An improved inverse-type Ca2+ indicator can detect putative neuronal inhibition in Caenorhabditis elegans by increasing signal intensity upon Ca2+ decrease

摘要

Sensory processing is regulated by the coordinated excitation and inhibition of neurons in neuronal circuits. The analysis of neuronal activities has greatly benefited from the recent development of genetically encoded Ca²⁺ indicators (GECIs). These molecules change their fluorescence intensities or colours in response to changing levels of Ca²⁺ and can, therefore, be used to sensitively monitor intracellular Ca²⁺ concentration, which enables the detection of neuronal excitation, including action potentials. These GECIs were developed to monitor increases in Ca²⁺ concentration; therefore, neuronal inhibition cannot be sensitively detected by these GECIs. To overcome this difficulty, we hypothesised that an inverse-type of GECI, whose fluorescence intensity increases as Ca²⁺ levels decrease, could sensitively monitor reducing intracellular Ca²⁺ concentrations. We, therefore, developed a Ca²⁺ indicator named inverse-pericam 2.0 (IP2.0) whose fluorescent intensity decreases 25-fold upon Ca²⁺ binding in vitro. Using IP2.0, we successfully detected putative neuronal inhibition by monitoring the decrease in intracellular Ca²⁺ concentration in AWC^ON and ASEL neurons in Caenorhabditis elegans. Therefore, IP2.0 is a useful tool for studying neuronal inhibition and for the detailed analysis of neuronal activities in vivo.