Comparison of two voltage-sensitive dyes and their suitability for long-term imaging of neuronal activity

One of the key approaches for studying neural network function is the simultaneous measurement of the activity of many neurons. Voltage-sensitive dyes (VSDs) simultaneously report the membrane potential of multiple neurons, but often have pharmacological and phototoxic effects on neuronal cells. Yet, to study the homeostatic processes that regulate neural network function long-term recordings of neuronal activities are required. This study aims to test the suitability of the VSDs RH795 and Di-4-ANEPPS for optically recording pattern generating neurons in the stomatogastric nervous system of crustaceans with an emphasis on long-term recordings of the pyloric central pattern generator. We demonstrate that both dyes stain pyloric neurons and determined an optimal concentration and light intensity for optical imaging. Although both dyes provided sufficient signal-to-noise ratio for measuring membrane potentials, Di-4-ANEPPS displayed a higher signal quality indicating an advantage of this dye over RH795 when small neuronal signals need to be recorded. For Di-4-ANEPPS, higher dye concentrations resulted in faster and brighter staining. Signal quality, however, only depended on excitation light strength, but not on dye concentration. RH795 showed weak and slowly developing phototoxic effects on the pyloric motor pattern as well as slow bleaching of the staining and is thus the better choice for long-term experiments. Low concentrations and low excitation intensities can be used as, in contrast to Di-4-ANEPPS, the signal-to-noise ratio was independent of excitation light strength. In summary, RH795 and Di-4-ANEPPS are suitable for optical imaging in the stomatogastric nervous system of crustaceans. They allow simultaneous recording of the membrane potential of multiple neurons with high signal quality. While Di-4-ANEPPS is better suited for short-term experiments that require high signal quality, RH795 is a better candidate for long-term experiments since it has only minor effects on the motor pattern.