Regulation of NMDA receptor activation by postsynaptic nanostructure

描述(由申请人提供)：由于突触功能的变化是许多精神和神经疾病的标志，了解是什么调节突触强度的变化是非常有意义的。在谷氨酸能突触，AMPA和NMDAR(NMDAR)通过滞留在突触后密度(PSD)内而定位于突触前活动区的对面。众所周知，改变突触后受体的数量是突触可塑性的一个关键机制。然而，理论和计算模型表明，除了受体数量的重要性外，PSD内受体的分布可能通过控制释放的神经递质激活受体的可能性对突触生理产生重大影响。在我的实验室里，PSD的单分子图谱(使用一种名为Palm的成像技术)已经导致在PSD中识别出包含相对高密度的支架蛋白PSD95和NMDAR亚单位GluN2B的纳米结构域。这些数据表明，突触的纳米级组织可能在突触功能中发挥关键作用。因此，我假设PSD-95的纳米结构域通过GluN2B的突触下定位来控制受体的激活。此前，由于缺乏成像分辨率，在活细胞中测试这一想法是不可行的。然而，通过将我们最近发表的分析与我开发的一种新方法相结合，我建议测试这一新功能如何影响突触传递。为了确定PSD纳米级组织和NMDAR激活之间的关系，我将光激活版本的PSD-95和遗传编码的钙指示剂GCaMP6f共同转染培养的海马神经元。这使得我可以将突触纳米结构的手掌成像与测量相同单个脊椎中微小的自发钙瞬变结合起来。这一方法揭示了NMDAR介导的单棘处的钙瞬变与位于纳米结构域内的PSD的分数区域之间的显著相关性。突触NMDAR数目与PSD大小的关系不明显，与以往的观察结果一致。为了更好地理解这种关系的机制，我将首先结合成像和电生理方法对突触的NMDAR功能进行测量，这些突触的纳米结构通过Palm进行监测。其次，GluN2A和GluN2B受体亚型被预测在其激活的时空方面有所不同，并且GluN2B优先富含在PSD-95高密度纳米结构域中。因此，我将探讨每个亚基在受体激活与PSD纳米结构之间的关系中的相对贡献。最后，我将使用不能结合NMDAR的多肽或PSD-95突变体来评估NMDAR与PSD-95相互作用的作用，我预计每一种都会取消或削弱纳米结构和NMDAR激活之间的关系。通过对受体分布的纳米级操纵来微调NMDAR的激活可能会对突触生理学和突触可塑性的诱导产生深远的影响。

DESCRIPTION (provided by applicant): Understanding what regulates variations in synaptic strength is of great interest, since changes in synaptic function are a hallmark of many psychiatric and neurological diseases. At glutamatergic synapses, AMPA and NMDA receptors (NMDARs) are positioned across from presynaptic active zones by retention within the postsynaptic density (PSD). It is well established that altering the number of postsynaptic receptors is a key mechanism of synaptic plasticity. However, theory and computational modeling suggest that aside from the importance of receptor number, the distribution of receptors within the PSD may have a dramatic impact on synapse physiology, by governing the likelihood that released neurotransmitter will activate the receptors. In my lab, single-molecule mapping of the PSD (using an imaging technique called PALM) has led to the identification of nanodomains within the PSD that contain a relatively high density of the scaffold protein PSD95 and the NMDAR subunit GluN2B. These data suggest that the nanoscale organization of the synapse could play a critical role in synaptic function. Thus, I hypothesize that nanodomains of PSD-95 control receptor activation through subsynaptic localization of GluN2B. Previously, it has not been feasible to test this idea in live cells due to the lack of imaging resolution. However, by combining our recently published assays with a new approach I have developed, I propose to test this how this novel feature influences synaptic transmission. To determine the relationship between PSD nanoscale organization and NMDAR activation, I co-transfected cultured hippocampal neurons with a photoactivatable version of PSD-95 and the genetically encoded Ca2+ indicator GCaMP6f. This permitted me to combine PALM imaging of synapse nanostructure with measurement of miniature spontaneous Ca2+ transients in the same individual spines. This approach revealed a significant correlation between NMDAR- mediated Ca2+ transients at single spines and the fractional area of the PSD that was within nanodomains. There was no relationship between the Ca2+ transient and PSD area, consistent with previous observations that synaptic NMDAR number is only weakly correlated with PSD size. To better understand the mechanism responsible for this relationship, I will first use a combination of imaging and electrophysiological measures of NMDAR function at synapses whose nanostructure is monitored via PALM. Second, GluN2A and GluN2B receptor subtypes are predicted to vary in the spatiotemporal aspects of their activation, and GluN2B is preferentially enriched in PSD-95 high-density nanodomains. Therefore, I will probe the relative contribution of each subunit to the relationship between receptor activation and PSD nanostructure. Finally, I will assess the role of NMDAR interaction with PSD-95 using peptides or PSD-95 mutants that cannot bind NMDARs, each of which I expect to abolish or weaken the relationship between nanostructure and NMDAR activation. Fine tuning of NMDAR activation through nanoscale manipulations of receptor distribution could have a profound impact on synaptic physiology and the induction of synaptic plasticity.