Dynamic Imaging of Synaptic Inhibition

Duke University, Durham NC

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Abstract

While many methods are available for functional imaging of excitatory processes in the brain, until now there has been no practical way to image synaptic inhibition in the brain. The goal of this project is to adopt clomeleon, a genetically-encoded indicator protein, for imaging Cl-dependent synaptic inhibition in the brain. This indicator was produced by fusing the chloride-sensitive yellow fluorescent protein with the chloride-insensitive cyan fluorescent protein; the ratio of fluorescence resonance energy transfer (FRET) dependent emission of these two fluorophores varies in proportion to the intracellular concentration of chloride ions ([Cl-]i). The proposed experiments will use a genetic strategy to target expression of clomeleon to subsets of neurons in the mouse brain. This should allow many novel experimental analyses of the physiological functions of Cl-. During the Phase I experiments proposed here, fluorescence imaging methods will be use to look for neuron-specific variations in resting [Cl-]i and changes in [Cl-]i associated with activation of inhibitory synaptic pathways in neurons of hippocampal, cerebellar, and cortical tissue slices. In Phase II, these experimental procedures will be extended to in vivo conditions to image the temporal and spatial patterns of synaptic inhibition in neural networks of the intact brain. This new technology should provide the first experimental views of the dynamics of synaptic inhibition in the brain and offers the promise of elucidating many important features of brain activity during normal function and as a consequence of drug abuse.