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

Modulation of synaptic function in retinal amacrine cells

Amacrine cells are interneurons that have diverse functions in retinal signal processing. In order to study signaling and modulation in retinal amacrine cells, we employ a simplified culture system containing identifiable GABAergic amacrine cells. Immunocytochemistry experiments indicate that GABAergic amacrine cells express metabotropic glutamate receptor 5 (mGluR5), a group I mGluR usually linked to the IP3 signaling pathway. Ca2+ imaging experiments using an mGluR5-specific agonist indicate that these receptors are functional and when activated, can stimulate temporally diverse Ca2+ elevations. To begin to establish the role of these receptors in modulating amacrine cell function, we have used electrophysiological methods to ask whether ion channels are the targets of mGluR5-dependent modulation. Here we discuss our results indicating that activation of mGluR5 leads to enhancement of currents through GABAA receptors. This enhancement is dependent upon elevations in cytosolic Ca2+ and activation of protein kinase C (PKC). To explore the consequences of Ca2+ elevations in another context, we have used nitric oxide (NO) donors to mimic the effects of activating the Ca2+-dependent synthetic enzyme for NO, neuronal nitric oxide synthase. We find that exposure to NO donors also enhances the amplitude of currents through GABAA receptors. Together, these results indicate that glutamate from presynaptic bipolar cells has the potential to work through multiple mechanisms to regulate the function of amacrine-to-amacrine cell GABAergic synapses

Nitric oxide promotes GABA release by activating a voltage-independent Ca\u3csup\u3e2+\u3c/sup\u3e influx pathway in retinal amacrine cells

© 2017 the American Physiological Society. Retinal amacrine cells express nitric oxide (NO) synthase and produce NO, making NO available to regulate the function of amacrine cells. Here we test the hypothesis that NO can alter the GABAergic synaptic output of amacrine cells. We investigate this using whole cell voltage clamp recordings and Ca2 imaging of cultured chick retinal amacrine cells. When recording from amacrine cells receiving synaptic input from other amacrine cells, we find that NO increases GABAergic spontaneous postsynaptic current (sPSC) frequency. This increase in sPSC frequency does not require the canonical NO receptor, soluble guan-ylate cyclase, or presynaptic action potentials. However, removal of extracellular Ca2+ and buffering of cytosolic Ca2+ both inhibit the response to NO. In Ca2+ imaging experiments, we confirm that NO increases cytosolic Ca2+ in amacrine cell processes by activating a Ca2+ influx pathway. Neither the increase in sPSC frequency nor the cytosolic Ca2+ elevations are dependent upon Ca2+ release from stores. NO also enhances evoked GABAergic responses. Because voltage-gated Ca2+ channel function is not altered by NO, the increased evoked response is likely due to the combined effect of voltage-dependent Ca2+ influx adding to the NO-dependent, voltage-independent, Ca2+ influx. Insight into the identity of the Ca2+ influx pathway is provided by the transient receptor potential canonical (TRPC) channel inhibitor clemizole, which prevents the NO-dependent increase in sPSC frequency and cytosolic Ca2+ elevations. These data suggest that NO production in the inner retina will enhance Ca2+-dependent GABA release from amacrine cells by activating TRPC channel(s). NEW & NOTEWORTHY Our research provides evidence that nitric oxide (NO) promotes GABAergic output from retinal amacrine cells by activating a likely transient receptor potential canonical-mediated Ca2+ influx pathway. This NO-dependent mechanism promoting GABA release can be voltage independent, suggesting that, in the retina, local NO production can bypass the formal retinal circuitry and increase local inhibition

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

TRPC5 is required for the NO-dependent increase in dendritic Ca \u3csup\u3e2+\u3c/sup\u3e and GABA release from chick retinal amacrine cells

© 2018 American Physiological Society. All rights reserved. GABAergic signaling from amacrine cells (ACs) is a fundamental aspect of visual signal processing in the inner retina. We have previously shown that nitric oxide (NO) can elicit release of GABA independently from activation of voltage-gated Ca 2+ channels in cultured retinal ACs. This voltage-independent quantal GABA release relies on a Ca 2+ influx mechanism with pharmacological characteristics consistent with the involvement of the transient receptor potential canonical (TRPC) channels TRPC4 and/or TRPC5. To determine the identity of these channels, we evaluated the ability of NO to elevate dendritic Ca 2+ and to stimulate GABA release from cultured ACs under conditions known to alter the function of TRPC4 and 5. We found that these effects of NO are phospholipase C dependent, have a biphasic dependence on La 3+ , and are unaffected by moderate concentrations of the TRPC4-selective antagonist ML204. Together, these results suggest that NO promotes GABA release by activating TRPC5 channels in AC dendrites. To confirm a role for TRPC5, we knocked down the expression of TRPC5 using CRISPR/Cas9-mediated gene knockdown and found that both the NO-dependent Ca 2+ elevations and increase in GABA release are dependent on the expression of TRPC5. These results demonstrate a novel NO-dependent mechanism for regulating neurotransmitter output from retinal ACs. NEW & NOTEWORTHY Elucidating the mechanisms regulating GABAergic synaptic transmission in the inner retina is key to understanding the flexibility of retinal ganglion cell output. Here, we demonstrate that nitric oxide (NO) can activate a transient receptor potential canonical 5 (TRPC5)-mediated Ca 2+ influx, which is sufficient to drive vesicular GABA release from retinal amacrine cells. This NO-dependent mechanism can bypass the need for depolarization and may have an important role in processing the visual signal by enhancing retinal amacrine cell GABAergic inhibitory output

A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl \u3csup\u3e–\u3c/sup\u3e from acidic organelles in amacrine cells

© 2017 the American Physiological Society. γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligandgated ion channels support the influx of Cl – . However, the electrochemical gradient for Cl – across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl – levels support inhibition, whereas higher levels of cytosolic Cl – can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl – from acidic organelles and transiently elevates cytosolic Cl – , making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl – . We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage- clamp recordings of currents through GABA-gated Cl – channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl – . To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl – from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl – from acidic organelles. NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl – from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl – levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission

Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double-labeling experiments, most metabotropic glutamate receptor 1-positive cell bodies in the inner nuclear layer also label with anti-GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca2+ elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca2+ elevations were enhanced by metabotropic glutamate receptor 1-selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca2+ signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells