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

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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.