不良，其作用標的與其影響小腦顆粒細胞上的持續性抑制的機轉仍需進一步的探討。

關鍵字

小腦 ； 持續性抑制 ； α6-γ-胺基丁酸A型受體

並列摘要

In a case report, we found a pediatric patient with Tourette syndrome (TS) with motor tics reluctant to multiple clinically anti-tic therapies. After taking the leaf extract of Clerodendron inerme (L.) Gaertn, her symptom of tics got dramatic alleviation. KLP-1 is an active constituent from this herb plant and has been found to inhibit the hyperlocomotor activity and the disruption of the prepulse inhibition (PPI) of the acoustic startle response induced by methamphetamine (MAPH) in our laboratory.
KLP-1, rather than benzodiazepine, has been proved to be a positive allosteric modulator of cloned GABAA receptors involving α6 subunit-containing GABAA receptors (α6GABAAR), which are exclusively expressed in cerebellar Golgi-granule synapses and mediate tonic inhibitory control in the cerebellar cortex.
In the first part of this study, we verified the effect of KLP-1 on the PPI. We then found that whether administered by intraperitoneal (i.p.) or intra-cerebellar (i.cb.) injection of KLP-1, the disrupted PPI induced by ketamine- and MK-801, two NMDA receptor blockers, both of which were confirmed to disrupt PPI, were reinstated. Then, i.cb. injection of Ro-154513, which activates α6GABAAR, effectively reinstated ketamine-induced disruption of PPI. While i.cb. injection of diazepam, which is insensitive to α6GABAAR, did not reinstated ketamine-induced disruption of PPI. Otherwise, i.cb. injection of furosemide, which selectively antagonizes α6GABAAR, significantly inhibited the reinstatement of KLP-1 and Ro-154513 on ketamine-induced disruption of PPI. Consequently, the reinstatement of KLP-1 on disrupted PPI may via activating α6GABAAR at cerebellar granule cells.
In the second part, we utilized visual patch clamp whole-cell recording technique to record the α6GABAAR-mediated tonic current, which can be inhibited either by bicuculline or furosemide. Unexpectedly, we found the effect of various concentration of KLP-1 on the tonic current was unremarkable, even to reverse the bicuculline-inhibited tonic current. Then, we tried to potentiate this tonic current by low dose of THIP or NO-711, a α6GABAAR agonist and GABA transporter inhibitor, preceding the application of KLP-1. However KLP-1 still exhibited an unremarkable effect on the tonic current, which has been increased by THIP or NO-711.
In summary, the contribution of KLP-1 on the reinstatement of the methamphetamine-, ketamine- and MK-801-induced disruption of PPI is well-investigated, while its central mechanism, the possible effect on the α6GABAAR-mediated tonic inhibition at cerebellar granule cells, remains to be further investigated.

並列關鍵字

cerebellum ； tonic inhibition ； α6GABAAR

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