Calmodulin Bidirectionally Regulates Evoked and Spontaneous Neurotransmitter Release at Retinal Ribbon Synapses

CaM bidirectionally regulates evoked and spontaneous neurotransmitter release from RBs. A1, Confocal images showing immunofluorescence double labeling of CaM (green) and PKCα (magenta), a specific cell marker of RBs, in a frozen retinal slice. In the merged image (green + magenta), expression of CaM could be clearly seen in the axon terminals (arrow) and somata (asterisk) of RBs. Scale bar: 10 μm. ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. A2, No labeling was observed in the negative control where the anti-CaM antibody was preincubated with the CaM immunopeptide. DAPI staining showed the three major cell body layers in the retina. Scale bar: 10 μm. A3, Preadsorption of the anti-CaM antibody with the CaBP5 immunopeptide did not change the staining pattern for CaM. Scale bar: 10 μm. B1, Two-millisecond flashes of 470-nm LED were presented to stimulate ChR2 in Pcp2-cre::Ai32 mice with L-AP4 and ACET in the bath to block synaptic transmission between photoreceptors and bipolar cells; all the inhibitory connections were also blocked. The evoked responses (eEPSCs) and the small responses induced by spontaneous release before light onset (mEPSCs; see gray background area) in AII amacrine cells were recorded. V_hold = −80 mV. Individual traces showed that the CaM antagonist, W-7 (50 μm) strongly increased mEPSC frequency and reduced eEPSC amplitude. B2, Average traces of eEPSCs recorded in the same AII in B1. B3, Statistics of the effects of 25 μm (n = 6) and 50 μm (n = 9) W-7 on eEPSC amplitude. The amplitudes were normalized to the amplitude at time 0 in each cell before averaging across cells. C1, Statistics of the effects of 10 μm (n = 8), 25 μm (n = 13), and 50 μm (n = 15) W-7 on mEPSC frequency. The frequencies were normalized to the frequency at time 0 in each cell before averaging across cells. C2, Statistics of the effects of 10 μm (n = 8), 25 μm (n = 13), and 50 μm (n = 15) W-7 on mEPSC amplitude. The amplitudes were normalized to the amplitude at time 0 in each cell before averaging across cells. D1, Individual traces showing that W-7 (50 μm) had no inhibitory effect on AMPA receptor-mediated currents recorded in an AII evoked by glutamate (1 mm) applied onto the AII dendrites at the border of the IPL and GCL. V_hold = −80 mV. D2, Magnification of the traces in the dashed line frames of D1, showing increase of mEPSC frequency by W-7. D3, Statistics of the effects of 50 μm W-7 (n = 7) on the amplitude of glutamate-evoked currents. The amplitudes were normalized to the amplitude at time 0 in each cell before averaging across cells. E, Summary data showing the effects of 50 μm W-7 (circles), 100 μm CMZ (triangles), another CaM antagonist, and 1 μm CALP1 (squares), a CaM agonist, on eEPSC amplitude after bath application for 15 min. The amplitudes were normalized to the amplitude at time 0 in each cell before averaging across cells. The data were also illustrated as mean ± SEM. Wilcoxon signed-rank tests were used (control vs W-7, n = 9, p = 0.0039; control vs CMZ, n = 12, p = 0.0005; control vs CALP1, n = 5, p = 0.6250); **p < 0.01, ***p < 0.001; ns: not statistically different. Note that CMZ reduced eEPSC amplitude too, but CALP1 did not enhance eEPSC amplitude under control conditions. F, Summary data showing the effects of 50 μm W-7 (circles), 100 μm CMZ (triangles), and 1 μm CALP1 (squares) on mEPSC frequency. The frequencies were normalized to the frequency at time 0 in each cell before averaging across cells. The data were also illustrated as mean ± SEM. Wilcoxon signed-rank tests were used (control vs W-7, n = 15, p < 0.0001; control vs CMZ, n = 12, p = 0.6377; control vs CALP1, n = 9, p = 0.0273); *p < 0.05, ****p < 0.0001; ns: not statistically different. Note that CMZ did not change the mEPSC frequency, but activation of CaM by CALP1 slightly reduced mEPSC frequency.

CaM is ubiquitously expressed at sites both near and away from ribbons in RB axon terminals. Confocal images showing immunofluorescence double labeling of CaM (green) and RIBEYE (magenta), a ribbon-specific protein, in the axon terminals of RBs. In the merged image (green + magenta), expression of CaM can be seen near ribbons (arrow) and away from ribbon sites. IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar: 10 μm.

