A novel modulatory role for nitric oxide in retinal amacrine cells

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 (100’s 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

Role of pH in a nitric oxide-dependent increase in cytosolic Cl \u3csup\u3e-\u3c/sup\u3e in retinal amacrine cells

Nitric oxide (NO) synthase-expressing neurons are found throughout the vertebrate retina. Previous work by our laboratory has shown that NO can transiently convert inhibitory GABAergic synapses onto cultured retinal amacrine cells into excitatory synapses by releasing Cl - from an internal store in the postsynaptic cell. The mechanism underlying this Cl -release is currently unknown. Because transport of Cl - across internal membranes can be coupled to proton flux, we asked whether protons could be involved in the NO-dependent release of internal Cl -. Using pH imaging and whole cell voltage-clamp recording, we addressed the relationship between cytosolic pH and cytosolic Cl - in cultured retinal amacrine cells. We found that NO reliably produces a transient decrease in cytosolic pH. A physiological link between cytosolic pH and cytosolic Cl - was established by demonstrating that shifting cytosolic pH in the absence of NO altered cytosolic Cl - concentrations. Strong buffering of cytosolic pH limited the ability of NO to increase cytosolic Cl -, suggesting that cytosolic acidification is involved in generating the NO-dependent elevation in cytosolic Cl -. Furthermore, disruption of internal proton gradients also reduced the effects of NO on cytosolic Cl -. Taken together, these results suggest a cytosolic environment where proton and Cl - fluxes are coupled in a dynamic and physiologically meaningful way. © 2011 the American Physiological Society

Nitric oxide transiently converts synaptic inhibition to excitation in retinal amacrine cells

Nitric oxide (NO) is generated by multiple cell types in the vertebrate retina, including amacrine cells. We investigate the role of NO in the modulation of synaptic function using a culture system containing identified retinal amacrine cells. We find that moderate concentrations of NO alter GABAA receptor function to produce an enhancement of the GABA-gated current. Higher concentrations of NO also enhance GABA-gated currents, but this enhancement is primarily due to a substantial positive shift in the reversal potential of the current. Several pieces of evidence, including a similar effect on glycine-gated currents, indicate that the positive shift is due to an increase in cytosolic Cl-. This change in the chloride distribution is especially significant because it can invert the sign of GABA- and glycine-gated voltage responses. Furthermore, current- and voltage-clamp recordings from synaptic pairs of GABAergic amacrine cells demonstrate that NO transiently converts signaling at GABAergic synapses from inhibition to excitation. Persistence of the NO-induced shift in ECl- in the absence of extracellular Cl- indicates that the increase in cytosolic Cl- is due to release of Cl- from an internal store. An NO-dependent release of Cl- from an internal store is also demonstrated for rat hippocampal neurons indicating that this mechanism is not restricted to the avian retina. Thus signaling in the CNS can be fundamentally altered by an NO-dependent mobilization of an internal Cl- store. Copyright © 2006 The American Physiological Society

Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue

Sphingosine-1-Phosphate Elicits Receptor-Dependent Calcium Signaling in Retinal Amacrine Cells

Evidence is emerging indicating that sphingosine-1-phosphate (S1P) participates in signaling in the retina. To determine whether S1P might be involved in signaling in the inner retina specifically, we examine the effects of this sphingolipid on cultured retinal amacrine cells. Whole cell voltage-clamp recordings reveal that S1P activates a cation current that is dependent on signaling through Gi and phospholipase C. These observations are consistent with the involvement of members of the S1P receptor family of G-protein-coupled receptors in the production of the current. Immunocytochemistry and PCR amplification provide evidence for the expression of S1P1R and S1P3R in amacrine cells. The receptor-mediated channel activity is shown to be highly sensitive to blockade by lanthanides consistent with the behavior of transient receptor potential canonical (TRPC) channels. PCR products amplified from amacrine cells reveal that TRPCs 1 and 3–7 channel subunits have the potential to be expressed. Because TRPC channels provide a Ca2+ entry pathway, we asked whether S1P caused cytosolic Ca2+ elevations in amacrine cells. We show that S1P-dependent Ca2+ elevations do occur in these cells and that they might be mediated by S1P1R and S1P3R. The Ca2+ elevations are partially due to release from internal stores, but the largest contribution is from influx across the plasma membrane. The effect of inhibition of sphingosine kinase suggests that the production of cytosolic S1P underlies the sustained nature of the Ca2+ elevations. Elucidation of the downstream effects of these signals will provide clues to the role of S1P in regulating inner retinal function