Excitatory synaptic transmission onto hippocampal interneurons

描述(申请人提供)：皮层回路的活动和功能取决于两组相互连接的细胞之间的相互作用：兴奋性主神经元和抑制性中间神经元。抑制性中间神经元在阻止导致癫痫样活动的回路兴奋性方面起着关键作用，也参与了许多其他过程，包括主神经元放电的调节和同步化，神经调制肽的释放，以及主神经元的躯体树突状细胞抑制。这些功能是由神经元间激活触发的，这种激活通过兴奋性突触传递到这些细胞上。这种兴奋性传递主要是谷氨酸能的，兴奋性突触后电流(EPSC)主要由AMPA受体(AMPAR)和海人藻酸受体(KAR)的离子亲性谷氨酸受体(AMPAR)亚型介导。我们研究了AMPAR/KAR EPSCs对位于放射层和腔隙分子层(SR/SLM)的海马神经元的影响。我们发现，由细胞外刺激产生的EPSC有两个动力学上不同的组成部分：由AMPAR介导的快速、大型EPSC，通常类似于遍及中枢的AMPAR EPSC；以及由KARS和AMPAR共同介导的缓慢、小型EPSC。这种缓慢的EPSC持续数百毫秒，这是完全意想不到的，基于异源表达的AMPAR/KARs对谷氨酸短暂脉冲的反应动力学。慢EPSC的AMPAR成分，而不是KAR成分，也可以通过抑制谷氨酸摄取而大量增强。这些结果表明，慢的EPSC的动力学必须由延长的谷氨酸瞬间决定，或者由中间神经元内的因素决定，与异源表达的受体相比，改变本地受体的动力学，答案可能因所涉及的受体亚型和摄取机制的有效性而不同。在这项拟议的研究中，我们将研究迟缓的EPSC背后的机制。我们将使用全细胞电压钳记录海马片中SR/SLM中间神经元来记录EPSC，以及影响谷氨酸释放、接收和摄取的各种药物和遗传操作。我们的具体目标是：1)测试慢的AMPAR EPSC是否是由谷氨酸溢出产生的；2)确定慢的EPSC背后的受体是否具有由受体亚单位组成或与支架蛋白相互作用决定的动力学；以及3)确定谷氨酸摄取机制在调节慢的EPSC中的作用。公共卫生相关性：该项目将确定调节海马区中间神经元活动的机制，以防止正常情况下的过度兴奋。这项研究将使我们能够评估开发治疗策略的可能性，通过操纵这些中间神经元上的兴奋性传递来控制癫痫的启动或传播。

DESCRIPTION (provided by applicant): The activity and function of cortical circuits depend on the interplay between two interconnected sets of cells: excitatory principal neurons and inhibitory interneurons. Inhibitory interneurons play a critical role in preventing the circuit excitability that leads to epileptiform activity, and also are involved in a number of other processes as well, including regulation and synchronization of firing in principal neurons, release of neuromodulatory peptides, and somatodendritic inhibition of principal neurons. These functions are triggered by interneuronal activation, which occurs through excitatory synaptic transmission onto these cells. This excitatory transmission is primarily glutamatergic, and the excitatory postsynaptic current (EPSC) is mediated largely by ionotropic glutamate receptors of the AMPA receptor (AMPAR) and kainate receptor (KAR) subtypes. We have studied the AMPAR/KAR EPSCs onto hippocampal interneurons located in stratum radiatum and stratum lacunosum-moleculare (SR/SLM). We have found that the EPSC generated by extracellular stimulation has two kinetically distinct components: a rapid, large EPSC that is mediated by AMPARs and is generally similar to AMPAR EPSCs seen throughout the CNS; and a slow, small EPSC that is mediated by both KARs and AMPARs. This slow EPSC lasts for hundreds of milliseconds, which is completely unexpected based on the kinetics of heterologously expressed AMPARs/KARs in response to brief pulses of glutamate. The AMPAR component of the slow EPSC, but not the KAR component, can also be massively potentiated by inhibition of glutamate uptake. These results suggest that the kinetics of the slow EPSC must be determined by a prolonged glutamate transient, or by factors within the interneuron that alter the kinetics of native receptors compared to heterologously expressed receptors, and that the answer may differ depending on the receptor subtype involved and the efficacy of uptake mechanisms. In the proposed research, we will examine the mechanisms underlying the slow EPSC. We will use whole-cell voltage-clamp recording of SR/SLM interneurons in hippocampal slices to record the EPSC, and a variety of pharmacological and genetic manipulations to affect glutamate release, reception, and uptake. Our specific aims are: 1) to test whether the slow AMPAR EPSC is generated by glutamate spillover; 2) to determine whether the receptors underlying the slow EPSC have kinetics that are dictated by receptor subunit composition or interactions with scaffolding proteins; and 3) to define the role of glutamate uptake mechanisms in regulating the slow EPSC. PUBLIC HEALTH RELEVANCE: This project will identify mechanisms of regulating the activity of hippocampal interneurons, which prevent hyperexcitability under normal conditions. This research will allow us to evaluate the possibility of developing therapeutic strategies to control seizure initiation or propagation by manipulating excitatory transmission onto these interneurons.