Melatonin potentiates glycine currents through a PLC/PKC signalling pathway in rat retinal ganglion cells

Abstract

In vertebrate retina, melatonin regulates various physiological functions. In this work we investigated the mechanisms underlying melatonin-induced potentiation of glycine currents in rat retinal ganglion cells (RGCs). Immunofluorescence double labelling showed that rat RGCs were solely immunoreactive to melatonin MT(2) receptors. Melatonin potentiated glycine currents of RGCs, which was reversed by the MT(2) receptor antagonist 4-P-PDOT. The melatonin effect was blocked by intracellular dialysis of GDP-beta-S. Either preincubation with pertussis toxin or application of the phosphatidylcholine (PC)-specific phospholipase C (PLC) inhibitor D609, but not the phosphatidylinositol (PI)-PLC inhibitor U73122, blocked the melatonin effect. The protein kinase C (PKC) activator PMA potentiated the glycine currents and in the presence of PMA melatonin failed to cause further potentiation of the currents, whereas application of the PKC inhibitor bisindolylmaleimide IV abolished the melatonin-induced potentiation. The melatonin effect persisted when [Ca(2+)](i) was chelated by BAPTA, and melatonin induced no increase in [Ca(2+)](i). Neither cAMP-PKA nor cGMP-PKG signalling pathways seemed to be involved because 8-Br-cAMP or 8-Br-cGMP failed to cause potentiation of the glycine currents and both the PKA inhibitor H-89 and the PKG inhibitor KT5823 did not block the melatonin-induced potentiation. In consequence, a distinct PC-PLC/PKC signalling pathway, following the activation of G(i/o)-coupled MT(2) receptors, is most likely responsible for the melatonin-induced potentiation of glycine currents of rat RGCs. Furthermore, in rat retinal slices melatonin potentiated light-evoked glycine receptor-mediated inhibitory postsynaptic currents in RGCs. These results suggest that melatonin, being at higher levels at night, may help animals to detect positive or negative contrast in night vision by modulating inhibitory signals largely mediated by glycinergic amacrine cells in the inner retina.

Figure 1. Expression of melatonin MT₂ receptors in rat retinal ganglion cells (RGCs)

Double immunofluorescence labelling was performed using the antibody against MT₁ or MT₂ receptors on acutely dissociated RGCs labelled retrogradely by injecting fluorogold (FG) into the superior colliculus bilaterally. A, Western blot of whole rat retinal extract using the antibody against MT₁, revealing a single band at the corresponding molecular weight of around 45 kDa. Ba–Bd, a RGC, double labelled by FG and MT₁. Ba is the DIC image of the cell. The cell was labelled by FG (Bb), but not by MT₁ (Bc). Bd is the merged image of Bb and Bc. C, Western blot of whole rat retinal extract using the antibody against MT₂, also revealing a single band at around 45 kDa, corresponding to the molecular weight of the native MT₂ receptor. Da–d, another RGC labelled by FG and MT₂. Da is the DIC image of the cell. Dd is the merged image of Db, showing labelling for FG, and Dc, showing labelling for MT₂. Note that the labelling for MT₂ is primarily located on the membrane of the soma, and some punctate labelling is also observed in the dendrites. Ea, a RGC labelled by FG. Eb, no immunoflorescence labelling for MT₂ receptors could be found when the MT₂ antibody was pre-absorbed with the immunizing antigen. Scale bars, 10 μm.

Figure 2. G-proteins mediating potentiation by melatonin of glycine-induced currents

A, average peak amplitudes of glycine currents of RGCs (n = 12) are plotted as a function of time, showing the time course of the potentiation of glycine currents following the application of 10 nm melatonin. Glycine (Gly) of 100 μm was applied for 5 s every 2 min (this protocol is also applicable to Figs 3–7 and Supplemental Fig. 1). Inset, recordings from a RGC obtained at different times. B, glycine current traces taken from a RGC, showing no potentiation of the current by 10 nm melatonin in the presence of 3 mm GDP-β-S. C, representative recordings, showing that no potentiation of glycine currents by 10 nm melatonin was observed in a pertussis toxin (PTX) 1 μg ml⁻¹ pretreated RGC. D, average peak amplitudes of glycine currents of RGCs as a function of time, showing no change in amplitudes by 10 nm melatonin in PTX-treated RGCs (n = 8). The data obtained in each cell were normalized to the control current of that cell (at 0 min), and the data collected from these cells in these experiments were then averaged, a procedure that was used to process all relevant data shown in Figs 2–7 and Supplemental Fig. 1. The data are presented as means ± s.e.m.

