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

Donal Hebb提出的记忆痕迹形成原理是指信息输入重合时功能性神经元连接的加强。这一原理在突触传递的长期增强（LTP）中得到了实证验证，LTP是突触记忆的基本模型。然而，我们对其细胞机制的理解仍然有限，主要是因为将LTP与完整大脑中的记忆痕迹联系起来的尝试从未实现单个突触的解析。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增加或突触前神经递质释放（突触保真度）的可能性增加，或两者兼而有之。目前的项目将利用最近出现的遗传编码指示器（GEIs），我们已经证明它可以提供从单个突触连接释放神经递质的强大监测。我们的试点数据和其他研究也表明，兴奋性神经递质谷氨酸可以逃避活跃的突触连接，在距离其突触释放位点微米的距离到达其受体。由于大脑中的突触之间的距离不到一微米，这表明兴奋性传递中存在重要的体积传递成分，这挑战了神经元网络中有线的一对一连接原则。记忆的形成是否涉及突触保真度的变化，体积传递的兴奋信号在完整的大脑中是否重要，以及它的神经计算含义是什么——这些问题是我们理解记忆机制的基础。解决这些问题是提出本建议的一个主要动机。因此，我们的总体目标是了解突触保真度的动态以及大脑记忆痕迹形成过程中突触外兴奋性信号的程度和作用。基于我们内部建立的创新方法和我们的初步结果，这一目标分解为几个具体目标。-确定LTP诱导有组织脑组织中突触释放概率的种群动态和关键的潜在机制。-在体内建立感觉刺激诱导LTP时丘脑皮质突触突触保真度的动力学。-评估体内兴奋性皮质突触中谷氨酸的突触外作用的程度和可塑性相关变化。-了解记录的突触外谷氨酸逃逸的生理意义和网络含义。我们将通过将单细胞电生理学与细胞靶向gei的高分辨率，高速多路双光子激发成像相结合来实现这些目标。离体制剂的对照试验将帮助我们优化体内实验，在我们测试的须刺激范例中，我们将监测个体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对大脑网络活动的观察的意义。我们迄今积累的既定实验方案和试点数据应确保所提议的研究策略在技术上的可行性。

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.