Presynaptic NMDA receptors in the CNS

NMDA受体（NMDARs）是位于中枢神经系统中绝大多数兴奋性突触突触后膜上的两种主要离子通道之一。它们不仅介导兴奋，而且由于它们对钙的渗透性，提高细胞内钙浓度，从而触发多种细胞内通路，导致突触强度的短期和长期变化。它们也会导致病理状态下的神经元死亡。最近的研究表明，NMDARs不仅位于突触后位点，也位于突触前释放位点，并且可以改变囊泡释放的可能性。由于这些受体在生理和病理条件下都很重要，确定它们的位置将有助于确定它们的作用。我们将测试NMDARs是否在突触前表达，结合钙成像，使用双光子显微镜和电生理学来测量对NMDAR激动剂和拮抗剂的整体和局部应用的突触前和突触后反应。我们选择了两种类型的神经元进行研究：小脑星状细胞和新皮层第5层锥体细胞。很明显，NMDAR激活改变了这两种细胞类型的递质释放，但尚不清楚是否涉及相同的机制。这些受体是通过直接增加释放位点的细胞内钙还是通过使释放位点去极化来改变释放尚不清楚。如果前者是正确的，那么NMDARs必须位于离释放位点非常近的地方，因为钙不会在细胞内扩散很远。然而，如果NMDARs对释放的调节次于它们的去极化影响，则它们不需要位于释放位点附近，因为去极化可以电张力传播数百微米。这两种机制并非相互排斥，而是可以相互加强的。nmdar介导的去极化本身可能对囊泡释放有多重影响：1)直接激活电压敏感钙通道（VSCCs），从而通过提高基线钙浓度增加释放；2)使轴突kv1型钾通道失活，导致动作电位扩大，增强VSCCs的激活，从而增加释放；4)电压依赖性钠通道和/或VSCCs的失活，从而阻断动作电位向释放区传播，减少钙内流，导致神经递质释放减少。我们将确定NMDARs是否位于突触前释放区，以及体细胞去极化是否通过一种或多种机制改变释放。

NMDA receptors (NMDARs) are one of two major types of ion channels located in the postsynaptic membrane at the vast majority of excitatory synapses in the CNS. They not only mediate excitation but, because of their permeability to calcium, raise intracellular calcium concentrations and thus trigger a variety of intracellular pathways leading to short and long term changes in synaptic strength. They also contribute to neuronal death in pathological conditions. Recent studies suggest that NMDARs are located not only at postsynaptic sites but also at presynaptic release sites and can modify the likelihood of vesicular release. As these receptors are important in both physiological and pathological conditions, determining where they are located will help define their effects. We will test whether NMDARs are expressed presynaptically with a combination of calcium imaging using two photon microscopy and electrophysiology to measure pre- and postsynaptic responses to global and local applications of NMDAR agonists and antagonists. We have chosen two types of neurons for this study: cerebellar stellate cells and neocortical layer 5 pyramidal cells. It is clear that NMDAR activation alters transmitter release from these two cell types but it is unclear if the same mechanisms are involved. Whether these receptors alter release by directly increasing intracellular calcium at release sites or rather by depolarizing release sites is not clear. If the former is true, NMDARs would have to be located very close to sites of release because calcium does not diffuse far intracellularly. If the regulation of release by NMDARs is secondary to their depolarizing influence, however, they need not be located near release sites because depolarizations can spread electrotonically for hundreds of micrometers. These two mechanisms are not mutually exclusive and could be mutually reinforcing. NMDAR-mediated depolarization by itself may have multiple effects on vesicular release by 1) directly activating voltage-sensitive calcium channels (VSCCs) and thereby increasing release by elevating baseline calcium concentrations, 2) inactivating axonal Kv1-type potassium channels leading to a broadening of action potentials, enhanced activation of VSCCs and thus greater release, 3) a calcium-independent, though uncharacterized, regulation of the release process and 4) inactivation of voltage-dependent sodium channels and/or VSCCs and therefore a blockade of action potential propagation to release zones and decreased calcium influx leading to decreased neurotransmitter release. We will determine whether NMDARs are located at presynaptic release zones and whether somatic depolarizations alter release by one or a combination of these mechanisms.