Cone pedicles and the mutant Cav1.4 channel are normally localized in the OPL of G369i KI mice but not Cav1.4 KO mice.

Confocal images of the ONL and OPL of WT, G369i KI, and Cav1.4 KO mice labeled with antibodies against cone arrestin (CAR), CtBP2, and Cav1.4. a, Inverted images of CAR labeling. Lower panels correspond to boxed region of the upper panels and depict pedicles labeled by CAR antibodies (dotted outlines). Cone pedicles remain within the OPL of WT and G369i KI retina (arrows) but are misshapen and retracted in the ONL of the CaV1.4 KO retina (arrowheads). b, High magnification, deconvolved images show CaV1.4 labeling near cone ribbons in WT and G369i KI pedicles (arrows) and ribbon spheres without CaV1.4 labeling in the CaV1.4 KO pedicle (arrowheads).

A low-voltage activated ICa is present in cones of G369i KI and CaV1.4 KO but absent in WT mice.

a,b, Representative traces of ICaevoked by voltage ramps (a) and voltage steps (b). c,d, I-V (c) and G-V (d) were plotted against test voltage for ICa evoked by 50-ms voltage steps from a holding voltage of -90 mV. Numbers of cells: WT, n = 13; G369i KI, n = 9; CaV1.4 KO, n = 3. e, Representative ICa traces (top) and voltage-protocol (bottom) for steady-state inactivation. ICa was evoked by a conditioning pre-pulse from -90 mV to various voltages for 500 ms followed by a test pulse to -30 mV for 50 ms. f, I/Imax represents the current amplitude of the test pulse normalized to current amplitude of the pre-pulse and was plotted against pre-pulse voltage. Numbers of cells: WT, n = 8; G369i KI, n = 8; Cav1.4 KO, n = 3. In graphs in c,d, and f, smooth lines represent Boltzmann fits, symbols and bars represent mean ± SEM, respectively. In graph in f, line without symbols represents G-V curve for G369i KI cones replotted from d. Shaded region indicates window current for G369i KI cones.

Comparison of parameters from electrophysiological recordings of cones.

Pharmacological characterization of ICa in cones of WT and G369i KI mice and ground squirrel.

a,b, Analysis of mouse cones. Left, representative traces for ICa evoked by voltage ramps in cones of WT or G369i KI mice before (baseline) and after exposure to 1 μM of isradipine (ISR, a, WT, n = 4; G369i KI, n = 5) or 5 μM ML 218 (b, WT, n = 5; G369i KI, n = 5). Right, ICa amplitudes before (-) and during (+) perfusion of the blocker on the same cells. Each point represents a different cell. *, p < 0.05 by paired t-test. c,d, Analysis of ground squirrel cones. Representative traces corresponding to baseline-corrected ICa evoked by voltage ramps (c,d, left) and corresponding G-V plot (c, right) before and during application of ML 218 (c) or ISR alone or ISR+ML218 (d). In d, ICa amplitudes are plotted before (-) and after (+) block for cones in the superior (sup.; n = 7 cones), middle (mid.; n = 5 cones), and inferior (inf.; n = 5 cones) thirds of the retina. The ICa blocked by ISR alone was measured at the peak of the control I-V curve between -40 and -20 mV. The ICa amplitude blocked by adding ML218 was measured as the average current between -50 and -45 mV before ML218 addition relative to zero current following the addition of ML218. The changes produced by ML218 were small but nonetheless significant (sup., n = 7, ISR: p < 0.0001, ML218: p = 0.0047; mid., n = 5, ISR: p < 0.0001, ML218: p = 0.0001; inf., n =5, ISR: p = 0.0019, ML218: p = 0.0460; two-tailed t test).

Immunofluorescence characterization of cone synapses in WT and G369i KI mice.

a, Confocal images of the OPL of WT and G369i KI mice labeled with antibodies against cone arrestin (CAR) and proteins that are presynaptic (CtBP2, bassoon) or postsynaptic (GPR179, TRPM1, mGluR6). Every other panel shows high-magnification, deconvolved images of single pedicles labeled with cone arrestin (rod spherule-associated signals were removed for clarity). Arrows indicate ribbon synapses, which appear enlarged in the G369i KI pedicles. b-d, Violin plots represent volume occupancy of labeling for each synaptic protein normalized to their respective CAR-labeled pedicles. e-i, Dependence of synapse size on pedicle size. Volumes corresponding to labeling of CtBP2 (e: p = 0.051, r = 0.4 for WT; p < 0.0001, r = 0.88 for G369i KI), CaV1.4 (f: p = 0.8, r = 0.06 for WT; p = 0.002, r = 0.88 for G369i KI), bassoon (g: p = 0.1, r = 0.34 for WT; p < 0.0001, r = 0.75 for G369i KI), mGluR6 (h: p = 0.32, r = 0.35 for WT; p = 0.002, r = 0.73 for G369i KI) and TRPM1 (i: p = 0.32, r = 0.35 for WT; p = 0.007, r = 0.66 for G369i KI) are plotted against pedicle volume. Dashed and solid lines represent fits by linear regression for WT and G369i KI, respectively.

