| Summary: | Nitric oxide is a gaseous signaling molecule that is produced by subsets of each cell type in the vertebrate retina. Though there is evidence that nitric oxide (NO) can affect multiple cellular processes in the retina, much remains unknown, especially with respect to its function in the inner retina. We have used a simplified system of cultured amacrine cells (interneurons that signal in the inner retina) to explore the role of nitric oxide in amacrine cell signaling. We find that physiological concentrations (100s of nM low μM) of nitric oxide (NO) transiently invert the sign of voltage responses mediated by GABA or glycine receptors by shifting the equilibrium potential for chloride (ECl-) to more positive values. The direction of the shift in ECl- is consistent with a transient elevation of intracellular chloride. The physiological consequence of this shift is that NO can switch inhibitory synapses into excitatory synapses. Manipulations of extracellular chloride demonstrate that the shift in ECl- is not caused by the transport of chloride across the plasma membrane into the cytosol. Instead, NO mediates a release of chloride from an internal compartment. Analysis of cellular pH using the pH indicator dye, SNARF-1AM, reveals that NO also induces a transient acidification of the cytosol that displays a similar time course to the cytosolic chloride elevation. Using measurements of chloride reversal potential (ECl-) to monitor changes in intracellular chloride levels, we found that alkalinization of the cytosol with NH4Cl resulted in a negative shift in ECl-, consistent with a decrease in internal chloride. Acidification of the cytosol with amiloride induced a positive shift in ECl-, consistent with a low cytosolic pH-driven increase in internal chloride. Furthermore, NO-induced positive shifts in the ECl- were reduced in a basic cellular environment. Finally, when we strongly buffered cytosolic pH with 125 mM HEPES in the recording pipet, we found that the ability of NO to alter cytosolic chloride levels was reduced. These results indicate that NO-induced changes in cellular pH are both sufficient and necessary to alter chloride distribution across internal membranes in neurons. The discovery that this redistribution can change the sign of central synapses has potentially broad implications for our understanding of the role of this signaling molecule in the CNS.
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