Excitatory synaptic transmission onto hippocampal interneurons

描述（由申请人提供）：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防止导致癫痫样活动的电路兴奋性方面起着关键作用，并且还参与许多其他过程，包括主神经元放电的调节和同步、神经调节肽的释放和主神经元的体突抑制。这些功能是由神经元间激活触发的，通过兴奋性突触传递到这些细胞。这种兴奋性传递主要是谷氨酸能传递，兴奋性突触后电流（EPSC）主要由AMPA受体（AMPAR）和kainate受体（KAR）亚型的嗜离子性谷氨酸受体介导。我们研究了AMPAR/KAR EPSCs在海马辐射层和空隙层分子（SR/SLM）间神经元上的作用。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、大的EPSC，通常与整个中枢神经系统中可见的AMPAR EPSC相似；以及由KARs和ampar介导的缓慢的小EPSC。这种缓慢的EPSC持续数百毫秒，这是完全出乎意料的，基于异源表达的AMPARs/KARs响应谷氨酸短脉冲的动力学。缓慢EPSC的AMPAR组分，而不是KAR组分，也可以通过抑制谷氨酸摄取而大量增强。这些结果表明，缓慢EPSC的动力学必须由长时间的谷氨酸短暂性决定，或者由中间神经元内与异源表达受体相比改变天然受体动力学的因素决定，并且答案可能取决于所涉及的受体亚型和摄取机制的有效性。在本研究中，我们将研究慢速EPSC的机制。我们将使用全细胞电压钳记录海马切片中SR/SLM中间神经元的EPSC，以及各种影响谷氨酸释放、接收和摄取的药理学和遗传学操作。我们的具体目的是：1)检验慢速AMPAR EPSC是否由谷氨酸溢出产生；2)确定慢速EPSC的受体是否具有由受体亚基组成或与支架蛋白相互作用决定的动力学；3)明确谷氨酸摄取机制在调节慢速EPSC中的作用。公共卫生相关性：本项目将确定调节海马中间神经元活动的机制，在正常情况下防止过度兴奋。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的兴奋性传递来控制癫痫发作的开始或传播。

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.