Cone synapses form incorrect pairings with postsynaptic partners in G369i KI mice.

3D reconstructions of WT and G369i KI pedicles (n = 2 each) were obtained by SBFSEM. a, 3D renderings showing ribbons (magenta) within cone pedicles (gray) from WT and G369i KI mice. b,b’, 3D renderings (b) show ribbon sites in a WT cone pedicle contacting one horizontal (HC1; yellow) and three bipolar cells (BC1-3; purple). The raw image (b’) shows a single plane example of BC1-2 and HC1 contacting the ribbon site. c-d’, Single plane raw images (left panels, c; d) and 3D reconstructions (right panels, c; d’) show ribbon sites within the G369i KI cone pedicle contacting in c: horizontal cells (HC1-2) only, CBCs only (BC1-2); and in d,d’: glia (the G369i KI cone pedicle contacts a glial cell (orange) and an unknown partner); the glial cell completely envelops the pedicle. Inset in d’ shows glial-contacting ribbon site (arrow). In other panels, arrows indicate points of contact between ribbons and other postsynaptic elements.

Comparison of parameters for cone synapse organization

Light responses of horizontal cells are impaired in G369i KI mice.

a-b, Representative traces from whole-cell patch clamp recordings of horizontal cells held at -70 mV (a) and quantified data (b) for currents evoked by 1 s pulses of light (λ = 410 nm) plotted against light intensities. In b, peak current amplitudes during (ON current) and after (OFF current) the light stimuli were plotted against photon flux per μm2 (Φq/μm2). Data represent mean ± SEM. WT, n = 8; G369i KI, n =9. c-d, Representative traces from horizontal cells held at -70 mV (c) and quantified data (d) for currents evoked by 1-s pulses of light (λ = 410 nm, 1.2 x 105 Φq/μm2) before, during, and after washout of DNQX (20 μM). In d, symbols represent responses from individual cells, n = 5 cells for WT and 3 cells for G369i KI, bars represent mean ± SEM. **, p < 0.01 by paired t-tests.

Photopic vision is reduced but not lost in G369i KI mice.

a, Representative traces of photopic ERGs recorded in the presence of background green light (20 cd · s/m2) in WT, G369i KI and CaV1.4 KO mice. Flash intensities are shown at left. Arrows and arrowheads depict the a-and b-waves, respectively. b, a-wave (left) and b-wave (right) amplitudes are plotted against light intensity. Symbols and bars represent mean ± SEM. WT, n = 7; G369i KI, n = 5; CaV1.4 KO, n = 6. For a-waves, there was a significant effect of light intensity (p < 0.001) and genotype (p = 0.0086) by a mixed-effects model; by post-hoc Tukey test, there was no significant difference between WT and G369i KI at any light intensity. For b-waves at the highest light intensity, WT vs G369i KI, p = 0.0001; WT vs. CaV1.4 KO, p < 0.0001; G369i KI vs. CaV1.4 KO, p = 0.0038, Two-way ANOVA with Tukey’s post hoc analysis. c,d, Representative traces (c) and quantified data (d) for 10 Hz flicker responses evoked by white light flashes of increasing luminance (from -4 to 2 log cd· s/m2). Arrows in c depict inverted waveform responses in G369i KI mice that are absent in CaV1.4 KO mice. Symbols and bars represent mean ± SEM. WT, n = 4; G369i KI, n = 5; CaV1.4 KO, n = 5. At each of the 3 highest light intensities there was a significant difference (p < 0.05) in b-waves of WT vs G369i KI and G369i KI vs Cav1.4 KO by Two-way ANOVA with Tukey’s post hoc analysis. e, Representative swim path traces of WT, G369i KI and CaV1.4 KO mice from the visible platform swim tests performed in the dark (upper traces) and light (lower traces). f, Quantified latency to platform. Symbols represent the average of the last 3 swim trials for each mouse of each genotype for both dark and light conditions. Dotted lines represent the mean. WT, n = 11; G369i KI, n = 10; CaV1.4 KO, n = 9. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; Kruskal-Wallis one-way ANOVA with Dunn’s post hoc analysis.

