Nitric oxide promotes GABA release by activating a voltage-independent Ca²⁺ influx pathway in retinal amacrine cells

Abstract

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 Ca 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 guanylate cyclase, or presynaptic action potentials. However, removal of extracellular Ca and buffering of cytosolic Ca both inhibit the response to NO. In Ca imaging experiments, we confirm that NO increases cytosolic Ca in amacrine cell processes by activating a Ca influx pathway. Neither the increase in sPSC frequency nor the cytosolic Ca elevations are dependent upon Ca release from stores. NO also enhances evoked GABAergic responses. Because voltage-gated Ca channel function is not altered by NO, the increased evoked response is likely due to the combined effect of voltage-dependent Ca influx adding to the NO-dependent, voltage-independent, Ca influx. Insight into the identity of the Ca 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 Ca elevations. These data suggest that NO production in the inner retina will enhance Ca-dependent GABA release from amacrine cells by activating TRPC channel(s). Our research provides evidence that nitric oxide (NO) promotes GABAergic output from retinal amacrine cells by activating a likely transient receptor potential canonical-mediated Ca 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.

Non-Cell-Autonomous Regulation of Optic Nerve Regeneration by Amacrine Cells

“…In chick ACs in vitro, NO transiently reverses GABA-and glycine-gated currents, converting inhibition of ACs into excitation, thereby increasing the firing of these cells and thus enhanced inhibitory drive on their synaptic partners (e.g., RGCs). This NO-induced shift in E Cl− is likely due to release of Cl − from intracellular stores (Hoffpauir et al, 2006;Krishnan and Gleason, 2015;Krishnan et al, 2017;Maddox and Gleason, 2017;Maddox et al, 2018). In addition, NO may drive synaptic glutamate release from BCs without membrane depolarization via a TRPC Ca 2+ influx-mediated pathway, as shown in the chick retina (Maddox et al, 2018), further depolarizing ACs.…”

Inhibition of endocytosis suppresses the nitric oxide-dependent release of Cl- in retinal amacrine cells

“…The involvement of SVs in the NOdrCl is especially relevant to AC function because ACs participate in serial [ 74 ] and reciprocal synapses [ 21 ] such that presynaptic Cl - can affect the sign of nearby incoming GABAergic synapses in a highly localized fashion [ 22 – 24 ]. We have also recently demonstrated that the NO donor SNAP can increase spontaneous and evoked GABA release via presynaptic activation of TRPC5 channels [ 41 , 75 ] providing an additional NO-dependent mechanism for enhancing GABAergic output from ACs.…”

“…For autaptic recordings, the ACs were not in contact with other neurons, ruling out synaptic input from unclamped cells. Charge transfer at autapses was calculated as previously described and reported as pC [ 41 ]. Inward Ca 2+ currents were often contaminated and obscured by outward synaptic current during the voltage step.…”

A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl− from acidic organelles in amacrine cells

“…Interestingly, two reports on the role of CFTR in neuronal cytosolic Cl Ϫ homeostasis demonstrate effects of CFTR inhibitors on cytosolic Cl Ϫ in the absence of stimulated PKA activity (Morales et al 2011;Ostroumov et al 2011), implying significant baseline CFTR (and possibly PKA) activity in those neurons. A third possibility is that the Ca 2ϩ elevations that we know are generated by NO in amacrine cells (Maddox and Gleason 2017) activate the Ca 2ϩ -sensitive adenylate cyclase, adenylate cyclase 1. CFTR activation via adenylate cyclase 1 has been previously demonstrated (Billet and Hanrahan 2013).…”