Optical responses evoked by cerebellar surface stimulation in vivo using Neutral Red

The pH sensitive dye, Neutral Red, was used with optical imaging techniques to map intracellular pH shifts elicited by cortical surface stimulation of the rat cerebellum. In the in vivo rat cerebellar cortex stained with Neutral Red, a brief stimulus train (three stimuli at 33 Hz) evoked a longitudinal beam of increased fluorescence (acidic shift) running parallel to the long axis of the folium within 100 ms of stimulation onset. A 5-10 s stimulus train (5-20 Hz) produced a biphasic optical response consisting of a beam of increased fluorescence (acidic shift) which returned to baseline in approximately 60 s, followed by a beam of decreased fluorescence (alkaline shift) for up to 120 s. A close spatial correspondence was observed between electrophysiological and optical maps of the response to surface stimulation. Application of acetazolamide enhanced the optical signals, acetabenzolamide-phenoxyethene had no effect, and the glutamate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, decreased the optical signals. Increased fluorescence was produced by superfusion of the cerebellar cortex with acidic Ringer solutions and a decrease in fluorescence by basic solutions. These fluorescence changes also occurred in the presence of several ion channel/receptor blockers. Increased fluorescence resulted from superfusion with Ringer solution containing sodium propionate and decreased fluorescence with the transition from 5% carbon dioxide to nominally carbon dioxide-free Ringer solution. Recovery from acid loading with ammonium chloride was prevented by amiloride, an inhibitor of the Na+/H+ transporter. Application of Ringer solution with high potassium concentration produced an increase in fluorescence but only a decrease in fluorescence was detected when neuronal blockers were present, an effect consistent with a glial contribution. This decrease in fluorescence was blocked by adding barium. No epifluorescent optical signals were obtained from unstained preparations or preparations stained with cell-permeant fluorescence markers, suggesting little contribution from activity-dependent volume changes and other intrinsic signals. These results demonstrate that the Neutral Red optical signals evoked by cerebellar surface stimulation are primarily pH based and include a significant component related to intracellular pH shifts. The large amplitudes of these optical signals are particularly useful for mapping neuronal activity. Furthermore, this technique provides a novel tool for the study of pH changes in vivo at both high spatial and temporal resolution.