elifeeLifeeLife2050-084XeLife Sciences Publications, Ltd9490810.7554/eLife.9490810.7554/eLife.94908.1reviewed preprint1.1NeuroscienceA non-conducting role of the Cav1.4 Ca2+ channel drives homeostatic plasticity at the cone photoreceptor synapseMaddoxJ. Wesley16OrdemannGregory J.16VázquezJuan de la Rosa1HuangAngie1GaultChristof1WisnerSerena R.23RandallKate1FutagiDaiki4DeVriesSteven H.4HoonMrinalini25http://orcid.org/0000-0001-8021-0443LeeAmy1*Dept of Neuroscience, University of Texas-Austin, Austin, TX 78712, USADept. of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, 53706, USANeuroscience Training Program, University of Wisconsin-Madison, Madison WI 53706 USADept. of Ophthalmology, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USAMcPherson Eye Research Institute, Madison WI 53706 USABroseNilsReviewing EditorMax Planck Institute of Experimental MedicineGöttingenGermanySwartzKenton JSenior EditorNational Institute of Neurological Disorders and StrokeBethesdaUnited States of America
In congenital stationary night blindness type 2 (CSNB2)—a disorder involving dysfunction of the Cav1.4 Ca2+ channel—visual impairment is relatively mild considering that Cav1.4 mediates synaptic transmission by rod and cone photoreceptors. Here, we addressed this conundrum using a Cav1.4 knockout (KO) mouse and a knock-in (KI) mouse expressing a non-conducting Cav1.4 mutant. Surprisingly, aberrant Cav3 currents were detected in cones of the KI and KO but not wild-type mice. Cone synapses, which fail to develop in KO mice, are present but enlarged in KI mice. Moreover, light responses in cone pathways and photopic visual behavior are preserved in KI but not in KO mice. In CSNB2, we propose that Cav3 channels maintain cone synaptic output provided that the Ca2+-independent role of Cav1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel.
At the first synapse in the visual pathway, the light-dependent graded electrical signals produced in rod and cone photoreceptors gates the release of glutamate onto postsynaptic neurons. To accomplish this task, photoreceptor synapses are specialized with a ribbon organelle, which helps prime synaptic vesicles 1, 2 and postsynaptic dendrites from horizontal and bipolar cells that invaginate deep within the terminal 3. A variety of proteins interact with the ribbon and synaptic vesicles near release sites (i.e., active zones) 4. The importance of these proteins for vision is illustrated by the numerous inherited retinal diseases linked to mutations in their encoding genes 5.
One such gene is CACNA1F, which encodes the voltage-gated Ca2+ (Cav) channel expressed in retinal photoreceptors, Cav1.4 6–8. Among the sub-family of Cav1.x L-type channels, Cav1.4 exhibits unusually slow inactivation that is well-matched for supporting the tonic, Ca2+-dependent release of glutamate from photoreceptor synaptic terminals in darkness 9,10. More than 200 mutations in CACNA1F cause vision disorders including congenital stationary night blindness type 2 (CSNB2) 11, 12. These mutations are broadly categorized as producing a gain of function or loss of function in Cav1.4 13. How these mutations in CACNA1F lead to the variable clinical phenotypes of CSNB2 is largely unknown. Symptoms may include strabismus, low visual acuity, and in many cases, night blindness 14, 15. The latter suggests a primary defect in rod pathways, which is surprising given that knockout (KO) mice are completely blind and lack any evidence of either rod or cone synaptic responses 6, 7, 16. A major caveat is that rod and cone synapses fail to form in Cav1.4 KO mice 7, 16–18. Thus, Cav1.4 KO mice are not suitable for studies of how CACNA1F mutations differentially affect rod and cone pathways or for efforts to uncover how the biophysical properties of Cav1.4 shape photoreceptor synaptic release properties.
Here, we overcome this hurdle with a knock-in mouse strain (G369i KI) expressing a non-conducting mutant form of Cav1.4 19. We show that cone ribbon synapses in G369i KI mice are largely preserved and that downstream signaling through cone pathways, although greatly impaired, can support visual function. This novel mechanism requires the ability of the Cav1.4 protein, independent of its Ca2+ conductance, to nucleate the assembly of cone ribbon synapses and involves an aberrant Cav3 (T-type) conductance that appears when Cav1.4 Ca2+ signals are compromised.