Inhibition of CaM strongly reduces evoked release from RBs by suppressing Ca²⁺ influx into their axon terminals. A1, Average traces showing that EPSCs recorded in an AII, evoked by puffing LY 341495 (LY), an mGluR6 antagonist, onto the dendrites of RBs located at the OPL, were strongly reduced by application of 50 μm W-7. V_hold = −80 mV. A2, LY-evoked EPSCs decreased over time with application of 50 μm W-7 (n = 7). The peak amplitudes and integrals of EPSCs were normalized to the amplitude and integral at time 0, respectively, in each cell before averaging across cells. B, LY-evoked EPSCs were completely abolished by removing extracellular Ca²⁺ (0 Ca). C, The EPSCs recorded in an AII, evoked by brief flashes of 470-nm LED in a Pcp2-cre::Ai32 mouse, were abolished completely by removing extracellular Ca²⁺ (0 Ca). D1, Average traces showing that W-7 (50 μm) strongly suppressed the voltage step-generated Ca currents (I_Ca) in an RB. D2, Statistics of the effects of 50 μm W-7 (n = 7) and DMSO control on the peak amplitude and integral of RB I_Ca. The suppression of I_Ca recorded in RBs was closely related to the inhibition of eEPSCs recorded in AIIs (data adapted from Fig. 2B3, superimposed in gray). E1, Calcium imaging pictures from a Pcp2-cre::Ai38 mouse showing that LY-evoked changes of Ca²⁺ signals in RB axon terminals (white frames), detected by the Ca²⁺ indicator GCaMP3, were reduced strongly by application of 50 μm W-7. Scale bar: 1 μm. E2, Representative ΔF/F₀ traces showing suppression of Ca²⁺ signals in an RB axon terminal by 50 μm W-7. E3, Summary data showing that LY-evoked Ca²⁺ signals in RB axon terminals (n = 21 terminals from 3 retinal slices), measured as areas under the curve (AUCs) of ΔF/F₀ traces, were strongly suppressed by 50 μm W-7 over time. All the data were illustrated as mean ± SEM.

Both Ca²⁺-dependent and Ca²⁺-independent spontaneous release are enhanced, but to different extents, when CaM is inhibited. A–D, Representative traces showing mEPSCs recorded in AIIs under four experimental conditions in different combinations of removal of extracellular calcium (0 Ca), 10 μm BAPTA-AM, 1 μm Tg, and 1 μm YM-58483 (YM). The traces showing mEPSCs after bath application of 50 μm W-7 for 15 min under each condition were also presented. E, Summary data for AII mEPSC frequency under four experimental conditions in A–D. The data under control and W-7 conditions (adapted from Fig. 2C1, empty and full up triangles, respectively) were also presented for direct comparison. The frequencies were normalized to the frequency under control condition in each cell before averaging across cells. The data were also illustrated as mean ± SEM. Paired Student’s t tests were used (0 Ca vs 0 Ca + W-7, n = 12, p = 0.0001; 0 Ca + BAPTA-AM vs 0 Ca + BAPTA-AM + W-7, n = 13, p = 0.0002; 0 Ca + BAPTA-AM + Tg vs 0 Ca + BAPTA-AM + Tg + W-7, n = 11, p = 0.0004; 0 Ca + BAPTA-AM + Tg + YM vs 0 Ca + BAPTA-AM + Tg + YM + W-7, n = 7, p = 0.0062) except for comparison of control and W-7 data by Wilcoxon signed-rank test (n = 15, p < 0.0001); **p < 0.01, ***p < 0.001, ****p < 0.0001. F, Summary data for the relative effect of W-7 on AII mEPSC frequency under control and four experimental conditions. The change in each cell was calculated as the ratio of mEPSC frequencies before and 15 min after application of W-7. The data were also illustrated as mean ± SEM. Mann–Whitney tests were used (W-7 vs 0 Ca + W-7, p = 0.0214; W-7 vs 0 Ca + BAPTA-AM + W-7, p = 0.0954; W-7 vs 0 Ca + BAPTA-AM + Tg + W-7, p = 0.0045; W-7 vs 0 Ca + BAPTA-AM + Tg + YM + W-7, p = 0.0262); *p < 0.05, **p < 0.01; ns: not statistically different.

