Dye screening and signal-to-noise ratio for retrogradely transported voltage-sensitive dyes

Using a novel method for retrogradely labeling specific neuronal populations, we tested different styryl dyes in attempt to find dyes whose staining would be specific, rapid, and lead to large activity dependent signals. The dyes were injected into the ventral roots of the isolated chick spinal cord from embryos at days E9-E12. The voltage-sensitive dye signals were recorded from synaptically activated motoneurons using a 464 element photodiode array. The best labeling and optical signals were obtained using the relatively hydrophobic dyes di-8-ANEPPQ and di-12-ANEPEQ. Over the 24 h period we examined, these dyes bound specifically to the cells with axons in the ventral roots. The dyes responded with an increase in fluorescence of 1-3% (delta F/F) in response to synaptic depolarization of the motoneurons. The signal-to-noise ratio obtained in a single trial from a detector that received light from a 14 x 14 microns2 area of the motoneuron population was about 10:1. Nonetheless, signals on neighboring diodes were similar, suggesting that we were not detecting the activity of individual neurons. Retrograde labeling and optical recording with voltage-sensitive dyes provides a means for monitoring the activity of identified neurons in situations where microelectrode recordings are not feasible.