Knowing which neurons are active during behavior is a crucial step toward understanding how nervous systems work. Neuronal activation is generally accompanied by an increase in intracellular calcium levels. Therefore, intracellular calcium levels are widely used as a proxy for neuronal activity. Many types of synthetic components and bioluminescent or fluorescent proteins that report transient and long-term changes in intracellular calcium levels have been developed over the past 60 years. Calcium indicators that enable imaging of the dynamic activity of a large ensemble of neurons in behaving animals have revolutionized the field of neuroscience. Among these, transcription-based genetically encoded calcium indicators (transcriptional GECIs) have proven easy to use and do not depend on sophisticated imaging systems, offering unique advantages over other types of calcium indicators. Here, we describe the two currently available fly transcriptional GECIs-calcium-dependent nuclear import of LexA (CaLexA) and transcriptional reporter of intracellular calcium (TRIC)-and review studies that have used them. In the accompanying protocol, we present step-by-step details for generating CaLexA- and TRIC-ready flies and for imaging CaLexA and TRIC signals in dissected brains after experimental manipulations of intact free-moving flies.
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