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 GABA(A) 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 E(Cl-) 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.
Crousillac S, Colonna J, McMains E, Dewey JS, Gleason E. Sphingosine-1-phosphate elicits receptor-dependent calcium signaling in retinal amacrine cells. J Neurophysiol 102: 3295-3309, 2009. First published September 23, 2009 doi:10.1152/jn.00119.2009. 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 G i and phospholipase C. These observations are consistent with the involvement of members of the S1P receptor family of G-proteincoupled 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 Ca 2ϩ entry pathway, we asked whether S1P caused cytosolic Ca 2ϩ elevations in amacrine cells. We show that S1P-dependent Ca 2ϩ elevations do occur in these cells and that they might be mediated by S1P1R and S1P3R. The Ca 2ϩ 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 Ca 2ϩ elevations. Elucidation of the downstream effects of these signals will provide clues to the role of S1P in regulating inner retinal function.
Background Rho GTPases are small monomeric G-proteins that play key roles in many cellular processes. Due to their widespread expression and broad functions, analyses of Rho GTPase function during late development require tissue-specific modulation of activity. The GAL4/UAS system provides an excellent tool for investigating the function of Rho GTPases in vivo. With this in mind, we created a transgenic toolkit enabling spatial and temporal modulation of Rho GTPase activity in zebrafish. Results Transgenic constructs were assembled driving dominant-negative, constitutively-active, and wild-type versions of Cdc42, RhoA and Rac1 under 10XUAS control. The self-cleaving viral peptide F2A was utilized to allow bicistronic expression of fluorescent reporter and Rho GTPase. Global heat shock of hsp70l:gal4+ transgenic embryos confirmed GAL4-specific construct expression. Western blot analysis indicated myc-tagged Rho GTPases were expressed only in the presence of GAL4. Construct expression was confined to proper cells when combined with pou4f3:gal4 or ptf1a:gal4. Finally, transgene expression resulted in reproducible defects in lens formation indicating that the transgenes are functional in vivo. Conclusions We generated and validated ten transgenic lines, creating a versatile toolkit for the temporal-spatial modulation of Cdc42, RhoA and Rac1 activity in vivo. These lines will enable systematic analysis of Rho GTPase function in any tissue of interest.
Ϫ . Furthermore, disruption of internal proton gradients also reduced the effects of NO on cytosolic Cl Ϫ . Taken together, these results suggest a cytosolic environment where proton and Cl Ϫ fluxes are coupled in a dynamic and physiologically meaningful way.
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.
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