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Robert P. Malchow, Jason Jacoby, Simon T. Alford, Haohua Qian, Matthew A. Kreitzer; The pH-sensitive Dye HAF Reports An Extracellular Alkalinization Upon Stimulation Of Catfish Horizontal Cells: Testing The H+ Hypothesis Of Lateral Inhibition. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4304.
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Extracellular acidification mediated by horizontal cells has been suggested to mediate feedback inhibition onto vertebrate photoreceptors. This hypothesis predicts an extracellular acidification at the surface of horizontal cells upon challenge with depolarizing agents. Jouhou et al. (2007) claim such an extracellular acidification using the pH-sensitive indicator dye HAF, but Molina et al. (2004), Kreitzer et al. (2007) and Jacoby et al. (2011) find an extracellular alkalinization by glutamate and high extracellular K+ using self-referencing H+-selective microelectrodes. Confocal microscopy reveals that HAF is not localized exclusively to the extracellular surface of cells as previously assumed, but rather is present throughout the intracellular compartment, complicating the interpretation of HAF results. We sought to find conditions in which HAF on the extracellular surface of cells was enhanced relative to intracellular staining, and to examine changes in fluorescence when cells were challenged with depolarizing agents.
We varied HAF concentration and the duration of dye application and examined the distribution of dye in isolated catfish cone-driven horizontal cells using confocal microscopy. We also performed ratiometric imaging of HAF fluorescence upon application of the excitatory neurotransmitter glutamate.
Reducing the concentration and time of exposure to HAF led to conditions in which intracellular accumulation of HAF was reduced and extracellular staining was prominent. Epifluorescent imaging of cells displaying primarily extracellular fluorescence revealed that glutamate produced a change in HAF fluorescence consistent with alkalinization. In cells in which intracellular HAF was prominent, an acidification was detected. Under these same conditions, an extracellular alkalinization was seen using self-referencing H+-selective microelectrodes.
The alkalinization seen using a protocol that maximizes extracellular staining of HAF over intracellular staining is consistent with the extracellular alkalinization observed using H+-selective electrodes. These data argue strongly against the H+ hypothesis of lateral inhibition.
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