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 use Clomeleon, a genetically-encoded indicator protein, to image chloride-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 dependent emission of these two fluorophores varies in proportion to the intracellular concentration of chloride ions ([Cl]i). The proposed experiments are designed to optimize the Clomeleon technique for imaging the spatial and temporal dynamics of inhibitory circuits in living brain tissue. First, we will use mutagenesis to change the chloride binding properties of Clomeleon. This will enhance the ability of Clomeleon to report changes in [Cl]i in the range relevant for synaptic inhibition. Second, we will use two genetic strategies to target expression of Clomeleon to subsets of neurons in the mouse brain. Third, we will use fluorescence imaging methods to measure changes in [Cl]i associated with activation of inhibitory synaptic pathways in neurons of slices of the cerebellum, amygdala, and superior colliculus from these Clomeleon-expressing mice. While these experimental procedures will be used in vitro, it is hoped that our improvements in Clomeleon technology eventually will allow imaging of the temporal and spatial patterns of synaptic inhibition in neural networks of the intact brain. This new technology should provide the first spatially-resolved 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, during psychiatric and neurological disorders, and as a consequence of drug abuse.
