中国科学院昆明动物研究所机构知识库

突触传递是神经信息处理的重要环节，主要通过突触前神经终末的囊泡释放神经递质诱发递质受体介导的突触后兴奋性或抑制性反应而实现。在神经突触上，囊泡有自发释放和突触前的动作电位诱发的快速同步释放和慢速异步释放三种形式。突触囊泡的释放有着不同程度的钙离子依赖性。钙离子通过与不同的感受器蛋白结合可以调控不同形式的囊泡释放。已有的研究表明，Synaptotagmin 1, 2, 9是参与调节突触囊泡同步释放的钙离子感受器蛋白，而Synaptotagmin 7 和 Double C2 domain (Doc-2) 则可能参与调节突触囊泡异步释放过程。以往研究的主要方法之一就是利用钙离子传感蛋白对钙、锶两种二价阳离子的不同亲合力，以锶离子取代钙离子进行二价阳离子内流和突触囊泡动力学的观察。然而，由于研究方法的限制，对于调节突触囊泡自发释放的钙离子感受器蛋白目前仍不清楚。其主要的技术瓶颈在于无法对没有去极化的静息条件下神经突触终末的二价阳离子电流进行直接的观测和对突触囊泡纳米邻域内的钙离子浓度进行直接调控。本课题利用Calyx of Held突触在形态和电生理特性上的优势，用细胞贴附式膜片钳方法研究静息状态下的钙/锶离子单通道电流的特性和全细胞记录模式下囊泡自发释放受突触终末钙/锶离子浓度调控的规律。鉴于Calyx of Held突触上Synaptotagmin 2已被证实不参与调控突触囊泡自发释放，我们选取以Synaptotagmin-2敲除的小鼠的Calyx of Held突触为标本，进一步突显囊泡自发释放机制的作用。我们发现，静息状态下电压门控钙离子通道对锶离子的开放时间较之钙离子更长，但其电导不变，成功解释了电压门控钙离子通道的锶离子通透电流大于钙离子电流的机制；我们还发现锶离子结合钙离子感受器蛋白调控囊泡自发释放的能力却显著降低，揭示了调控自发释放的钙离子感受器蛋白与锶离子的相互作用及其调控囊泡自发释放的内在特征，为最终确认囊泡自发释放的钙离子传感器蛋白和进一步阐述囊泡自发释放的分子机制提供了重要证据。相当长时期以来，以电刺激并结合传统的电生理记录方法是用于检测神经细胞的特性和探索不同脑区的功能的主要手段。光敏感通道作为一种光遗传学手段，使对特定脑区与/或特定种类细胞的观测成为可能，已广泛用于神经环路乃至动物的行为学研究。同时，在神经细胞生物学上对光敏感通道的表达和诱发动作电位的功效和可靠性的研究非常有限。Calyx of Held突触是在定量研究突触传递上独具特色的标本。然而因其突触前很长的神经纤维来自于对侧耳蜗核，在脑片制作中极易被切断，传统电极刺激诱发突触后兴奋性电流的几率不高，直接影响了研究效率，因此不少实验室试图引入光遗传学方法。由于幼年期小鼠脑部立体定位困难和自身发育特点，这一方法一直没能成功应用。本课题中，我们通过用腺相关病毒感染的方法在腹侧耳蜗核区表达光遗传基因，特异性地在Calyx of Held突触前膜表达光敏感通道蛋白，成功并可靠地诱发了突触后电流。比较电刺激和光刺激诱发的突触后电流的相关参数。发现光遗传学方法除了存在延时效应外，二者没有明显的差异。该技术为在小鼠Calyx of Held突触，特别是在幼年期Calyx of Held突触上展开突触传递的研究提供了高效的诱发神经纤维动作电位的手段。同时也为进一步研究诱发动作电位以及神经突触可塑性的功效和可靠性提供了理想研究平台。 

Synaptic transmission is the process of neuronal communication whereby one neuron release neurotransmitter molecules that interact with receptors of the recipient neuron. It has been known calcium ions play an essential role in mediating different modes of neurotransmitter release via different sensing mechanisms. So far, the mechanism of calcium sensors that mediated the synchronous release and asynchronous release were well konwned. Comparatively, the calcium sensors for spontaneous release remain a mystery. In order to disclose the characterizaton of calcium sensors of spontaneous release, we employed pre-/post-synaptic whole-cell recording at the Calyx of Held synapses of Synaptotagmin-2 knock out mice to assay the calcium ions and strontium ions influx into the nerve terminal at resting potential and observed the effects of calcium ions and strontium ions on spontaneous neurotransmitter release. We used cell-attached patch recording the current influx by single voltage gated calcium channel. We found that same extracellular concentration of calcium ions and strontium ions have similar effect in regulating spontaneous release. But we found the dwell time of single voltage gated calcium channel opening increased around 3-fold for strontium ions than calcijm ions with the channel conductance unchanged; the divalent cation sensing machinery in regulating spontaneous release has much lower sensitivity to strontium ions than calcium ions. Thus, our study depicted the intrinsic properties of calcium sensor(s) of spontaneous transmitter release and provided an insight into the underlying mechanisms.Electrical stimulation in combination with electrophysiological recording has for long time been used as a major tool to study the functional property of different neurons and brained areas. Recently, optogenetic approach was applied and made it possible to activate specified neurons and/or at specific brain area and thus successfully used in the study of neural circuits and neural behavior. Relatively, the cell biological studies on efficiency and reliability of optogenetic initiation of action potential are much fewer. Calyx of Held synapse is a unigue preparation for the quantitative study of synaptic transmission. However, the success rate of inducing fiber stimulation evoke postsynaptic response is never high enough due to the shortened length of fiber during brain slice making. It has been long time for the researcher to applied optogenetic techniques to optically induce presynaptic action potential at Calyceal nerve terminal but no successful case has been achieved in mouse brain. Here we developed an approach with modified protocol to overexpress Channelrhodorpsin at Cayceal terminal via stereotaxic injection of Channelrhodorpsin packaged Adeno-associated virus into ventral cochlear nucleus. After side-by-side comparison, we determined that optogenetic stimulation can successfully induce presynaptic action potential and achieved the identical postsynaptic response in terms of EPSC kinetics, short term synaptic plasticity as well as readily releasable pool. Thus we developed an approach to study synaptic transmission at the Calyx of Held synase with high efficiency and provide an ideal platform for the studies of the efficiency and reliability of optogenetic initiation action potential in cell biological perspective.