Modulation and plasticity of neurotransmission: Presynaptic mechanisms

The synchronous release of neurotransmitters (NTs) occurring within 2 ms of an action potential is spatially restricted to synapses. This spatial confinement is thought to be due to the reliance of synaptic vesicle (SV) docking and priming on proteins that are enriched in presynaptic active zone cytomatrices. NT release may also be spatially constrained at the level of calcium-triggered fusion of SVs with the plasma membrane. Synchronous release is initiated by synaptotagmins with low calcium affinity that need to be in close proximity to open voltage-gated calcium channels to trigger SV exocytosis. In addition to synchronous release, neurons also release NTs asynchronously tens to hundreds of milliseconds following action potentials. This form of release is triggered by sensors with high calcium affinity. At present, it is unclear whether asynchronous release is limited to synapses. We have gathered evidence to suggest that asynchronous occurs in perisynaptic axonal regions as well as at synapses. Building on this intriguing finding, we aim to identify mechanisms serving to spatially restrict NT release and to study the implications of perisynaptic asynchronous NT release for neurotransmission and synaptic plasticity. In particular, we will: 1) Characterize the spatial distribution of asynchronous NT release. Using several molecular approaches, we will augment or suppress asynchronous NT release and assess the effects on the spatial constraints of SV exocytosis at hippocampal synapses to confirm our preliminary finding that SV exocytosis extends into perisynaptic axonal regions. 2) Address modulation of asynchronous release by calcium extrusion and buffering. Asynchronous release may be spatially restricted by mechanisms limiting calcium diffusion. Initial experiments suggest that calcium transients are highly localized to presynaptic active zones with lateral diffusion into adjacent axonal segments corresponding to the observed extent of asynchronous release. To demonstrate the causal relationship, we will manipulate presynaptic calcium extrusion and buffering mechanisms and assess the effect of these manipulations on asynchronous release. 3) Examine differences in the recruitment of glutamate receptors by asynchronous and synchronous release and the role of asynchronous release on synaptic plasticity. We will test the hypothesis that asynchronous release of glutamate in response to burst stimulation activates extrasynaptic NMDA receptors and metabotropic receptors and modulates long-term synaptic plasticity in the hippocampus. Taken together, our research will further our understanding of mechanisms that spatially confine neurotransmitter release at synapses and will demonstrate the importance of extrasynaptic asynchronous release in the activation of extrasynaptic glutamate receptors. It will highlight the importance of this non-synaptic form of neuronal communication in the modulation of synaptic plasticity.