ResultsCa<sup>2+</sup> currents in cones are mediated by Ca<sub>v</sub>3 channels upon Ca<sub>v</sub>1.4 loss-of-function
A prevailing yet unsupported hypothesis regarding the relatively mild visual phenotypes in CSNB2 is that additional Cav subtypes may compensate for Cav1.4 loss of function in cones. If so, then Ca2+ currents (ICa) mediated by these subtypes should be evident in cones of Cav1.4 KO and G369i KI mice. The G369i mutation is an insertion of a glycine residue in a transmembrane domain, which prevents Ca2+ permeation through the channel 19. Rods of G369i KI mice lack any evidence of ICa, despite the normal presynaptic clustering of the mutant channel 19. To test if this might differ in G369i KI cones, we first analyzed the localization of the mutant G369i Cav1.4 channels in cones by immunofluorescence with antibodies against Cav1.4, as well as cone arrestin (CAR) and CtBP2 to label cone terminals (i.e., pedicles) and ribbons, respectively (Fig. 1). In Cav1.4 KO mouse retinas, cone pedicles were shrunken and retracted into the outer nuclear layer (ONL, Fig. 1a) and had malformed ribbons (Fig. 1b). In contrast, cone pedicles in G369i KI mice were normally localized in the outer plexiform layer (OPL, Fig. 1a) and were populated by multiple ribbons (Fig. 1b). As in WT mice, labeling for Cav1.4 was clustered near elongated ribbons in cones of G369i KI mice (Fig. 1b). Thus, unlike in Cav1.4 KO mice, the mutant Cav1.4 protein is normally localized and supports the integrity of cone pedicles and ribbons in G369i KI mice.
Cone pedicles and the mutant Ca<sub>v</sub>1.4 channel are normally localized in the OPL of G369i KI mice but not Ca<sub>v</sub>1.4 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).
We next compared patch clamp recordings of cones in retinal slices of adult WT, G369i KI, and Cav1.4 KO mice under conditions designed to isolate ICa (Fig. 2). In WT cones, there was a large, sustained ICa that activated around -50 mV and peaked near -20 mV, consistent with the properties of Cav1.4 (Fig. 2a-d). A small-amplitude ICawas detected in cones of G369i KI and Cav1.4 KO mice but activated at significantly more negative voltages than in WT cones (Fig. 2a-d; Table 1). Moreover, ICa in the G369i KI and Cav1.4 KO cones activated around -60 mV and peaked near -35 mV. This aberrant ICa was not sustained as in WT cones but inactivated rapidly during 50-ms (Fig. 2b) and 500-ms (Fig. 2e) step depolarizations. Overlay of the conductance-voltage (Fig. 2d) and inactivation curves (Fig. 2f) revealed a sizeable window current. These features of ICa in G369i KI and Cav1.4 KO cones resembled those of Cav3 T-type channels rather than Cav1.4 20.
A low-voltage activated <italic>I<sub>Ca</sub></italic> is present in cones of G369i KI and Ca<sub>V</sub>1.4 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.
Although Cav3 channels were reported in patch clamp recordings of cone pedicles in WT mouse cones 21, we did not observe a low voltage-activated component of ICa in the I-V curve from our recordings of WT mouse cone somas, which would be indicative of a Cav3 subtype (Fig. 2a,c). Moreover, ICa in WT cones was blunted by the Cav1 antagonist isradipine (Fig. 3a) but not the Cav3 antagonist, ML 218 (Fig. 3b; ML 218 caused a minor suppression of ICa in some WT cones, which could be attributed to weak activity on Cav1.4, Supp.Fig. S1). In contrast, ICain G369i KI cones was significantly suppressed by ML 218 but not by isradipine (Fig. 3a,b). To further test for a Cav3 contribution to ICa, we analyzed cones of ground squirrel retina, where the large amplitude of ICa facilitates pharmacological analyses. Consistent with its actions on Cav1.4 in WT mouse cones (Fig. 3c) and transfected HEK293T cells (Supp.Fig. S1), ML 218 caused an insignificant inhibition of peak ICa (-7.0 ± 20.8%, n = 6 cones) as well as a negative shift in the voltage-dependence of activation in ground squirrel cones (Supp.Fig. S2). As a positive control, we confirmed that ML 218 blocked a prominent Cav3-type current in ground squirrel type 3a OFF cone bipolar cells (Supp.Fig. S2). Application of isradipine followed by ML 218 resulted in a time-and voltage-dependent suppression of the residual ICa, which is a hallmark of Cav1 inhibition by dihyropyridine antagonists such as isradipine (Fig. 3d, Supp.Fig. S2)22. Finally, we also performed patch clamp recordings of cone terminals in macaque retina where there was no evidence of a Cav3 current (Supp.Fig. S3). We conclude that Cav3 channels contribute significantly to ICa in cones only when Cav1.4 Ca2+ signals are absent.
Pharmacological characterization of <italic>I<sub>Ca</sub></italic> 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).
Cone synaptogenesis relies on the Ca<sub>v</sub>1.4 protein but not its Ca<sup>2+</sup> conductance
As shown in previous studies 7, 16–18, CtBP2 labeled stubby, sphere-like structures resembling immature ribbon material in Cav1.4 KO mice (Fig. 1). The presence of elongated ribbons in G369i KI mice suggested that, as in rods 19, the non-conducting mutant Cav1.4 protein may support the molecular assembly of cone synapses. To this end, we analyzed cone synapses by immunofluorescence and confocal microscopy. Along with the major constituents of the ribbon, CtBP2 and RIBEYE 23, presynaptic proteins such as bassoon and members of the postsynaptic signaling complex in depolarizing (ON) cone bipolar cells (GPR179, mGluR6, and TRPM1 24) were enriched near cone ribbons in G369i KI mice as in WT mice (Fig. 4a). Compared to WT mice, the labeled structures occupied a larger volume (Fig. 4b,c), albeit at a much lower density (Fig. 4d), which increased linearly with the volume of the pedicle in G369i KI mice (Fig. 4e-i), perhaps as a homeostatic response to Cav1.4 loss-of-function.
