Melatonin Modulates Glycine-induced Currents Of Retinal Ganglion Cells In Rat

Melatonin is a hormone participating in modulation of various physiological functions via binding to specific melatonin receptors.In the retina melatonin is synthesized in and released by photoreceptors and may play a neuromodulatory role. In the present work we studied modulation by melatonin of glycine-induced currents of isolated rat retinal ganglion cells(RGCs).Expression pattem of melatonin receptors on isolated RGCs was examined by immunofluoreseence labeling with the antibodies against MT1/MT2 and the retrograde labeling of fluorogold.It was shown that the melatonin receptor MT2,but not MT1,was expressed in isolated RGCs.Isolated RGCs,retrogradely labeled by rhodamine-B-isothiocyanate(RITC),were picked up for whole-cell recording.Using patch clamp techniques,we recorded glycine-induced currents from isolated RGCs voltage-clamped at -60 mV.These currents were strychnine-sensitive,with an EC₅₀of 71.0±8.3μM and a reversal potential near 0 mV.Peak amplitude of the glycine response was potentiated in the presence of 10 nM melatonin in 54 out of 163 cells(33%)in a time-dependent manner. Our experiment focused on these RGCs.The melatonin effect was completely blocked by 10 nM 4-P-PDOT,a specific MT2 receptor antagonist,as well as by intracellular infusion of 3 mM GDP-β-S,a nonhydrolyzable GDP analogue.This is in accordance with the evidence that MT2 receptor belongs to the G protein-coupled receptor superfamily.Extracellular application of 1 mM 8-Br-cAMP or 1 mM 8-Br-cGMP had no effect on the glycine currents.Moreover,the melatonin-caused potentiation of glycine currents was altered by neither 2μM H-89,a PKA inhibitor,nor 20μM KT5823,a PKG inhibitor.Therefore,cAMP and cGMP pathways were not involved.The melatonin effect was not observed in the presence of 30μM D609,a phosphatidylcholine-specific phospholipase(PC-PLC)inhibitor,which decreased the glycine-caused cun'ents,suggesting that PC-PLC may be involved.Furthermore,the PKC activator phorbol 12omyristate 13-acetate(PMA)potentiated glycine-induced currents,thus mimicking the melatonin effect.When glycine currents had been potentiated by 10 nM melatonin,no further increase was observed by coapplication of 1μM PMA and 10 nM melatonin.The PKC inhibitor Bisâ…£(5μM)decreased glycine-induced currents,an effect that is just opposite to the effect of melatonin,and Bisâ…£completely blocked the melatonin effect.These results strongly support the participation of the PKC pathway in the melatonin effect.To determine if melatonin changes intracellular calcium concentration([Ca²⁺]_i)of RGCs,calcium imaging was used.Application of 20 nM melatonin did not change [Ca²⁺]_i,represented as the ratio(340/380).However,application of 60 mM KC1 caused a big change in[Ca²⁺]_i.These results indicated no change in[Ca²⁺]_i after melatonin application.All these results suggest that melatonin,through activation of the MT2 receptor, could be involved in visual information processing in the inner retina by modulating strychnine-sensitive glycine receptors of RGCs via the PLC-PKC pathway.