Molecular mechanisms of Synaptotagmin 1 mediated synaptic vesicle fusion

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The goal of this project, which falls into the Bioscience and Biotechnology priority area, is to obtain new fundamental insights into the molecular mechanism of synaptic transmitter release. This project features the development of new experimental tools (with Scientifica) via integration of optogenetics, patch-clamp electrophysiology and fluorescence imaging.The timing of synaptic vesicle fusion is precisely controlled by vesicular Ca2+ sensor synaptotagmin (Syt1, 2, and 9 in different synapses), yet the mechanism of its action remains enigmatic. We propose to test the hypothesis that formation of Ca2+-sensitive Syt1 oligomeric rings plays a major role in synchronisation of evoked vesicular release. We will capitalise on recent findings of our collaborator J. Rothman (Yale) who produced a Syt1 mutant (F349A) that exerts a dominant-negative effect on Syt1 oligomersation in biochemical assays. If Syt1 oligomeric rings are essential for synchronisation of transmitter release then overexpression of the F349A mutant in presynaptic terminals should desynchronise vesicle fusion.Testing this hypothesis requires measurements of evoked vesicle release with a submillisecond precision. This is difficult to achieve in randomly connected neurons in culture and thus requires the use of organised brain tissue. We will use transgenic mice where EYFP tagged channelrhodopsin-2 (ChR2) is expressed in pyramidal neurons under the control of CaMKII promoter (the colony is established in our lab). We will inject lentiviral constructs carrying either WT or F349A Syt1 fused with mCherry into mouse neocortex in vivo. Several weeks later we will prepare acute brain slices and use yellow and red fluorescence to identify ChR2 expressing cells which also express recombinant Syt1 constructs. Identified individual neurons will be stimulated with 477 nm laser and optically evoked post-synaptic currents (EPSCs) will be recorded from a post-synaptic cell. In this way, we will compare the effects of F349A and WT Syt1 constructs on fast evoked transmitter release and will establish whether Syt1 oligomerisation is required for synchronisation of vesicle fusion.These experiments pose several technical challenges that are squarely within the expertise of our industrial partner. First, we need to restrict photostimulation to a single presynaptic neuron avoiding spike generation in the neighbouring neurons and their fibres. To address this, the student will develop new software capabilities that will implement precise control of the spatio-temporal stimulation patters in the recently marketed Scientifica Laser Applied Stimulation and Uncaging (LASU) system.Second, we need to ensure that EPSCs are generated by spiking of a single neuron. Imaging of spikes in neurons co-expressing a genetically encoded Ca2+ indicator (e.g. GCaMP6) potentially provides a powerful control for the specificity of photostimulation. Therefore, along with Syt1 constructs we will co-inject GCaMP6 viral constructs. The challenge here is the overlap between the excitation spectra of ChR2 and GCaMP6. To overcome this we will integrate an EM-CCD camera and an LED-based epifluorescence imaging system with the LASU. The use of EM-CCD will allow us to reduce the intensity of GCaMP6 excitation light below the ChR2 activation threshold.The use of the above approach should allow parallel stimulation of Syt1 expressing neurons. As a contingency we will also use paired whole-cell patch-clamp recordings.