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.
The cone synapse is structurally complex, with the dendritic tips of two horizontal cells and an intervening ON cone bipolar cell invaginating deeply into the pedicle near the ribbon 3. To test how the switch in Cav subtypes might affect this arrangement of postsynaptic partners, we generated 3D reconstructions of cone synapses by serial block-face scanning electron microscopy (SBFSEM; Fig. 5). As with the enlarged synaptic contacts (Fig. 4), there was some evidence of structural modifications in G369i KI pedicles. Compared to WT pedicles, ribbons appeared disorganized in G369i KI pedicles which extended telodendria laterally rather than apically (Fig. 5a). In addition, a slightly larger fraction of synaptic sites in the G369i KI pedicle (35% vs 14% in WT) formed incorrect postsynaptic partnerships (Fig.5b-d’; Table 2). Nevertheless, the number of ribbons is normal in G369i KI cones and about half of the ribbons making invaginating contacts with the appropriate cell types (i.e., both horizontal cells and cone bipolar cells; Table 2). Therefore, while necessary for the structural refinement of the cone synapses, Cav1.4 Ca2+ signals are largely dispensable for cone synapse assembly.
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
Cone signaling to a postsynaptic partner is intact in G369i KI mice
The preservation of cone synapses in G369i KI mice allowed the unique opportunity to test whether a Cav subtype other than Cav1.4 could support ribbon-mediated synaptic release. To this end, we compared synaptic transmission between cones and horizontal cells (HCs, Fig. 6) in retinal slices of WT and G369i KI mice. In darkness, glutamate released from cones depolarizes horizontal cells via activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA)/kainate receptors 25, 26. The resulting excitatory postsynaptic current (EPSC) undergoes a decline in response to light stimuli that hyperpolarize cones 27. In WT HCs, a 1 s light pulse (λ=410 nm) inhibited the standing EPSC, which is reflected as an outward (hyperpolarizing “ON”) current (Fig. 6a). Upon termination of the light pulse, an inward (depolarizing “OFF”) current signaled the resumption of the EPSC (Fig. 6a). Both the ON and OFF components of the EPSCs in WT HCs increased with light intensity, reflecting the impact of luminance on the presynaptic membrane potential of cones and the subsequent change in glutamate release from their terminals (Fig. 6b). The ON and OFF components of EPSCs in G369i KI HCs were measurable, although lower in amplitude than in WT (Fig. 6a,b). The EPSC in darkness and the light response were abolished by the AMPA/kainate receptor antagonist, DNQX, in both WT and G369i KI horizontal cells (Fig. 6c,d). Thus, while greatly impaired, cone synapses can transmit light information to postsynaptic partners in the retina of G369i KI mice.
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.
Light responses of bipolar cells and visual behavior is spared in G369i KI but not Ca<sub>v</sub>1.4 KO mice
While horizontal cells act as inhibitory interneurons that modulate photoreceptor output, the vertical dissemination of visual information from cones to the inner retina is relayed by glutamate to ON and OFF cone bipolar cells (CBCs) which express mGluR6 and AMPA/kainate receptors, respectively. To test whether cone synaptic transmission to CBCs is intact in G369i KI mice, we recorded electroretinograms (ERGs) under light-adapted conditions using Cav1.4 KO mice as a negative control (Fig. 7a-d). In these recordings, the light-induced response of photoreceptors and the postsynaptic response of ON CBCs corresponds to the a-and b-waves, respectively. Unlike in Cav1.4 KO mice, the a-waves of G369i KI mice were like those in WT mice, which indicates that cones do not degenerate in this mouse strain. While reduced in amplitude, the b-wave was measurable in G369i KI mice and significantly larger than in Cav1.4 KO mice at the highest light intensities (Fig. 7a,b). We also recorded flicker ERGs using 10 Hz light stimuli that can isolate cone pathways involving both ON and OFF CBCs. In WT mice, flicker responses exhibited two peaks, one at a lower irradiance (-2 log cd·s/m2) and one at higher irradiance (0.5 log cd·s/m2). The peak at the lower irradiance is attributed to responses in both rod and cone pathways, and the peak at higher irradiance is attributed to responses exclusively in cone pathways (Fig. 7c,d)28, 29. While significantly lower in amplitude than in WT mice, flicker responses in G369i KI mice showed a similar non-monotonic relation. Compared to Cav1.4 KO mice, G369i KI mice showed peak flicker responses that did not differ at -2 log cd·s/m2, but were significantly higher at 0.5 log cd·s/m2 (Fig.7d). The inverted flicker responses at higher illuminations in G369i KI mice (Fig. 7c) were absent in Cav1.4 KO mice and may result from the hyperpolarizing contribution of cone-to-OFF CBC transmission. These results suggest that cone-to-CBC signaling is intact in G369i KI mice.
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.
