Extracellular pH Dynamics of Isolated Retinal Horizontal Cells

Lateral inhibition is a fundamental building block of neuronal processing. Despite the passage of over sixty years, the precise molecular mechanism(s) that generates lateral inhibition in the outer retina is still unknown and contentiously debated. Surprisingly, two separate papers using two different techniques were published that came to completely opposite conclusions while examining the extracellular H+ dynamics of horizontal cells following cell activation. One group attempted to monitor extracellular changes in H+ from isolated horizontal cells using the pH-sensitive dye 5-hexadecanoylamino-fluorescein (HAF); depolarization of horizontal cells resulted in an extracellular acidification, consistent with the H+ hypothesis of lateral inhibition. Using H+-selective microelectrodes in a self-referencing format, another research group reported that the depolarization of isolated horizontal cells induced an alkalinization of the extracellular milieu; the H+ hypothesis of lateral inhibition predicts an acidification instead. I was able to show that despite being performed in same experimental conditions, opposite results were still obtained from the two techniques. Laser-scanning confocal microscopy revealed that HAF was not exclusively isolated to the extracellular surface of the plasma membrane as previously assumed, but instead was found to be present extensively throughout the intracellular compartment. We confirmed that HAF is not reporting an extracellular acidification, but rather an acidification taking place within the cell’s interior. A new staining protocol was established whereby intracellular dye accumulation was minimized and membrane-associated staining was prevalent, creating a functional extracellular pH sensor. Horizontal cells exhibiting membrane-associated HAF now reported an extracellular alkalinization following activation by glutamatergic agents. By breaking the imaged cell down to a single-pixel matrix to examine the spatial distribution of the changes in extracellular H+, an overall global alkalinization around the cell was observed with no signs of discrete microdomains of acidification. Our data suggests that protons released from horizontal cells do not appear to be the mediating agent responsible for facilitating inhibitory feedback onto photoreceptor synaptic terminals. Rather, activation of calcium-dependent plasmalemma Ca2+/H+ antiporters (PMCA) on horizontal cells may take up protons from within the synaptic cleft, working to disinhibit voltage-gated calcium channels and thereby enhancing the effects of glutamate onto second order neurons.