Figure 3. No involvement of cAMP-PKA and cGMP-PKG signalling pathways in the potentiation by melatonin of glycine currents

A and B, representative recordings made in two different cells, showing that the glycine currents were almost unchanged by application of 1 mm 8-Br-cAMP (A) or 1 mm 8-Br-cGMP (B). C, plot of average peak current amplitudes as a function of time, showing that either 8-Br-cAMP or 8-Br-cGMP hardly affected the glycine currents of RGCs (n = 7 for 8-Br-cAMP and n = 10 for 8-Br-cGMP). D and E, recordings made in two different RGCs showing that application of 2 μm H-89 (D) or 20 μm KT5823 (E) did not block the potentiation by melatonin of the glycine currents. F, plot of average peak current amplitudes as a function of time, showing the time course of the effects of 10 nm melatonin on glycine currents in the presence of H-89 (n = 5) or KT5823 (n = 5). *P < 0.05 and **P < 0.01, as compared to the current amplitudes before melatonin application (control).

Figure 4. Involvement of PC-PLC pathways in melatonin-induced potentiation of glycine currents

A, representative recordings from a RGC, showing the effect of melatonin on glycine currents in the presence of 60 μm D609. Note that D609 itself reduced the current amplitude (4 min). Addition of 10 nm melatonin in the presence of D609 did not increase the current (14 min). When D609 was removed from the external solution, the peak current amplitude was significantly increased (24 min). B, plot of average peak amplitudes of glycine currents (n = 4) as a function of time, showing the change in amplitudes. *P < 0.05 and **P < 0.01 as compared to that recorded in Ringer solution (at 0 min). C, current traces, showing the effect of melatonin on glycine currents in a RGC dialysed with 5 μm U73122. In the presence of U73122, melatonin still caused a potentiation of the glycine current. D, average peak glycine current amplitudes (n = 6) changed as a function of time in the presence of 5 μm U73122, showing that melatonin persisted to cause a current potentiation. *P < 0.05 and **P < 0.01 (vs. control).

Figure 5. PKC is involved in melatonin-caused potentiation of glycine currents

A, current traces from a RGC, showing that extracellular application of 1 μm PMA gradually increased the current amplitude, and in the presence of PMA 10 nm melatonin did not cause a further increase. B, cumulative data from nine cells showing the effects of PMA and melatonin on glycine currents. The data were taken at different time points as shown in A (0 min for control; 12 min for PMA; 24 min for PMA+melatonin). *P < 0.05 vs. control. C, current traces recorded at different times, showing that application of 5 μm Bis IV reduced the glycine current of a RGC, and in the presence of Bis IV 10 nm melatonin failed to potentiate the current, but potentiation of the glycine current was clearly observed when Bis IV was withdrawn but melatonin remained. D, average peak amplitudes of glycine currents (n = 6) are plotted as a function of time during such experiments. *P < 0.05 and **P < 0.01, as compared to that in Ringer solution (0 min).

Figure 6. Intracellular Ca²⁺ is not involved in melatonin-induced potentiation of glycine currents

A, a continuous recording of [Ca²⁺]_i in a RGC, represented by the ratio of fura-2 AM fluorescence at 340 nm and 380 nm (340/380). Application of 20 nm melatonin hardly changed [Ca²⁺]_i. After washout with Ringer solution for 4 min, application of 60 mm KCl induced a dramatic increase of [Ca²⁺]_i. Inset, three CCD images of a RITC-labelled RGC loaded with fura-2 AM, taken before (left) and after (middle) 6 min perfusion of 20 nm melatonin, and in the presence of 60 mm KCl (right). B, bar chart summarizing the changes of [Ca²⁺]_i induced by melatonin (20 nm) and KCl (60 mm) in RGCs (n = 13, **P < 0.01 vs. control). C, current traces recorded at different times from a RGC, showing that elimination of [Ca²⁺]_i by 10 mm BAPTA did not change the potentiation by melatonin of the glycine current. D, average peak amplitudes of glycine currents are plotted as a function of time, showing the effect of melatonin on glycine currents when 10 mm BAPTA-filled recording pipettes were used (n = 5, **P < 0.01, as compared to the currents recorded at 0 min).

Figure 7. Melatonin potentiates light-evoked glycine receptor-mediated IPSCs in RGCs of rat retinal slices

A, light evoked current traces from a RGC, recorded in the presence of d-APV, CNQX, and bicuculline. Application of 50 nm melatonin increased the peak amplitude, and the effect was reversed by co-application of 4-P-PDOT. The current was eliminated by application of 5 μm strychnine. The cell was held at +20 mV. B, bar chart summarizing the changes of peak amplitudes of light-evoked glycine currents induced by melatonin and co-application with 4-P-PDOT. n = 8, ***P < 0.001 vs. control.

Figure 8. A schematic diagram showing the possible signalling pathway mediating the potentiation by melatonin of glycine currents in rat RGCs

By challenging PTX-sensitive G_i/o-coupled MT₂ receptors on rat RGCs, melatonin potentiates glycine currents via a distinct intracellular PC-PLC/PKC pathway. Mel: melatonin; GlyR: glycine receptor; PC-PLC: phosphatidylcholine-specific phospholipase C; PC: phosphatidylcholine; DAG: diacylglycerol; PKC: protein kinase C; ℗, phosphorylation.