Variation in Neuroligin Concentration and Presynaptic Functional Development

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DESCRIPTION (provided by applicant): Schizophrenia and autism are devastating, complex brain disorders whose causes are poorly understood. Extensive genetic evidence links autism and schizophrenia to mutations in the cell adhesion molecule neuroligin. Disease-linked mutations alter neuroligin levels at the postsynaptic membrane. Abnormal neuroligin levels are associated with immature synapse formation, including immaturity of mechanisms to release neurotransmitter and recycle synaptic vesicles. These findings raise the possibility of treating autism and schizophrenia by targeting mechanisms of vesicular release and recycling to effect the mature presynaptic phenotype. We will use fluorescent markers of synaptic vesicle exocytosis to study the effect of varying neuroligin concentrations on synapse development and function. To model abnormal neuroligin concentration, we will attach neuroligin to coverslips in microislands roughly the size of a synapse and culture dissociated hippocampal neurons on them. Previous studies have shown that neuroligin is sufficient to induce neurons to form presynaptic boutons. Our preliminary experiments suggest that neurons indeed form presynaptic specializations on neuroligin-patterned glass. Unlike current technologies, this novel cell culture substrate allows us to control "postsynaptic" neuroligin levels precisely and disentangle neuroligin's effects on presynaptic and postsynaptic development. The geometry of the presynaptic terminals that form against the glass will allow us to apply high-resolution total internal reflection (TIRF) microscopy as well as epifluorescence at the same terminals. The work proposed here would be the first application of TIRF to the presynaptic compartment of maturing small central neurons. The detailed view of the presynaptic terminal offered by TIRF imaging has broad implications for understanding normal and pathological presynaptic function. The technology described here could be further developed to rapidly screen therapeutic agents that target abnormal synapse development, vesicle recycling, and neurotransmitter release.