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

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.