Distinctive patterns of alterations in proton efflux from goldfish retinal horizontal cells monitored with self-referencing H⁺-selective electrodes

Abstract

The H(+) hypothesis of lateral feedback inhibition in the outer retina predicts that depolarizing agents should increase H(+) release from horizontal cells. To test this hypothesis, self-referencing H(+) -selective microelectrodes were used to measure extracellular H(+) fluxes from isolated goldfish horizontal cells. We found a more complex pattern of cellular responses than previously observed from horizontal cells of other species examined using this technique. One class of cells had an initial standing signal indicative of high extracellular H(+) adjacent to the cell membrane; challenge with glutamate, kainate or high extracellular potassium induced an extracellular alkalinization. This alkalinization was reduced by the calcium channel blockers nifedipine and cobalt. A second class of cells displayed spontaneous oscillations in extracellular H(+) that were abolished by cobalt, nifedipine and low extracellular calcium. A strong correlation between changes in intracellular calcium and extracellular proton flux was detected in experiments simultaneously monitoring intracellular calcium and extracellular H(+) . A third set of cells was characterized by a standing extracellular alkalinization which was turned into an acidic signal by cobalt. In this last set of cells, addition of glutamate or high extracellular potassium did not significantly alter the proton signal. Taken together, the response characteristics of all three sets of neurons are most parsimoniously explained by activation of a plasma membrane Ca(2+) ATPase pump, with an extracellular alkalinization resulting from exchange of intracellular calcium for extracellular H(+) . These findings argue strongly against the hypothesis that H(+) release from horizontal cells mediates lateral inhibition in the outer retina.

Figure 1.

Responses of cells with standing initial positive (acidic) H⁺ signals in response to application of glutamate and kainate measured using self-referencing H⁺ selective microelectrodes. (A) Representative trace showing the alkalinization response to application of 1mL bolus of 100 μM glutamate from one horizontal cell. The alkalization was reversed with additional application of 1mL of a solution containing glutamate (100 μM) and the ionotropic glutamate receptor blocker CNQX (75 μM). Asterisk in this and other figures shows control recording 200 μm away from the cell. (B) Average result from 6 cells before glutamate, initially after glutamate, 100 sec after glutamate, and following co-application of CNQX. (C) Representative trace from a different horizontal cell. 1mL bath application of additional Ringer’s solution (R) by itself did not change the standing differential recording. Addition of 1mL of kainate induced an extracellular alkalization. (D) Average result from 8 cells before kainate, initially after kainate, and 100s after kainate.

Figure 2.

The alkalinization induced by high external potassium in cells with a standing acidic initial flux is markedly reduced by nifedipine and cobalt (Co²⁺). (A) Representative trace from one horizontal cell showing the alkalinizing effect of high extracellular potassium, and its subsequent abolishment with co-application of nifedipine. (B) Average results from 6 cells prior to stimulation, initially after the addition of 43 mM extracellular potassium, 100 sec after high potassium addition, and following co-application of 10 μM nifedipine. (C) Representative trace from a second horizontal cell showing the extracellular alkalization induced by high extracellular potassium and its elimination upon co-application of Co²⁺. (D) Average results from 9 cells showing signals prior to stimulation, initially after the addition of 43 mM extracellular potassium, 100 sec after KCl, and following co-application of 4 mM Co²⁺. (E) Representative trace from a third horizontal cell showing the extracellular alkalinization induced by high extracellular potassium. In this experiment cells were bathed in a solution in which 45 mM NMDG replaced 45mM Na⁺. High potassium was then added by an equimolar exchange of NMDG rather than reducing the extracellular sodium concentration. (F) Average signals from 6 cells prior to stimulation, initially after the addition of high extracellular potassium, and 100 sec after KCl.

Figure 3.

Spontaneous extracellular H⁺ oscillations and their abolishment by nifedipine, cobalt, nominally 0mM extracellular Ca²⁺, and kainate. (A) Representative trace from one cell showing spontaneous extracellular H⁺ oscillations; addition of 10 μM nifedipine abolished the oscillations and resulted in a standing acidic H⁺ signal. (B) Representative trace showing oscillations from a second cell that were abolished reversed upon addition of 4 mM Co²⁺; a standing extracellular acidification was observed following cobalt application. (C) H⁺ oscillations observed in a third cell were eliminated by applying a solution containing nominally 0 mM external Ca²⁺. A continuous extracellular acidification was observed following the addition of the nominally 0 mM external calcium solution. (D) H⁺ oscillations in the final cell were quieted in an extracellular alkalinized state following the addition of 20 μM kainate.

Figure 4.

Spontaneous oscillations in intracellular calcium in isolated goldfish horizontal cells as revealed by the calcium-sensitive dye Oregon Green. (A) Traces show changes in Oregon Green fluorescence in three different cells as a function of time. Two of the cells in the same field of view displayed oscillations in fluorescence indicative of calcium oscillations, but the timing and length of oscillations differed between the two cells (lower two traces). The top trace shows the fluorescence of a third horizontal cell that did not display any oscillations. Traces have been offset by a constant amount for clarity. (B) A montage of oscillations in Oregon Green fluorescence from the cell whose data was plotted as the lowest trace in (A). Numbers on the image correspond with the numbers on the oscillations in the lowest trace.

Figure 5.

Simultaneous measurements of changes in the fluorescence of the calcium indicator dye (top trace) and alterations in extracellular H⁺ from a single goldfish horizontal cell. Dye fluorescence is presented as intensity normalized to the beginning of the trace prior to the initial oscillation; the trace has been shifted vertically for clarity. The lower trace represents the differential output of the self-referencing H⁺ electrode with the 10 point running averaging removed.

Figure 6.

Responses of cells with standing initial negative (alkaline) H⁺ signals in response to (A) cobalt, (B) high extracellular potassium, (C) glutamate, and (D) carboxyeosin. The calcium channel blocker cobalt reduced the standing alkalinization and turned it into a standing acidic flux, while glutamate and high extracellular potassium were without effect on the standing alkaline flux. Similar to calcium channel blockers, the plasma membrane calcium ATPase blocker, carboxyeosin, also flipped the standing alkalinization to a standing acidic flux.

Figure 7.

Confocal microscopy reveals HAF staining within the intracellular compartment of isolated retinal neurons. Image taken from an optical section approximately in the center of the Z axis of (A) a horizontal cell stained with HAF showing high fluorescent labeling through the interior of the cell and (B) a horizontal cell stained with FM 1-43. The center slice through the horizontal cell stained with HAF shows extensive intracellular staining in the cell; flanking the center optical section, above and to the right are orthographic projections showing extensive intracellular staining throughout the cells’ interior. The optical slice and orthographic projections obtained from the horizontal cell stained with FM 1-43 displays staining located primarily to the plasmalemma Scale bar, 20 μm.