Analysis of neural activity with fluorescent protein biosensors

Abstract Fluorescent calcium sensors provide a means of detecting and analyzing cytoplasmic calcium levels in embryos and larvae. Conventional RNA injection of eggs results in expression of protein sensors throughout larval tissues. Larvae are immobilized for wide field or confocal recordings and video records reveal recurrent fluctuations in cytoplasmic calcium levels in several cell types. Neurons can be identified by location and form, and continuous records made of their activity. Confocal image stacks are registered and Z -axis, fluorescence intensity profiles of individual neurons generated to provide time/activity plots. These optogenetic methods enable analysis in intact larvae of the activity of identified neurons or effectors, such as muscles or ciliary band cells.