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

Vernon K Dunn,Evanna Gleason,Alexander G Obukhov

doi:10.1371/journal.pone.0201184

Abstract

Our lab has previously shown that nitric oxide (NO) can alter the synaptic response properties of amacrine cells by releasing Cl- from internal acidic compartments. This alteration in the Cl- gradient brings about a positive shift in the reversal potential of the GABA-gated current, which can convert inhibitory synapses into excitatory synapses. Recently, we have shown that the cystic fibrosis transmembrane regulator (CFTR) Cl- channel is involved in the Cl- release. Here, we test the hypothesis that (acidic) synaptic vesicles are a source of NO-releasable Cl- in chick retinal amacrine cells. If SVs are a source of Cl-, then depleting synaptic vesicles should decrease the nitric oxide-dependent shift in the reversal potential of the GABA-gated current. The efficacy of four inhibitors of dynamin (dynasore, Dyngo 4a, Dynole 34–2, and MiTMAB) were evaluated. In order to deplete synaptic vesicles, voltage-steps were used to activate V-gated Ca2+ channels and stimulate the synaptic vesicle cycle either under control conditions or after treatment with the dynamin inhibitors. Voltage-ramps were used to measure the NO-dependent shift in the reversal potential of the GABA-gated currents under both conditions. Our results reveal that activating the synaptic vesicle cycle in the presence of dynasore or Dyngo 4a blocked the NO-dependent shift in EGABA. However, we also discovered that some dynamin inhibitors reduced Ca2+ signaling and L-type Ca2+ currents. Conversely, dynasore also increased neurotransmitter release at autaptic sites. To further resolve the mechanism underlying the inhibition of the NO-dependent shift in the reversal potential for the GABA-gated currents, we also tested the effects of the clathrin assembly inhibitor Pitstop 2 and found that this compound also inhibited the shift. These data provide evidence that dynamin inhibitors have multiple effects on amacrine cell synaptic transmission. These data also suggest that inhibition of endocytosis disrupts the ability of NO to elicit Cl- release from internal stores which may in part be due to depletion of synaptic vesicles.

Highlights

The transmission of information through neuronal circuits depends on the function and regulation of synapses
We have demonstrated that cystic fibrosis transmembrane regulator (CFTR) expression and function is required for the NO-dependent release of Cl- (NOdrCl) [7]
The Pearson’s R-value from 48 regions of interest in amacrine cell processes was 0.38 ± 15 (p

Summary

Introduction

The transmission of information through neuronal circuits depends on the function and regulation of synapses. Chemical synapses are especially important because the diversity of physiological types of synapses and their flexibility can alter the conveyance of information. Amacrine cells (ACs) form primarily gamma-aminobutyric acid (GABA)ergic and glycinergic synapses with ganglion cells, bipolar cells, and other ACs. GABA and glycine bind to ionotropic postsynaptic receptors and activate Cl- channels. The excitatory or inhibitory effect of the postsynaptic response to these neurotransmitters is dependent upon the electrochemical gradient for Cl- across the postsynaptic plasma membrane. Understanding the regulation of cytosolic Cl- at synapses is fundamental to understanding the full flexibility of neuronal circuitry

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