$BFbMF!'(JClomeleon is a genetically encoded indicator that uses FRET to provide an absolute measure of the internal Cl^- concentration ([Cl^-]_i;). We have developed transgenic mice with Clomeleon expression targeted selectively to neurons by employing a neuron-specific promoter, Thy1. This transgene was introduced into several lines of transgenic mice, each of which express Clomeleon in specific subsets of neurons.

$B!!(JThe ability of Clomeleon to report changes in [Cl^-]i associated withphasic synaptic activity was tested in vitro by preparing acute slices from the brains of these transgenic mice. [Cl^-]_i was imaged in brain slices from the cerebellum, amygdala and hippocampus of 14$B!](J20-day-old mice. In all of these regions, synaptic activation caused detectable rises in [Cl^-]_i, although relatively large inhibitory events are required to produce an adequate signal/noise. These results indicate that Clomeleon can report changes in [Cl^-]_i associated with phasic synaptic inhibition, We also used Clomeleon to examine the properties of tonic inhibition of cerebellar granule cells. Application of the GABAA receptor antagonists reduced resting [Cl^-]_i in these cells, due to blockade of tonic activation of GABA_A receptors. Cl- imaging allowed us to examine the spatial distribution of tonic inhibition in the cerebellum. GABA_A antagonists decreased [Cl^-]_i in the molecular layer, where granule cell axons (parallel fibers) reside, and also in some mossy fiber terminals. These results indicate that Clomeleon can be used to image tonic release of GABA and that this release causes tonic inhibition at several loci within the cerebellar cortex. In summary, Clomeleon permits imaging of both phasic and tonic inhibition of central neurons in vitro. This ability should enable imaging of the spatiotemporal dynamics of inhibitory circuits in the brain.

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