To validate these results with respect to vision-guided behavior, we used a swim test that assesses the ability of mice to identify a visible platform 30. In darkness, G369i KI and Cav1.4 KO mice took significantly longer to find the platform compared to WT mice (Fig. 7e,f). These results are consistent with our flicker response assays (Fig.7c,d) as well as the absence of ICa in rods and rod-to-rod bipolar cell synaptic transmission in G369i KI mice 19. In daylight conditions, G369i KI mice but not Cav1.4 KO mice performed as well as WT mice (Fig. 7e,f). Thus, G369i KI mice are unique in retaining visual function under photopic but not scotopic conditions. Collectively, our results suggest that Cav3 channels can support cone synaptic responses and visual behavior in G369i KI but not Cav1.4 KO mice.
Discussion
The nervous system has remarkable abilities to adapt to pathological perturbations in neuronal activity. In the retina, ablation or degeneration of photoreceptors triggers various postsynaptic mechanisms that maintain some level of visual function in rod or cone pathways. These include remodeling of bipolar cell dendrites and their synapses31–33 as well as changes in the sensitivity of bipolar cells to photoreceptor input and inhibitory modulation33, 34. To our knowledge, this study provides the first evidence for a presynaptic form of homeostatic plasticity that originates within photoreceptors. Using Cav1.4 KO and G369i KI mice, we identify the upregulation of a Cav3 conductance as a common response to Cav1.4 loss-of-function in cones. However, Cav3 channels can only compensate for Cav1.4 loss-of-function when cone synapse structure is maintained. Thus, our results also highlight a crucial, non-conducting role for the Cav1.4 protein that allows cone synapses to function in the absence of Cav1.4 Ca2+ signals.
A non-canonical role for Ca<sub>v</sub>1.4 in regulating cone synapse assembly
A major finding of our study is that cone synapse formation requires the Cav1.4 protein but not Cav-mediated Ca2+ influx. As shown for rod synapses 19, ribbons and other components of the pre-and post-synaptic complex assemble normally at cone synapses in G369i mice (Figs. 1,4,5). Cav3 Ca2+ signals are dispensable for this process since their presence in Cav1.4 KO cones is not accompanied by any semblance of ribbon synapses (Fig.1B). An intimate relationship between Cav1.4 and ribbons is supported by the colocalization of Cav1.4 and RIBEYE puncta resembling ribbon precursor spheres in the developing OPL7. Moreover, light adaptation, which decreases the size of the ribbon, leads to a reduction in Cav1.4 labeling in mouse retina2. While evidence for the binding of RIBEYE to Cav1.4 is lacking, such an interaction could support the oligomerization of RIBEYE A and B domain within a macromolecular complex 35. Alternatively, Cav1.4 could pioneer sites of ribbon assembly perhaps by serving as a docking or nucleation site for the active zone. Regardless of the mechanism, our findings show that the formation of photoreceptor synaptic complexes does not require Ca2+ influx through Cav1.4 channels.
Despite a normal molecular organization, G369i KI cone synapses were enlarged and made errors in postsynaptic partner selection. While I-V curves predict similar peak ICa amplitudes in cones of WT and G369i KI mice near the membrane potential of cones in darkness (-45 to -50 mV 36, Fig.1c), the strong inactivation of Cav3 channels predicts greatly diminished Ca2+ signals in G369i KI pedicles. Paradoxically, reductions in presynaptic Ca2+ in rod photoreceptors are thought to cause illumination-dependent shrinkage of ribbons in vivo and in vitro2, 37, 38. However, an inverse correlation between presynaptic Ca2+ influx and ribbon synapse size is seen in sensory hair cells in zebrafish 39. The mechanism involves decreased mitochondrial Ca2+ uptake and increase in the redox state of nicotinamide adenine dinucleotide (NAD+/NADH ratio) 40. If a similar mechanism applied in cones, Cav3 Ca2+ signals in G369i KI cones may decay too quickly to enable mitochondrial Ca2+ uptake mechanisms that trim ribbons. The size of ribbons and the postsynaptic specialization increased linearly with pedicle volume in G369i KI but not in WT mice (Fig. 4e-i), which could represent a form of homeostatic synaptic scaling as has been demonstrated at the neuromuscular junction 41, 42. Similarly, the presence of some non-invaginating contacts with incorrect partner pairings in G369i KI mice (Fig.5, Table 2) could represent a compensatory response to weakened synaptic output. Considering that only a subset of CBC subtypes re-wire correctly following partial ablation of cones in immature mice 32, the structurally normal cone synapses of G369i KI mice could involve contacts with CBCs that are unusually resilient to loss of presynaptic input.
