Regulation of extrasynaptic GABA-A receptors in health and disease

 描述(由申请人提供)： 抑制性神经递质GABA激活几种类型的受体，这些受体是癫痫和其他神经精神疾病的治疗靶点。GABAA受体与氯离子通道相连，在突触激活时产生电信号。GABAB受体是G蛋白偶联受体，调节细胞内信号通路。除了突触GABAA受体外，还有定位于突触外部位的独特的GABAA受体。这些高亲和力的突触外GABAA受体被大脑中环境中低水平的GABA激活。突触外GABAA受体产生的紧张性抑制形式对细胞和网络的兴奋性有重要影响。新的数据表明，突触外GABAA受体在几个大脑区域受突触后GABAB受体的调节。由于受GABAB受体影响的细胞内信号通路被许多GPCRs所共享，其他神经递质受体也可能调节突触外的GABAA受体。这项建议描述了三个假说来研究突触外GABAA受体在健康和疾病中的调节。假设通过GPCRs作用的多种神经递质调节突触外GABA受体。采用标准的全细胞技术，在急性海马脑片的齿状回颗粒细胞(DGGCs)和CA1区锥体神经元上记录突触外GABAA受体产生的紧张性电流。我们将研究细胞内信号通路对紧张性电流的影响。使用选择性的激动剂和拮抗剂，可以确定其他GPCRs(即肾上腺素受体、D2多巴胺受体和5-HT2受体)对紧张性电流的影响。据预测，抑制PKA将增强紧张性电流 (D2受体)和PKC激活(5HT2受体)。通过肾上腺素受体激活PKA可以减少紧张性电流。将进行生化研究，以确定细胞内信号通路是否通过增加GABAA受体的表面表达来增强紧张性电流。自发的生理或病理生理神经活动可导致环境中GABA水平的周期性和一过性升高。推测环境中GABA的周期性升高将激活突触后GABAB受体，增强突触外GABAA受体功能。在定期增加环境中的GABA之前和过程中，将重复测量张力电流。将使用三种方法来产生周围环境中GABA的一过性增加：1.周期性地应用外源性GABA，2.刺激内源性GABA的释放(电刺激，高K)，以及3.用4-AP诱发自发发作间放电。将证实这些过程是由突触后用GABAB受体拮抗剂CGP55845和含有G蛋白抑制剂的吸管溶液(Gdp-ç-S)激活的结果。创伤性脑损伤(TBI)改变 GABAA受体在海马区发挥作用。推测GPCRs对实验性脑损伤后突触外GABAA受体具有调节作用。我们将确定颅脑损伤对基础紧张性电流的影响以及GPCRs对它们的调节。具体地说，假设脑损伤将增强DGGC的张力电流，部分原因是GPCRs的激活增加。

 DESCRIPTION (provided by applicant): The inhibitory neurotransmitter GABA activates several types of receptors that are therapeutic targets for the treatment of epilepsy and other neuropsychiatric illness. GABAA receptors are linked to a Cl ion channel and produce electrical signals when activated at synapses. GABAB receptors are G- protein coupled receptors (GPCRs) that regulate intracellular signaling pathways. In addition to synaptic GABAA receptors, there are unique GABAA receptors localized to extrasynaptic sites. These high-affinity extrasynaptic GABAA receptors are tonically activated by the low levels of ambient GABA in the brain. The tonic form of inhibition produced by extrasynaptic GABAA receptors importantly affects cellular and network excitability. Emerging data indicate that extrasynaptic GABAA receptors in several brain regions are regulated by postsynaptic GABAB receptors. Because the intracellular signaling pathways affected by GABAB receptors are shared by many GPCRs, other neurotransmitter receptors may also regulate extrasynaptic GABAA receptors. This proposal describes three hypotheses to investigate the regulation of extrasynaptic GABAA receptors in health and disease. It is hypothesized that multiple neurotransmitters acting through GPCRs regulate extrasynaptic GABA receptors. Tonic currents produced by extrasynaptic GABAA receptors will be recorded from dentate gyrus granule cells (DGGCs) and CA1 pyramidal neurons in acute hippocampal brain slices using standard whole cell techniques. The effects of intracellular signaling pathways on tonic currents will be investigated. Using selective agonists and antagonists, the effects of other GPCRs (i.e. ß- adrenoreceptors, D2 dopamine receptors, and 5-HT2 receptors) on tonic currents will be determined. It is predicted that tonic currents will be enhanced by PKA inhibition (D2 receptors) and PKC activation (5HT2 receptors). Activation of PKA by ß-adrenoreceptors is predicted to reduce tonic currents. Biochemical studies will be done to determine if intracellular signaling pathways enhance tonic currents by increasing surface expression of GABAA receptors. Spontaneous physiological or pathophysiological neural activity can produce periodic and transient increases in ambient GABA levels. It is hypothesized that periodic increases in ambient GABA will activate postsynaptic GABAB receptors and enhance extrasynaptic GABAA receptor function. Tonic currents will be repeatedly measured before and during procedures to periodically increase ambient GABA. Three approaches will be used to produce transient increases in ambient GABA; 1. periodic application of exogenous GABA, 2. stimulating release of endogenous GABA (electrical stimulation, high K+), and 3. inducing spontaneous interictal discharges with 4-AP. It will be confirmed that the effects of these procedures resulted from postsynaptic GABAB receptor activation using the GABAB receptor antagonist CGP55845 and pipette solutions containing a G protein inhibitor (GDP-ß-s). Traumatic brain injury (TBI) alters GABAA receptor function in the hippocampus. It is hypothesized that GPCRs regulate extrasynaptic GABAA receptors following experimental TBI. We will determine the effects of TBI on basal tonic currents and their regulation by GPCRs. Specifically, it is hypothesized that tonic currents of DGGCs will be enhanced by TBI, due in part to increased activation of GPCRs.