An evaluation of in vivo voltage-sensitive dyes: pharmacological side effects and signal-to-noise ratios after effective removal of brain-pulsation artifacts.

In the current study, we investigated pharmacological side effects and signal-to-noise ratios (SNRs) of two commonly used voltage-sensitive dyes (VSDs): the blue dye RH-1691 (1 mg/ml) and the red dye di-4-ANEPPS (0.1 mg/ml), applied in vivo to the rat barrel cortex. Blue dyes are often favored over red dyes in in vivo studies due to their apparent superior SNR, partly because their fluorescence spectrum is farther away from the hemoglobin absorption spectrum, making them less prone to heartbeat-associated brain-pulsation artifacts (BPA). We implemented a previously reported template-based BPA removal algorithm and evaluated its applicability to di-4-ANEPPS before comparing characteristics of the two dyes. Somatosensory-evoked potentials (SEPs) were also recorded. Whereas SEPs recorded before and after application of di-4-ANEPPS failed to exhibit demonstrable differences, RH-1691 caused a significant and prolonged increase in SEP amplitude for several hours. In contrast, neither dye influenced the spontaneous cortical activity as assessed by the spectral content of the EEG. Both dyes turned out to be strikingly similar with respect to changes in fractional fluorescence as a function of SEP response amplitude, as well as regarding shot noise characteristics after removal of the BPA. Thus there is strong evidence that the increased SNR for RH-1691 is a consequence of an artificially increased signal. When applying an appropriate BPA removal algorithm, di-4-ANEPPS has proven to be suitable for single-trial in vivo VSD imaging (VSDI) and produces no detectable neurophysiological changes in the system under investigation. Taken together, our data argue for a careful re-evaluation of pharmacological side effects of RH-1691 and support the applicability of di-4-ANEPPS for stable single-trial in vivo VSDI recordings.