While it is well-established that Cav1.4 is the main Cav subtype in mouse cones, Cav3 channels, in particular Cav3.2, have been reported to be expressed in mouse cones by electrophysiology 21 and single cell RNA-seq studies 21, 43, 44. By pharmacological and other criteria, we found no evidence for a functional contribution of Cav3 in our recordings of cones in WT mice (Figs. 2,3), ground squirrels, or macaque (Supp.Figs. S2,S3). A caveat of using dihydropyridine antagonists such as isradipine to isolate the contribution of Cav1 from other Cav subtypes is the relatively low sensitivity of Cav1 channels to these drugs and their strong voltage-dependence. At a holding voltage of -90 mV, isradipine at 1 μM causes only ∼80% inhibition of Cav1.4 with greater block at depolarized voltages 22, which agrees with our recordings of cones in WT mice (Fig.3a) and ground squirrels (Fig.3d, Supp.Fig.S2). Moreover, Cav3 blockers such as Z944 45 and ML218 (Supp.Fig. S2) have additional activity on Cav1 channels at micromolar concentrations including effects on current amplitude and activation voltage. Thus, currents mediated by Cav1.4 that are sensitive to Cav3 blockers (e.g, >5 µM ML218) and spared by dihydropyridines at negative voltages may be mistaken as being mediated by Cav3. In our experiments, the biophysical properties of the isradipine-sensitive ICain cones of WT mice and ground squirrels resembled only those of Cav1, whereas those of the ML218-sensitive ICa in cones of G369i KI mice resembled only those of Cav3. Therefore, we favor the interpretation that Cav3 contributes to ICa in mouse cones only upon silencing of the Cav1.4 Ca2+ conductance.
Consistent with the diminutive Cav3-mediated ICa, light responses of HCs were evident but smaller in G369i KI than in WT mice. Due to their slow activation and strong inactivation 46, Cav3 channels may have a reduced ability to fuel the Ca2+ nanodomains that support fast and sustained components of release, both of which occur only at ribbon sites in cones147. Cav3 channels may also be located further from the ribbon than Cav1.4, thus lowering the efficiency of coupling to exocytosis. Unfortunately, it was not possible to test this with commercially available antibodies against Cav3.2, which yielded identical patterns of immunofluorescence in the OPL of WT and Cav3.2 KO mice (data not shown). A detailed analysis of the Cav3 subtype(s) and their subcellular localization in G369i KI cones is required to unravel the shortcomings of Cav3 channels with respect to cone synaptic release.
Based on extremely heterogeneous clinical presentations, CSNB2 manifests as a spectrum of visual disorders that originate from various mutations in CACNA1F14, 48. Even though some CSNB2 mutant Cav1.4 channels may traffic normally to the plasma membrane, many of these mutations are expected to produce non-functional, non-conducting Cav1.4 channels.49, 50 Yet, the visual phenotypes of CSNB2 patients are not as severe as the complete blindness in Cav1.4 KO mice, which lack any Cav1.4 protein expression and exhibit no signs of visual behavior (Fig.7e,f)51. Collectively, our results suggest that G369i KI mice accurately model Cav1.4 channelopathies in CSNB2 patients that are characterized by a greater impairment in rod than in cone pathways. This interpretation is supported by our findings that G369i KI mice exhibit horizontal cell responses to bright but not dim illumination (Fig. 6), ERG responses under conditions of light adaptation (Fig.7a-d) but not dark-adaptation 19, and visual behavior under photopic but not scotopic conditions (Fig. 7d). Together with the enlargement of synaptic sites, modest levels of synaptic release from cones of G369i KI mice may be sufficient to support nominal transmission of visual information through cone pathways. Furthermore, we acknowledge that G369i KI mice could also exhibit homeostatic alterations in the inner retina, which are known to support visual function when photoreceptor input is severely compromised 32–34. Future studies of the retinal circuitry and visual behavior of G369i KI mice could identify compensatory pathways that are recruited upon Cav1.4 loss-of-function and how they might be targeted in novel therapies for CSNB2 and related disorders.
Acknowledgements
J.W.M., G.J.O., J.D.V., A.H., K.R., and A.L. were supported by NIH grants EY026817 (to A.L.), EY029953 (to J.W.M.), and startup funds from the University of Texas-Austin (to A.L.); S.R.W. and M.H. were supported by NIH grant EY031677 (to M.H.), an unrestricted grant from RPB to UW Madison Dept. of Ophthalmology and a McPherson ERI professorship to M.H.; D.F. and S.H.D. were supported by NIH grants EY012141 and EY032506, an unrestricted grant from RPB to the Northwestern University Dept. of Ophthalmology, and an RPB International Travel Award. The authors thank S. Knecht and R. Wong for assistance with serial EM image collection, E. Seidemann and B. Shukla for the generous donation and collection of macaque retinal tissue, respectively, and M. McCall and F. Vinberg for advice on ERGs.
All mouse and macaque experiments were performed in accordance with guidelines approved by the National Institutes of Health and the Institutional Animal Care and Use Committees at the University of Texas at Austin. All ground squirrel (Ictidomys tridecemlineatus) procedures performed at Northwestern University were approved by the Institutional Animal Care and Use Committee. The G369i KI 19 and Cav1.4 KO mouse strains were bred on the C57BL6/J background strain for at least 10 generations. Adult male and female mice were used (6-12 weeks old), and aged-matched C57BL6/J mice were used as the control (WT) animals.
