Understanding the function of synapses is key to understanding the nervous system, and yet, while synaptic transmission is ubiquitously controlled by presynaptic G protein coupled receptors (GPCRs), the means by which these receptors modify release is contentious. Central nervous diseases, in which GPCR function is treated by pharmacological intervention, cover conditions ranging from aggression, depression and schizophrenia. Inhibitory presynaptic GPCRs alter synaptic function by many mechanisms. These may utilize the G?? G protein subunit to modify presynaptic ion channels and then synaptic secretion, or to modify the very machinery of vesicle fusion, the SNARE complex. Alternatively, these signaling systems may act indirectly through, other second messengers. We have focused on direct mechanisms by which G?? inhibits transmitter release and have demonstrated that G?? can inhibit neurosecretion by an interaction with the fusion machinery - the SNARE complex. This interaction occurs late in the activation of the synapse, after priming, in vesicles whose SNARE complex is formed. We hypothesize that G?? binds to the ternary SNARE complex to compete with Ca2+-dependent synaptotagmin binding. In this way G?? might be thought of as means to interfere with the final switch leading to neurosecretion. We propose that this action of G?? also confers a Ca2+ dependency on G?? -mediated presynaptic inhibition because high presynaptic Ca2+ concentrations allow synaptotagmin to compete more effectively with the machinery of vesicle fusion. The final outcome of this G?? competition with synaptotagmin allows a fine-tuned control of secretion. G?? causes kiss-and-run fusion of the exocytosing vesicle. This reduces the peak synaptic concentration of neurotransmitter, adding a complex twist to the method by which GPCRs alter synaptic function. We propose to investigate competition between the switch that activates synapses - Ca2+-synaptotagmin - and an inhibitor of this process - G??. We hypothesize that G?? and synaptotagmin share an interaction site on the SNARE complex. To demonstrate this, we will investigate G?? competition with synaptotagmin at the SNARE complex and compare effects of Ca2+ on this competition to similar manipulations in a simple reconstituted model for fusion and at the lamprey giant axon in situ. We will then modify in situ SNARE complex proteins using intracellularly applied Botulinum toxins to compare similar truncations in vitro. We will also use fluorescence measurements of exogenously applied G?? model systems of the protein components of vesicle fusion to determine whether Ca2+ evokes similar effects on binding and on neurotransmitter release in situ. These experimental paradigms will then be reproduced with dynamically fluctuating Ca2+ concentrations to mimic a real world activity of central synapses. These experiments will afford insight into the dynamic modulation possible with a simple protein-protein interaction and explain some of the complexities of presynaptic GPCR-mediated presynaptic modulation and their roles in neuropathologies.
In the central nervous system, G protein coupled receptors are ubiquitous and pathologies related to dysfunction of this widely distributed group of receptors lead to devastating illnesses of the mind, including Parkinson's disease, Schizoprenia, depression and compulsive disorders. However, at the basic unit of nervous system communication - the synapse - we understand little of the way G proteins function. We wish to understand how these mechanisms modify chemical communication using one such receptor, the 5-HT1B receptor, as a model in a simple vertebrate synapse. We will determine how this receptor acts and how it modulates chemical communication throughout the brain.
|Ramachandran, Shankar; Alford, Simon (2014) Acute dissociation of lamprey reticulospinal axons to enable recording from the release face membrane of individual functional presynaptic terminals. J Vis Exp :e51925|
|Hamid, Edaeni; Church, Emily; Wells, Christopher A et al. (2014) Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex. J Neurosci 34:260-74|
|Ryczko, Dimitri; Gratsch, Swantje; Auclair, Francois et al. (2013) Forebrain dopamine neurons project down to a brainstem region controlling locomotion. Proc Natl Acad Sci U S A 110:E3235-42|
|Alpert, Michael H; Alford, Simon (2013) Synaptic NMDA receptor-dependent Ca²? entry drives membrane potential and Ca²? oscillations in spinal ventral horn neurons. PLoS One 8:e63154|