Presynaptic NMDA receptors in the CNS

NMDAR是中枢神经系统兴奋性突触的两种主要离子通道之一，位于突触后膜上。它们不仅介导兴奋，而且由于它们对钙的通透性，提高细胞内钙浓度，从而触发各种细胞内通路，导致突触强度的短期和长期变化。在病理条件下，它们也会导致神经元死亡。最近的研究表明，NMDAR不仅位于突触后部位，也位于突触前释放部位，并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。我们将使用双光子显微镜和电生理学结合钙成像来测试NMDAR是否在突触前表达，以测量突触前和突触后对NMDAR激动剂和拮抗剂全局和局部应用的反应。我们为这项研究选择了两种类型的神经元：小脑星状细胞和新皮质第五层锥体细胞。很明显，NMDAR激活改变了这两种细胞的递质释放，但是否涉及相同的机制尚不清楚。目前尚不清楚这些受体是通过直接增加释放部位的细胞内钙离子，还是通过去极化释放部位来改变释放。如果前者是真的，NMDARs必须位于非常靠近释放部位的地方，因为钙不会在细胞内扩散太远。然而，如果NMDAR对释放的调节仅次于它们的去极化影响，它们就不需要位于释放地点附近，因为去极化可以通过电子传播数百微米。这两个机制并不是相互排斥的，可以相辅相成。NMDAR介导的去极化本身可能通过以下方式对囊泡释放产生多种影响：1)直接激活电压敏感钙通道(VSCCs)，从而通过提高基线钙浓度来增加释放；2)灭活轴突KV1型钾通道，导致动作电位扩大，增强VSCCs的激活，从而增加释放；3)钙非依赖性的释放过程的调节；4)电压依赖性钠通道和/或VSCCs的失活，从而阻断动作电位的传播，释放区，减少钙内流，导致神经递质释放减少。我们将确定NMDAR是否位于突触前释放区，以及体细胞去极化是否通过这些机制中的一种或一种组合来改变释放。

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.