Immunofluorescence
Mice between 6-8 weeks of age were anesthetized using isoflurane and euthanized by cervical dislocation. Eyes were enucleated and hemisected. The eye cups with retina were fixed on ice in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 30 min. Fixed eye cups were then washed three times with 0.1 M PB containing 1% glycine followed by infusion of 30% sucrose at 4°C overnight. The eye cups were orientated along their dorsal-ventral axis and frozen in a 1:1 (wt/vol) mixture of Optimal Cutting Temperature compound and 30% sucrose in a dry ice/isopentane bath. Eye cups were cryosectioned at 20 µm on a Leica CM1850 cryostat (Leica Microsystems), mounted on Superfrost plus Micro Slides (VWR), dried for 5 to 10 min at 42°C, and stored at -20°C until used. Slides with mounted cryosections were warmed to room temperature, washed with 0.1 M PB for 30 min to remove the OCT/sucrose mixture and blocked with dilution solution (DS, 0.1 M PB/10% goat serum/0.5% Triton-X100) for 15 min or overnight at room temperature. All remaining steps were carried out at room temperature. All primary antibodies and appropriate secondary antibodies were diluted in DS at concentrations specified in the Key Resources Table (Table 3). Sections were incubated with primary antibodies for 1 h or overnight and then washed five times with 0.1 M PB. Sections were then incubated with secondary antibodies for 30 min and then washed five times with 0.1 M PB. Trace 0.1 M PB was removed, and sections were then mounted with #1.5H coverslips (ThorLabs) using ProLong Glass Antifade Mountant with or without NucBlue (Thermo Fisher Scientific).
Key resources used in this study.
For double labeling with other rabbit polyclonal antibodies, CAR antibodies were conjugated with the CF647 fluorophore (CAR-647) using the Mix-n-Stain antibody labeling kit according to the manufacturer’s protocol (Biotium). Sections were processed first with rabbit polyclonal Cav1.4 or EAAT2 antibodies and corresponding secondary antibodies as described above. To prevent CAR-647 from binding to any available sites on the anti-rabbit secondary antibodies previously added to the sections, CaV1.4 or EAAT2 antibodies were readded to the sections and incubated for 30 min. After washing, the sections were incubated with CAR-647 for 1 h, followed by wash steps and cover glass mounting.
Immunofluorescence in labeled retinal sections was visualized using an Olympus FV3000 confocal microscope (Tokyo, Japan) equipped with an UPlanApo 60x oil HR objective (1.5 NA). Images were captured using the Olympus FLUOVIEW software package. Acquisition settings were optimized using a saturation mask to prevent signal saturation prior to collecting 16 -bit. All confocal images presented are maximum z-projections. Images (256 x 256 pixels) used in analyses of synaptic proteins were collected using a 30X optical zoom, 0.6 Airy disk aperture, and voxel size of 0.028 μm × 0.028 μm × 0.2 μm (X × Y × Z). Amira segmentation was used for generating 3D binary masks, and Amira 3D label analysis was used for quantification of immunofluorescent and masked images. Non-deconvolved images were used for all analyses. 3D binary masks of individual CAR-labeled pedicles were made by setting the threshold 1 standard deviation above mean fluorescence intensity of each 3D image. 3D binary masks of presynaptic labels (CtBP2, CaV1.4, and Bassoon) or postsynaptic labels (GPR179, mGluR6, and TRPM1) within or associated with the pedicle, respectively, were made by setting the threshold 3 standard deviations above the mean fluorescence intensity. To aid in presentation, high-mag images displayed in Figs.3f, 4a, and 6f were deconvolved using cellSens software (Olympus), and any immunofluorescence corresponding to rod synaptic proteins was subtracted from the image.
Molecular biology and transfection
The cDNAs for CaV1.4 (GenBank: NM_019582), β2X13 (GenBank: KJ789960), and α2δ-4 (GenBank: NM_172364) were previously cloned into pcDNA3.1 52. The cDNA for CaV3.2 (GenBank: AF051946) was a gift from Dr. Edward Perez-Reyes, University of Virginia. All constructs were verified by DNA sequencing before use. Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco’s Modified Eagle’s Medium with 10% FBS at 37°C in 5% CO2. At 70–80% confluence, the cells were co-transfected with cDNAs encoding human Cav1.4 (1.8 μg) β2X13 (0.6 μg), α2δ-4 (0.6 μg), and enhanced GFP in pEGFP-C1 (Clonetech, 0.1 μg) or CaV3.2 (2 μg) and pEGFP-C1 (0.1 μg) using FuGENE 6 transfection reagent according to the manufacturer’s protocol. Cells treated with the transfection mixture were incubated at 37°C for 24 hr, dissociated using Trypsin-EDTA, and replated at a low density to isolate single cells. Replated cells were then incubated at 30°C or 37°C for an additional 24 hr before beginning experiments.
Solutions for patch clamp recordings
HEK293T extracellular recording solution contained the following (in mM): 140 Tris, 20 CaCl2, 1 MgCl2, pH 7.3 with methansulfonic acid, osmolarity 309 mOsm/kg. HEK293T internal recording solution contained the following: 140 NMDG, 10 HEPES, 2 MgCl2, 2 Mg-ATP, 5 EGTA, pH 7.3 with methansulfonic acid, osmolarity 358 mOsm/kg.
