伽瑪-胺基丁酸B型受體-胞外信號調節激酶途徑在藍斑核神經元之角色探討

Abstract

藍斑核(LC)-正腎上腺素性神經元在中樞神經系統中扮演著許多重要的生理功能，包括情緒、鎮痛以及睡眠清醒週期等。在實驗室前人的研究顯示，藍斑核神經元的自發性放射動作電位是受到伽瑪-胺基丁酸B型受器 (GABABR) 所調控。然而，對於藍斑核神經元中GABABR的調控機制尚不明瞭。 代謝型GABABR調控在眾多大腦神經活性並且扮演重要功能，它在突觸前及突觸後神經元皆有表現，透過抑制電位依賴型鈣離子通道與開啟鉀離子通道抑制神經活性。除此之外，我們發現活化的GABABR能夠誘導胞外信號調節激酶(ERK1/2)的表現，ERK1/2也被認為是調控正腎上腺素性神經元以改變動物行為的角色之一。 本研究採用全細胞膜片箝制紀錄、細胞接觸型紀錄、細胞組織免疫化學呈色法及西方點墨法，以此探討伽瑪-胺基丁酸B型受器-胞外信號調節激酶途徑在藍斑核細胞上的調控機制。型態上的結果發現在藍斑核細胞中磷酸化的ERK1/2信號表現具有生理節律週期，此節律近似於藍斑核胞外的GABA濃度變化週期，也和藍斑核的活性變化節律相反•我們將藍斑核組織從處理過GABABR激活劑巴氯芬(baclofen)的腦幹切片分離出來，接著使用西方點墨法偵測蛋白表現，結果發現磷酸化的ERK1及ERK2表現量上升。此外，巴氯芬造成磷酸化的ERK1/2的增加效果，可以被GABABR抑制劑 (CGP54626)給去除。有趣的是，在全細胞紀錄下，給予藍斑核巴氯芬誘導出一電流，電流波形顯示此電流具有緩慢且部分去敏感化的特性。將腦切片預先給予ERK1/2抑制劑 (U0126 or FR180204)而不是ERK1抑制劑(PD98059)處理，巴氯芬誘導的電流波形顯示更快速且顯著的去敏感化特性。除了去敏感化之外，G蛋白偶合受體的膜上膜內運輸也是控制GABABR重要因素之一。使用細胞接觸型紀錄發現ERK1/2的活性也會影響GABABR回送到藍斑核細胞膜上的運輸，更進一步改變藍斑核的放射動作電位頻率。 綜合以上結果顯示GABABR的活化激發ERK1/2信號途徑並且自我調控，以延緩GABABR的去敏感化且能夠回復藍斑核細胞膜上的GABABR。因此GABABR的持續性抑制效果能夠維持且穩定藍斑核的放射動作電位頻率。

Locus Coeruleus (LC)-noradrenaline system plays important roles in many brain functions, including decision making, arousal, emotion, antinociception and sleep-wake cycle. The previous data of our group showed that the spontaneous firing rate (SFR) of LC neurons is regulated by GABAB receptors (GABABRs) in a tonic inhibition manner. However, the mechanism of this GABABR regulation is still unknown. The metabotropic GABAB receptor (GABABR) plays important roles in regulating neuronal excitability in the brain. GABABR is well known to exert both pre- and postsynaptic inhibition through inhibiting voltage-gated Ca2+ channel and activating K+ (GIRK) channel, respectively. Beside these effects, here we found that GABABR activation caused an increase in phosphorylated extracellular signal- regulated kinase 1/2 (pERK1/2) level in LC, which consists of noradrenergic neurons and play divers roles in behavior. Our work applies whole cell patch-clamp and cell-attachment electrophysiology, cellular immunohistochemistry staining and western blot to study the GABABR-pERK1/2 signaling pathway mechanism on LC neurons in rats. Morphological results show that pERK1/2 signal in LC neurons exhibits circadian cycle which is similar to ambient GABA level, opposite to LC activity. Using western-blot analysis, LC tissues isolated from brainstem slices bathed in baclofen, a GABABR agonist, showed an increase in pERK1 and pERK2 compared to tissue from slices bathed in normal medium. Furthermore, this effect was specific to GABABR activation as it was not observed in LC tissue from slices bathed with baclofen and CGP54626, a GABABR antagonist. More interestingly, in whole cell recording, bath application of baclofen for 15 min induced a CGP54626 sensitive baclofen-induced current in LC neuron (Vm -70 mV) that underwent slow and partial desensitization. In slices pretreated with ERK1/2 blockers, U0126 or FR180204, but not in ERK1 blocker, PD98059, baclofen-induced current showed a faster and more prominent desensitization. Besides to desensitization, balance of GPCR trafficking is also important for controlling the GABAB receptor functions. Using cell-attachment recording, we found that ERK1/2 activity is involved in restoration of GABAB receptor on cell surface of LC neurons, and further affects the average firing rate of LC neurons. Together, the above results show that GABABR activation recruits ERK1/2-signaling pathway for an autoregulation that prevents GABABR from quick desensitization and restores cell surface GABABR of LC neurons, therefore maintains tonic inhibition, an important mechanism for tuning FR of LC neurons.