Regulation of NMDA receptor activation by postsynaptic nanostructure

描述（由申请人提供）：了解是什么调节了突触强度的变化是非常有趣的，因为突触功能的变化是许多精神和神经疾病的标志。在谷氨酸突触中，AMPA和NMDA受体（NMDARs）通过保留在突触后密度（PSD）内而位于突触前活性区对面。突触后受体数量的改变是突触可塑性的一个重要机制。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而对突触生理学产生巨大影响。在我的实验室里，PSD的单分子定位（使用一种称为PALM的成像技术）已经导致PSD内纳米结构域的鉴定，其中包含相对高密度的支架蛋白PSD95和NMDAR亚基GluN2B。这些数据表明，突触的纳米级组织可能在突触功能中起关键作用。因此，我假设PSD-95的纳米结构域通过GluN2B的亚突触定位来控制受体的激活。在此之前，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新方法相结合，我建议测试这种新特征如何影响突触传递。为了确定PSD纳米级组织与NMDAR激活之间的关系，我用光激活版本的PSD-95和基因编码的Ca2+指示物GCaMP6f共转染了培养的海马神经元。这使我能够将突触纳米结构的PALM成像与同一个体脊柱中微型自发Ca2+瞬态的测量相结合。该方法揭示了NMDAR介导的单棘Ca2+瞬态与纳米结构域内PSD的分数面积之间的显著相关性。Ca2+瞬态与PSD面积之间没有关系，这与之前的观察一致，即突触NMDAR数量与PSD大小仅呈弱相关。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测量突触的NMDAR功能，这些突触的纳米结构是通过PALM监测的。其次，GluN2A和GluN2B受体亚型在激活的时空方面存在差异，GluN2B优先富集于PSD-95高密度纳米结构域。因此，我将探讨每个亚基在受体激活和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的肽或PSD-95突变体来评估NMDAR与PSD-95相互作用的作用，我希望每一种突变体都能消除或削弱纳米结构与NMDAR激活之间的关系。通过纳米尺度的受体分布调控NMDAR的激活可能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.