For recordings of ICa in mouse retina, extracellular recording solution contained the following (in mM): 115 NaCl, 2.5 KCl, 22.5 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 5 HEPES, 5 CsCl, 5.5 Glucose, osmolarity 290 mOsm/kg. Mouse cone intracellular solution contained the following (in mM): 105 CsMeSO4, 20 TEA-Cl, 1 MgCl2, 11 HEPES, 10 EGTA, 4 Mg-ATP, 10 phosphocreatine, 0.3 Na-GTP, pH 7.4 with CsOH, osmolarity 300 mOsm/kg.
For recordings of ICa in ground squirrel retina, extracellular recording solution contained the following (in mM): 10 HEPES, 85 NaCl, 3.1 KCl, 2.48 MgSO4, 6 Glucose, 1 Na-succinate, 1 Na-malate, 1 Na-lactate, 1 Na-pyruvate, 2 CaCl2, 25 NaHCO3, and 20 TEA-Cl, osmolarity 285 ± 5 mOsm/kg. Intracellular solution contained the following (in mM): 80 CsCl, 10 BAPTA, 2 MgSO4, 10 HEPES, 20 TEA-Cl, 5 Mg-ATP, and 0.5 Na-GTP, pH 7.35 with CsOH, osmolarity 285 ± 5 mOsm/kg. For recordings of IAglu, extracellular recording solution contained (in mM): 125 NaCl, 3.0 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 3 dextrose, 3 sodium pyruvate, 0.1 picrotoxin and 0.02 DNQX, and in indicated experiments 0.0013 TFB-TBOA. Intracellular IAglu contained (in mM): 125 KSCN, 10 TEA-Cl, 10 HEPES, 1 CaCl2, 2 MgCl2, 0.3 Na-GTP, 4 Mg-ATP, 10 K2 phosphocreatine, 0.02 ZD7288. Mouse and ground squirrel extracellular slice recording solutions were equilibrated with 5% CO2 / 95% O2 to a pH of ∼7.5.
For recordings of light responses in horizontal cells of mouse retina, extracellular recording solution consisted of Ames’ media supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 22.6 mM NaHCO3, osmolarity 280 ± 5 mOsm/kg. The intracellular recording solution contained the following (in mM): 135 K-Aspartate, 10 KCl, 10 HEPES, 5 EDTA, 0.5 CaCl2, 1 Mg-ATP, 0.2 Na-GTP, pH 7.35 with KOH, osmolarity 305 ± 5 mOsm/kg.
Patch clamp electrophysiology
Whole-cell voltage clamp recordings of transfected HEK293T cells were performed 48 to 72 hrs after transfection using an EPC-10 amplifier and Patchmaster software (HEKA Elektronik, Lambrecht, Germany). Patch pipette electrodes with a tip resistance between 4 and 6 MΩ were pulled from thin-walled borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) using a P-97 Flaming/Brown Puller (Sutter Instruments, Novato, CA). A reference Ag/AgCl wire was placed into the culture dish mounted on an inverted Olympus IX70 microscope. Recordings were performed at room temperature. A pressurized perfusion pencil multi-barrel manifold controlled with Valve Bank II (AutoMate Scientific, Inc., Berkeley, CA) was used to deliver extracellular solutions. ML218 of different concentrations (0.5, 1, 5, 25 and 100 mM) was added to the extracellular solution the day of experiments. Series resistance was compensated up to 70%, and passive membrane leak subtraction was conducted using a P/−4 protocol. Whole-cell Ca2+ currents (ICa) of transfected HEK293T cells were evoked for 50 ms with incremental +5 mV steps from -80 mV to +65 mV. Current-Voltage (IV) data were fit with a single Boltzmann equation: ICa = Gmax(Vm-Vr)/(1+exp[-(Vm-Vh))/k]), where Gmax is the maximal conductance, Vm is the test voltage, Vris the Ca2+ reversal potential, Vh is the membrane potential required to activate 50% of Gmax, and k is the slope factor. Data were sampled at 100 kHz, filtered at 3 kHz, and analyzed using custom programs written in IgroPro (WaveMetrics).
Whole-cell voltage clamp recordings of mouse cones, macaque cone terminals, and mouse horizontal cells were performed using an EPC-10 amplifier and Patchmaster software (HEKA). Patch pipette electrodes with a tip resistance between 10 and 14 MΩ for cones and 6-8 for horizontal cells were pulled from thick-walled borosilicate glass (1.5 mm outer diameter; 0.84 mm inner diameter; World Precision Instruments).
