Modulation and plasticity of neurotransmission: Presynaptic mechanisms

神经递质(NTS)的同步释放发生在动作电位的2ms内，在空间上仅限于突触。这种空间限制被认为是由于突触小泡(SV)的对接和启动依赖于富含在突触前活动区细胞上的蛋白质。NT的释放也可能在钙触发的SVS与质膜融合的水平上受到空间限制。同步释放是由低钙亲和力的突触素启动的，这些突触聚集素需要靠近开放的电压门控钙通道来触发SV胞吐。除了同步释放外，神经元还在动作电位后几十到几百毫秒的时间内异步释放NTS。这种释放形式是由具有高钙亲和力的传感器触发的。目前，尚不清楚异步释放是否仅限于突触。我们收集的证据表明，非同步发生在突触周围轴突区域以及突触。基于这一有趣的发现，我们的目标是确定在空间上限制NT释放的机制，并研究突触周围非同步NT释放对神经传递和突触可塑性的影响。具体地说，我们将：1)表征非同步释放的空间分布。我们将使用几种分子方法，增强或抑制NT的异步化释放，并评估对海马突触SV胞吐的空间限制的影响，以证实我们的初步发现，SV胞吐延伸到突触周围轴突区域。2)钙离子挤出和缓冲对非同步释放的地址调制。异步化释放可能受到限制钙扩散的机制的空间限制。初步实验表明，钙瞬变高度定位于突触前活动区，并与观察到的非同步释放程度相对应地横向扩散到相邻的轴突节段。为了证明因果关系，我们将操纵突触前钙释放和缓冲机制，并评估这些操纵对异步释放的影响。3)研究非同步释放和同步释放对谷氨酸受体募集的影响，以及非同步释放对突触可塑性的影响。我们将测试这一假说，即突触刺激反应中谷氨酸的异步释放激活突触外NMDA受体和代谢性受体，并调节海马区长期突触的可塑性。综上所述，我们的研究将进一步加深我们对突触神经递质释放的空间限制机制的理解，并将证明突触外异步释放在突触外谷氨酸受体激活中的重要性。它将强调这种非突触形式的神经元通信在突触可塑性调节中的重要性。

The synchronous release of neurotransmitters (NTs) occurring within 2 ms of an action potential is spatially restricted to synapses. This spatial confinement is thought to be due to the reliance of synaptic vesicle (SV) docking and priming on proteins that are enriched in presynaptic active zone cytomatrices. NT release may also be spatially constrained at the level of calcium-triggered fusion of SVs with the plasma membrane. Synchronous release is initiated by synaptotagmins with low calcium affinity that need to be in close proximity to open voltage-gated calcium channels to trigger SV exocytosis. In addition to synchronous release, neurons also release NTs asynchronously tens to hundreds of milliseconds following action potentials. This form of release is triggered by sensors with high calcium affinity. At present, it is unclear whether asynchronous release is limited to synapses. We have gathered evidence to suggest that asynchronous occurs in perisynaptic axonal regions as well as at synapses. Building on this intriguing finding, we aim to identify mechanisms serving to spatially restrict NT release and to study the implications of perisynaptic asynchronous NT release for neurotransmission and synaptic plasticity. In particular, we will: 1) Characterize the spatial distribution of asynchronous NT release. Using several molecular approaches, we will augment or suppress asynchronous NT release and assess the effects on the spatial constraints of SV exocytosis at hippocampal synapses to confirm our preliminary finding that SV exocytosis extends into perisynaptic axonal regions. 2) Address modulation of asynchronous release by calcium extrusion and buffering. Asynchronous release may be spatially restricted by mechanisms limiting calcium diffusion. Initial experiments suggest that calcium transients are highly localized to presynaptic active zones with lateral diffusion into adjacent axonal segments corresponding to the observed extent of asynchronous release. To demonstrate the causal relationship, we will manipulate presynaptic calcium extrusion and buffering mechanisms and assess the effect of these manipulations on asynchronous release. 3) Examine differences in the recruitment of glutamate receptors by asynchronous and synchronous release and the role of asynchronous release on synaptic plasticity. We will test the hypothesis that asynchronous release of glutamate in response to burst stimulation activates extrasynaptic NMDA receptors and metabotropic receptors and modulates long-term synaptic plasticity in the hippocampus. Taken together, our research will further our understanding of mechanisms that spatially confine neurotransmitter release at synapses and will demonstrate the importance of extrasynaptic asynchronous release in the activation of extrasynaptic glutamate receptors. It will highlight the importance of this non-synaptic form of neuronal communication in the modulation of synaptic plasticity.