Key resources used in this study.

Pharmacological drugs and their concentrations used in electrophysiological recordings of cones.

Effect of ML218 on HEK293T cells transfected with Cav1.4, β2x13, and α2δ−4 or Cav3.2.

a,b, Traces (a) and I-V plot (b) for ICa evoked by 50 ms depolarizations from -90 mv to various voltages without (0 μM) and in the presence of the indicated concentrations of ML218. c,d, Traces (c) and graph (d) depicting inhibitory effects of ML218 (5 μM) on Cav3.2-mediated ICa evoked by voltage ramp in individual transfected cells. **, p < 0.01 by paired t-test.

Adult ground squirrel cones lack a Cav3-like conductance.

a, Representative traces for ICa evoked by voltage steps from a continuous holding voltage of -85 mV to various voltages. Following recording of ICa bathed in control solution (baseline), cone terminals were perfused sequentially with ISR (2 μM) and then ISR (2 μM) + ML218 (5 μM). Voltage protocol is shown below current traces. Dashed line indicates zero current. b, Traces show the ICa sensitive to ISR (top) and ISR+ML218 (middle). ICa was evoked by steps from -85 mV to voltages between -65 and -5 mV in increments of +10 mV. The ICa recorded in ISR was subtracted from the baseline ICa (top). ICa recorded in ISR+ML218 was subtracted from ICa recorded in ISR (middle). The small amplitude and noninactivating properties of the current blocked by adding ML218 to ISR suggest that it is mediated by Cav1.4 channels that continue to undergo a time-dependent block at the concentration of ISR used here. c, Top, Current-voltage (I-V) plots of peak and steady-state ICa from b. Bottom, normalized conductance (G/Gmax) vs. voltage relationship (G-V) of the ICa blocked by ISR and ISR + ML218. Smooth lines represent Boltzmann fits; symbols and bars represent mean ± SEM, respectively; n = 4 cones. Due a negative shift in the activation properties caused by ML218 on a residual Cav1 current that is assumed to remain at the end of the experiment, the G-V curve of the ICa isolated through subtraction by applying ISR+ML218 underwent a statistically insignificant shift to the right. Data presented in a-c are from the same cone. d, Voltage ramp current response in an OFF cb3a bipolar cell before and after the application of ML218 (5 μM). V1/2 was shifted to the right by 13.1 ± 4.3 mV (n = 3 cb3a cells, mean ± SD) consistent with the block of a Cav3-like conductance. In this experiment, the amplitude of an ICa component that had a more depolarized activation range was slightly increased. A similar voltage ramp applied to OFF cb3b bipolar cells produced only a slight leftward shift in an ICa that had a more depolarized activation range, consistent with the exclusive expression a Cav1-type current (V1/2 was shifted by -1.9 ± 0.4 mV, n = 3 cb3b cells; data not shown). e, Neurobiotin filled OFF cb3a. Following recordings of cells in d, retina slices were fixed and co-immunolabeled for bassoon and choline acetyltransferase (CHAT) to depict synapses and the OFF and ON sublamina of the innerplexiform layer (IPL), respectively. Streptavidin was used to label the neurobiotin to depict the OFF cb3a axon stratification and morphology.

Characterization of ICa in cones of macaque retina.

(a-c) Representative current traces (a) and I-V (b), and G-V (b) for ICa evoked by 200-ms steps from a holding voltage (Vh) of -90 mV or -50 mV (which should isolate the Cav1-mediated ICa from any contribution of Cav3 channels). n=3 cells. (d) V1/2 and slope factor (k) obtained from Boltzmann fit of data in c. There was no significant effect of holding voltage on these parameters, arguing against a contribution of Cav3 channels to the whole-cell ICa. (e) Lack of effect of inactivating voltage on ICa. Test currents were evoked from a holding voltage of -100 V to -30 mV before (P1) and after (P2) a 500 ms step to -60 mV. Voltage protocol shown above overlay of P1 and P2 current traces. Boxed region is shown with expanded timescale; the similar activation kinetics of P1 and P2 currents supports the contribution of a single Cav subtype. Graph at right shows lack of significant difference in amplitudes of P1 and P2 currents, arguing against the contribution of Cav3 channels to the P1 current.