Understanding the connectivity of neural networks and its relationships to nervous system function and dysfunction remains a major challenge. Typical approaches to this problem include lesions or focal pharmacology; these methods are crude and non-specific. Recently, the mouse genome project and largescale expression studies, together with emerging transgenic technologies, have begun to allow selection of specific neuronal populations for intervention. But what should one target to intervene in neuronal function? Ideally a mechanism for intervention should be conditional, i.e. only interfere with function when prompted by the experimenter. Such induction should be rapid and rapidly reversible. Intervention should also be delicate, not requiring, for example, major surgical intervention. Finally, the intervention should be specific, in the sense that it interferes with a well-defined aspect of neuronal function without a myriad of secondary effects. We will design molecular genetic tools that allow conditional perturbation of synaptic transmission. Triggered by administration of pharmacological agents without endogenous targets, synaptic vesicles and/or proteins important for exocytosis will be mislocalized or immobilized, interfering with synaptic transmission. This will be accomplished in three steps. 1.) We will engineer modified versions of proteins involved in exocytosis by introducing domains (FK506 binding protein and target of rapamycin binding domain) that, while by themselves do not interfere with protein function, will confer on the fusions the ability to interact with small chemical crosslinkers (derivatives of FK506 and rapamycin). Addition of such cell-permeable dimerizers will induce protein-protein interactions that will sequester essential synaptic vesicle components away from their natural partners and/or the site of function, resulting in loss of synaptic transmission. 2.) Systems for inactivating synaptic transmission will be tested in cultured neurons and brain slices using optical techniques to monitor vesicle cycling and electrophysiology to measure synaptic currents. 3.) Well-characterized systems will be introduced into subpopulations of neurons in mice using viruses and transgenic approaches. Systems will be tested in vivo using intrinsic signal imaging and electrophysiological techniques.
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