Memory trace formation at and beyond individual synapses in the intact brain

Donal Hebb提出的记忆轨迹形成原理是指在信息输入重合时功能神经元连接的加强。这一原理在突触传递的长时程增强(LTP)中得到了经验验证，LTP是突触记忆的基本模型。然而，我们对其细胞机制的了解仍然有限，主要是因为试图将LTP与完整大脑中的记忆痕迹联系起来，从未实现对单个突触的解析。已发表的证据表明，代表LTP的神经元反应的增加可能是由于突触后电流的增加或突触前神经递质释放(突触保真度)的增加，或者两者兼而有之。本项目将利用最近出现的遗传编码指示器(GEI)，我们已经证明，它可以提供对单个突触连接的神经递质释放的强有力的监测。我们的试验数据和其他研究也表明，兴奋性神经递质谷氨酸可以在距离其突触释放部位微米的地方逃避主动的突触连接到达其受体。因为大脑中的突触之间的距离不到一微米，这表明兴奋性传递的一个重要的体积传递成分，挑战了神经网络中有线的一对一连接的原则。记忆的形成是否涉及突触保真度的变化，容量传递的兴奋信号在完整的大脑中是否意义重大，以及它可能带来的神经计算影响-这些问题是我们理解记忆机制的基础。解决这些问题一直是本提案的一个关键动机。因此，我们的总体目标是了解突触保真度的动态以及突触外兴奋信号在大脑记忆痕迹形成过程中的程度和作用。这一目标建立在我们内部确立的创新方法和初步结果的基础上，分为几个具体目标。-确定在LTP诱导过程中有组织脑组织中突触释放概率的群体动力学和关键的潜在机制。-在体内建立感觉刺激诱导的LTP期间丘脑皮质突触的突触保真度的动力学。-评估谷氨酸在体内兴奋性皮质突触的突触外作用的程度和可塑性相关的变化。-了解已记录的突触外谷氨酸逃逸的生理意义和网络影响。我们将通过将单细胞电生理学与细胞靶向GEI的高分辨率、高速多路复用双光子激发成像相结合来实现这些目标。体外准备中的对照测试将有助于我们优化体内实验，在这些实验中，我们将在我们测试的胡须刺激范例中监控个别识别的丘脑皮质突触的功能。我们将使用我们建立的高端计算机模型来评估我们观察到的大脑网络活动的意义。我们迄今积累的既定实验方案和试点数据应确保拟议研究战略的技术可行性。

The principle of memory trace formation formulated by Donal Hebb refers to the strengthening of functional neuronal connections upon coincidence of informative inputs. This principle has found its empirical validation in the long-term potentiation (LTP) of synaptic transmission, an elemental model of synaptic memory. However, our understanding of its cellular mechanisms remains limited, mainly because attempts to relate LTP to a memory trace in the intact brain have never achieved resolution of individual synapses. Published evidence suggests that the increase in neuronal responses representing LTP could arise form either increased postsynaptic current or from increased probability of presynaptic neurotransmitter release (synaptic fidelity), or both. The present project will take advantage of the recent emergence of genetically encoded indicators (GEIs), which we have shown could provide robust monitoring of neurotransmitter release from individual synaptic connections. Our pilot data and other studies also indicate that the excitatory neurotransmitter glutamate could escape the active synaptic connection reaching its receptors at micron distances from its synaptic release site. Because synapses in the brain are less than a micron apart, this suggests a significant volume-transmitted component of excitatory transmission, challenging the principle of wired, one-to-one connectivity in neuronal networks. Whether memory formation involves changes in synaptic fidelity, whether the volume-transmitted excitatory signal is significant in the intact brain, and what could be its neurocomputational implications - these questions are fundamental for our understanding of the memory machinery. Addressing them has been a key motive for the present proposal. Our overall aim is therefore to understand the dynamics of synaptic fidelity and the extent and role of extrasynaptic excitatory signalling during memory trace formation in the brain. Built on our in-house established innovative methods and our preliminary results, this aim breaks down into several specific objectives. - To determine population dynamics of synaptic release probability during LTP induction in organised brain tissue and the key underlying mechanisms. - To establish the dynamics of synaptic fidelity at thalamocortical synapses during a sensory stimulation induced LTP in vivo. - To evaluate the extent and plasticity-associated changes in the extrasynaptic actions of glutamate at excitatory cortical synapses in vivo. - To understand physiological significance and network implications of the documented extrasynaptic glutamate escape. We will achieve these objectives by combining single-cell electrophysiology with high-resolution, high-speed multiplexed two-photon excitation imaging of cell-targeted GEIs. Control tests in ex vivo preparations will help us to optimise experiments in vivo in which we will monitor function of individual identified thalamocortical synapses in our tested paradigm of whisker stimulation. We will use our established high-end computer models to evaluate the significance of our observations for brain network activity. The established experimental protocols and pilot data that we have accumulated to date should ensure technical feasibility of the proposed research strategy.