"Recent studies in the Calyx of Held, a specialized synapse in the auditory pathway, have now produced clear evidence of heterogeneity in release-‘willingness’ among releasable vesicles. The general aim of this project is to investigate heterogeneity of release willingness in typical CNS synapses and to characterize the molecular mechanisms that control this heterogeneity. We will first develop a method that allows single synapse recording in primary cultured hippocampal neurons using pharmacological isolation of single visually identified (fluorescently labelled) synapses. By combining electrophysiology, fluorescence microscopy and pharmacological tools (e.g. hyperosmotic sucrose to assess total synaptic release pool size), we will perform a detailed description of (the heterogeneity in) synaptic vesicle release from single synaptic boutons. In addition, the combined research methods permit simultaneous pre- and postsynaptic assessment of neurotransmission efficacy, allowing us to pioneer in segregating pre- and postsynaptic plasticity mechanisms. Furthermore, a recently developed EGTA-based calcium-cage (DMNPE-4) allows stringent control over presynaptic Ca2+ concentrations, thereby enabling detailed analysis of Ca2+-sensitivity of synaptic vesicles within a single presynaptic terminal. Similar analyses will be performed in neurons deficient for candidate molecules or expressing mutated candidate molecules, thereby illuminating their role in controlling release willingness and/or Ca2+ sensitivity of synaptic vesicles. A detailed understanding of these molecular mechanisms is a fundamental starting point for unravelling synaptic mechanisms behind learning and memory. In general, these efforts will provide new insights in synaptic processing and plasticity, bring us closer to the understanding of human pathogenic mechanisms in the terminal and may provide insights into the pathology of ‘synaptopathies’."