Neutral red as a hydrophobic probe for monitoring neuronal activity

Neutral red fluorescence has been used to monitor neuronal activity. Local changes in either pH or hydrophobicity are reported to increase neutral red fluorescence, but the mechanisms underlying the increases remain unclear. In this study, the pH-dependent fluorescence changes in the basic and acidic forms of neutral red preloaded in rat cerebellar slices were separately measured with two excitation wavelengths. Bath application of kainate, domoate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) at 1 mM increased fluorescence of the acidic form and decreased that of the basic form irreversibly, consistent with a pH-dependent mechanism. In contrast, application of lower concentrations of AMPA, (S)-3,5-dihydroxyphenylglycine or KCl increased fluorescence of both forms transiently, suggesting that a pH-independent mechanism may also increase neutral red fluorescence. Electrical stimulation in the molecular layer also increased fluorescence of both forms. The response to weak electrical stimulation decayed in about 1 s, while response to intense stimulation lasted longer than 1 min. Neutral red binding to phospholipids was also detected as fluorescent spots on thin-layer chromatography, suggesting that interaction with phospholipids enhances neutral red fluorescence. Thus, the short-lasting signal of neutral red suggests its usage as a hydrophobic probe for neuronal activity.