Inhibition of MLCK, but not other CaM targets, differentially regulates evoked and spontaneous release from RBs. A1, Five-millisecond flashes of 470-nm LED were presented to stimulate ChR2-expressing RBs in Pcp2-cre::Ai32 mice. The mEPSCs (in gray background area) and eEPSCs in AIIs were recorded. V_hold = −80 mV. Individual traces showed that a specific MLCK inhibitor, ML-9 (100 μm) strongly increased mEPSC frequency and reduced eEPSC amplitude. A2, Average traces of eEPSCs recorded in the same AII in A1. A3, Statistics of the effects of 50 μm (n = 5) and 100 μm (n = 10) ML-9 on eEPSC amplitude. The amplitudes were normalized to the amplitude at time 0 in each cell before averaging across cells. The data of 50 μm W-7 (adapted from Fig. 2B3, superimposed in magenta) were also included for direct comparison. B1, Statistics of the effects of 25 μm (n = 7), 50 μm (n = 13), and 100 μm (n = 10) ML-9 on mEPSC frequency. The frequencies were normalized to the frequency at time 0 in each cell before averaging across cells. The data of 50 μm W-7 (adapted from Fig. 2C1, superimposed in magenta) were also included for direct comparison. B2, Statistics of the effects of 25 μm (n = 7), 50 μm (n = 13), and 100 μm (n = 10) ML-9 on mEPSC amplitude. The amplitudes were normalized to the amplitude at time 0 in each cell before averaging across cells. C1, Individual traces showing that ML-9 (100 μm) had no inhibitory effect on AMPA receptor-mediated currents recorded in an AII evoked by glutamate (1 mm) applied onto the AII dendrites at the border of the IPL and GCL. V_hold = −80 mV. C2, Magnification of the traces in the dashed line frames of C1, showing increase of mEPSC frequency by ML-9. C3, Statistics of the effects of 100 μm ML-9 (n = 4) on the amplitude of glutamate-evoked currents. The amplitudes were normalized to the amplitude at time 0 in each cell before averaging across cells. D, Summary data showing the effects of bath application of W-7 (50 μm; full circles; adapted from Fig. 2E), ML-9 (100 μm; full down triangles), KN-62 (4 μm; empty circles), MMPX (40 μm; empty squares), and ascomycin (1 μm; empty up triangles), respectively, for 15 min on the amplitude of eEPSCs recorded in AIIs. In each group of data, the amplitudes were normalized to the amplitude before application of a drug in each cell before averaging across cells. The data were also illustrated as mean ± SEM. Wilcoxon signed-rank tests were used (control vs W-7, n = 9, p = 0.0039; control vs ML-9, n = 10, p = 0.0020; control vs KN-62, n = 10, p = 0.0586; control vs MMPX, n = 8, p = 0.4609; control vs ascomycin, n = 8, p = 0.0078); **p < 0.01, ****p < 0.0001; ns: not statistically different. E, Summary data showing the effects of bath application of W-7 (50 μm), ML-9 (100 μm), KN-62 (4 μm), MMPX (40 μm), and ascomycin (1 μm) for 15 min on mEPSC frequency. In each group of data, the frequencies were normalized to the frequency before application of a drug in each cell before averaging across cells. The data were also illustrated as mean ± SEM. Wilcoxon signed-rank tests were used (control vs W-7, n = 15, p < 0.0001; control vs ML-9, n = 10, p = 0.0020; control vs KN-62, n = 11, p = 0.2139; control vs MMPX, n = 8, p = 0.3828; control vs ascomycin, n = 15, p = 0.3591); **p < 0.01, ****p < 0.0001; ns: not statistically different.

Inhibition of MLCK strongly reduces evoked release by suppressing calcium currents in RBs. A1, Average traces showing that EPSCs recorded in an AII, evoked by puffing LY 341495 (LY), an mGluR6 antagonist, onto the dendrites of RBs located at the OPL, were strongly reduced by application of 100 μm ML-9. V_hold = −80 mV. A2, LY-evoked EPSCs decreased over time with bath application of 100 μm ML-9 (n = 5). The peak amplitudes and integrals of EPSCs were normalized to the amplitude and integral at time 0, respectively, in each cell before averaging across cells. All the data were illustrated as mean ± SEM. B1, Average traces showing that ML-9 (100 μm) strongly suppressed the voltage step-generated calcium currents (I_Ca) in an RB. B2, Statistics of the effects of 100 μm ML-9 (n = 10) on the peak amplitude and integral of RB I_Ca. The suppression of I_Ca recorded in RBs was closely related to the inhibition of eEPSCs recorded in AIIs (adapted from Fig. 6A3, superimposed in gray). All the data were illustrated as mean ± SEM.

Both Ca²⁺-dependent and Ca²⁺-independent spontaneous release are enhanced, but to different extents, when MLCK is inhibited. A, B, Representative traces showing AII mEPSCs under control, and different combinations of removal of extracellular calcium (0 Ca), 10 μm BAPTA-AM and 100 μm ML-9 conditions. C, Summary data for AII mEPSC frequency under two experimental conditions in A (empty and full circles) and B (empty and full squares). The data under control and ML-9 conditions (adapted from Fig. 6B1, empty and full triangles, respectively) were also presented for direct comparison. The frequencies were normalized to the frequency under control condition in each cell before averaging across cells. The data were also illustrated as mean ± SEM. Wilcoxon signed-rank tests were used (control vs ML-9, n = 10, p = 0.0020; 0 Ca + BAPTA-AM vs 0 Ca + BAPTA-AM + ML-9, n = 9, p = 0.0039) except for comparison of 0 Ca and 0 Ca + ML-9 data by paired Student’s t test (n = 10, p = 0.0001); **p < 0.01, ***p < 0.001. D, Summary data for changes of AII mEPSC frequency after bath application of 100 μm ML-9 for 15 min under control and two experimental conditions. The change in each cell was calculated as the ratio of mEPSC frequencies before and 15 min after application of ML-9. The data were also illustrated as mean ± SEM. Mann–Whitney tests were used (ML-9 vs 0 Ca + ML-9, p = 0.0232; ML-9 vs 0 Ca + BAPTA-AM + ML-9, p =0.0030); *p < 0.05, **p < 0.01.