Optogenetic control of phosphoinositide at mouse calyx of Held synapse

点击翻译按钮获取中文摘要

Synaptic transmission relies on effective transduction of presynaptic action potentials into Ca2+-triggered synaptic vesicle (SV) fusion within a millisecond. To achieve the speed, SVs morphologically attach to the plasma membrane and prepare to be fusion competent before the arrival of action potentials. This entire process occurs in a specialized structure called active zone that constitutes a complex molecular architecture and spatially organized lipid rafts, which mediates the speed, precision and the plasticity of synaptic transmission. Phosphatidylinositol 4,5-bisphosphate (PIP2) is one of the phospholipids that locates at the inner leaflet of the active zone membrane. Its unique property of carrying multiple negative charges is proposed to be important for recruiting downstream effector proteins during exocytosis and endocytosis. However, despite the extensive efforts over the years for studying its function, our understanding of how PIP2 regulates synaptic transmission is still very limited. This is largely due to the lack of spatiotemporal control that allows direct manipulation of its quantity at presynaptic membrane. Most of the methods relied on pharmacological or genetic perturbations of lipid kinase/phosphatase, however off-target or long-term compensatory effects often complicate the interpretation of PIP2’s role. To overcome this limitation, I aim to pioneer a novel optogenetic PIP2-deletion system (CIB1-CRY2) at a mammalian central nervous synapse called calyx of Held. This tool enables transient and reversible recruitment of phosphatase into the presynaptic membrane by using pulses of blue light, which leads to a rapid and local elimination of PIP2 within a few seconds. In combination with two-photon PIP2 live-imaging and pre- and post-synaptic patch-clamp recordings, this proposal aims to directly probe the functional link between the spatial distribution of PIP2 and SV fusion. Specifically, three major questions will be addressed in the present proposal: (1) Whether PIP2 regulates the gating and/or clustering of voltage-gated Ca2+ channel at presynaptic terminal? (2) Do PIP2-SV interactions determine the number of readily-releasable SVs at active zone? (3) Does PIP2 regulate synaptic short-term plasticity? These results are expected to gain unequivocal insights into the functional role of PIP2 in synaptic transmission and further lead weight on our understanding of the mechanisms of synaptic plasticity at mammalian central nervous synapse.