OPTICAL-RESPONSES EVOKED BY CEREBELLAR SURFACE STIMULATION IN-VIVO USING NEUTRAL RED

The pH sensitive dye, Neutral Red, was used with optical imaging techn iques to map intracellular pH shifts elicited by cortical surface stim ulation of the rat cerebellum. In the in vivo rat cerebellar cortex st ained 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 stimu lation onset. A 5-10 s stimulus train (5-20 Hz) produced a biphasic op tical 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 cl ose spatial correspondence was observed between electrophysiological a nd optical maps of the response to surface stimulation. Application of acetazolamide enhanced the optical signals, acetabenzolamide-phenoxye thene had no effect, and the glutamate antagonist, 6-cyano-7-nitroquin oxaline-2,3-dione, decreased the optical signals. Increased fluorescen ce was produced by superfusion of the cerebellar cortex with acidic Ri nger solutions and a decrease in fluorescence by basic solutions. Thes e fluorescence changes also occurred in the presence of several ion ch annel/receptor blockers. Increased fluorescence resulted from superfus ion with Ringer solution containing sodium propionate and decreased fl uorescence with the transition from 5% carbon dioxide to nominally car bon dioxide-free Ringer solution. Recovery from acid loading with ammo nium chloride was prevented by amiloride, an inhibitor of the Na+/H+ t ransporter. Application of Ringer solution with high potassium concent ration produced an increase in fluorescence but only a decrease in flu orescence was detected when neuronal blockers were present, an effect consistent with a glial contribution. This decrease in fluorescence wa s blocked by adding barium. No epifluorescent optical signals were obt ained from unstained preparations or preparations stained with cell-pe rmeant fluorescence markers, suggesting little contribution from activ ity-dependent volume changes and other intrinsic signals. These result s demonstrate that the Neutral Red optical signals evoked by cerebella r 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 ac tivity. Furthermore, this technique provides a novel tool for the stud y of pH changes in vivo at both high spatial and temporal resolution. (C) 1998 IBRO. Published by Elsevier Science Ltd.