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Robert P. Malchow, Jason Jacoby, Sophia E. Booth, Simon T. Alford, Haohua Qian, Matthew A. Kreitzer; Testing the H+ Hypothesis of Feedback Inhibition from Horizontal Cells to Vertebrate Photoreceptors: a Tale of Two Techniques. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2568.
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Alterations in extracellular H+ concentration have been hypothesized to mediate feedback inhibition from retinal horizontal cells onto vertebrate photoreceptors. If this hypothesis is correct, depolarization of isolated horizontal cells should lead to an extracellular acidification adjacent to the membrane of horizontal cells. Experiments to test this hypothesis have resulted in conflicting findings. Jouhou et al. (2007) claim an extracellular acidification from isolated carp horizontal cells measured using the pH-sensitive fluorescent indicator HAF, but Molina et al. (2004) and Kreitzer et al. (2007) report an extracellular alkalinization induced by glutamate and depolarization from skate and catfish horizontal cells measured using self-referencing H+-selective microelectrodes. These studies differed in the species used, the extracellular pH buffer employed (bicarbonate vs. HEPES) and the presence or absence of cobalt. In the present study, we used both techniques to examine H+ changes from isolated horizontal cells of the same species (catfish cone horizontal cells) under identical experimental conditions (1 mM HEPES and no cobalt).
We used both H+-selective electrodes and the pH-sensitive dye HAF (using the protocol of Johuou et al., 2007) to examine extracellular H+ next to isolated horizontal cells. We also examined the cellular distribution of HAF using confocal microscopy.
HAF fluorescence indicated an acidification when cells were challenged by glutamate or high extracellular potassium. However, a clear alkalinization of the extracellular milieu was found using H+-selective microelectrodes with cells under the same conditions. Confocal microscopy revealed that HAF was not localized exclusively to the external face of the plasma membrane of cells, but rather was detected throughout the interior of the cells. HAF staining co-localized in part with stains specific for mitochondria.
The presence of intracellular HAF makes it likely that this pH-sensitive fluorescent dye will report the intracellular acidification known to occur in horizontal cells when activated by extracellular glutamate (cf. Dixon, Takahashi & Copenhagen, 1993). We suggest that the acidifications detected using the dye HAF may well represent changes in intracellular acid levels rather than extracellular alterations in H+ concentration.
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