To prepare retinal slices, adult mice (6 – 8 weeks old) were anesthetized using isoflurane and euthanized by cervical dislocation. Eyes were enucleated, placed into cold Ames’ media slicing solution, and hemisected. Following removal of the vitreous, the eye cup was separated into dorsal and ventral halves using a scalpel. Ventral retina was isolated, molded into low-melt agarose and mounted in a Leica VT1200s vibratome (Leica Biosystems). Mouse retina slicing solution was continuously bubbled with 100% O2 and contained the following: Ames’ Medium with L-glutamine supplemented with (in mM) 15 NaCl, 10 HEPES, 10 U/mL penicillin, 0.1 mg/mL streptomycin, pH 7.4, osmolarity 300 mOsm/kg. Vertical (∼200 µm) or horizontal (∼160 µm) retinal slices were anchored in a recording chamber, placed onto a fixed stage, and positioned under an upright Olympus BX51WI microscope equipped with a 60X water-immersion objective (1.0 NA), and superfused with extracellular solution (flow rate of ∼1-2 ml/min) at room temperature. Slices were visualized using IR-DIC optics and an IR-2000 (Dage MTI, Michigan City, IN) or SciCam Pro (Scientifica, Uckfield, United Kingdom) CCD camera controlled by the IR-capture software package or µManager, respectively 53, 54. Drugs used in these experiments were added to the mouse extracellular solution the day of experiments at the concentration described in Table 4. A reference Ag/AgCl pellet electrode was placed directly into the recording chamber solution. Data from whole-cell recordings with a series resistance >20 MΩ were discarded.
Pharmacological drugs and their concentrations used in electrophysiological recordings of cones.
Cone somas were identified based on their morphology and location (outer ONL cell layer). Cone identity was confirmed by the whole-cell capacitance (∼3-4 pF), which is larger than rod whole-cell capacitance (∼0.7-1 pF). For cone voltage ramp recordings, cones were held at - 90 mV for 200 ms followed by a ramp of +0.5 mV/ms to +40 mV. To determine voltage activation of ICa, whole-cell Ca2+ currents in cones were evoked for 50 ms with incremental +5 mV steps from -80 mV to +40 mV. The activation voltage of ICa is reported as G/Gmax, where G is the conductance at each test voltage and Gmax is the maximum peak conductance for each cone. Conductance was calculated using the equation ICa = G(Vm-Vr), where Vr is +60 mV. To determine steady state inactivation of ICa, currents in cones were evoked for 500 ms with incremental +5 mV steps from -90 mV and -30 mV followed by a final step to -30 mV for 50 ms after each test voltage. The steady state inactivation of ICa is reported as I/Imax, where I is the peak current in the final voltage step to -30 mV and Imaxis the maximum peak current for each cone. Data were sampled between 20 and 60 kHz and filtered at 3 kHz.
For horizontal cell light responses, horizontal slices were prepared from central mouse retina. The identity of horizontal cells was determined based on their larger soma diameter (∼15 μm) compared to bipolar cell somas (∼6 μm). During whole cell patch clamp recordings, horizontal cells were held at -70 mV. Light stimuli (1 s) at 410 nm (630 x 830 μm) were presented onto the retina (at a minimum of 5 s intervals) through the microscope’s condenser using a Polygon1000 DMD pattern illuminator (Mightex, Pleasanton, CA, USA) and a custom-built light path. Light intensity (in watts) was measured at the point on the microscope stage where the retina is placed using a power meter (Thorlabs, Newton, New Jersey, USA). Photon flux Фq (photons/s) within the light stimulus area was calculated using the measured light intensities in the formula:
where h is Planck’s constant (J*s), c is the speed of light (m/s), and λ is the wavelength (m). Light stimulus intensity was increased in Log2 steps from 4.9x102 to 2.1x105 Фq/μm2. For each light intensity step, both ON and OFF current amplitudes were measured from baseline (averaged 5 ms of current prior to light onset) to the maximum positive (after light onset) or maximum negative (after light offset) current, respectively. To confirm the identity of these horizontal cell light responses as AMPA-mediated currents, 1 s light stimuli (410 nm, 1.2x105 Фq/μm2) were continuously delivered every 10 s before, during and after bath application of 20 μM DNQX.
For voltage clamp recordings of 13-lined ground squirrel cones, retinal slices were prepared as previously described 55. The eyecup was divided along the dorsal to ventral axis into superior, middle, and inferior parts. The dorsal area above the line of the optic nerve head was defined as superior, the central region with a width of about 5 mm just ventral to the optic nerve head was middle, and the remaining ventral area was inferior. Isradipine and ML218, alone or in combination, were applied from separate puffer pipettes whose orifices were aimed at the cone synaptic region. Recordings were made with an Axopatch 200B amplifier (Molecular Devices). Signals were electronically filtered at 5 kHz and digitized at a rate of 10 kHz. Additional Gaussian filtering was added (cutoff frequency of 500 Hz). Tissue was viewed through a 63x water immersion objective on a Zeiss Axioskop FS2 microscope and superfused with extracellular solution at room temperature. Drugs and their concentrations used during these experiments are described in Table 4. Membrane potential was continuously maintained at -85 mV. During ramp stimulation, the membrane potential was depolarized to -85 to +35 mV at a rate of 1 mV/ms.
For rhesus macaque cone terminal recordings, a 12-year-old male was sedated with ketamine (5 mg/kg I.M.) and dexmedetomidine (0.015 mg/kg I.M.). Post-sedation, the animal received buprenorphine (0.02 mg/kg I.M.), atropine (0.02 mg/kg I.M.), and maropitant citrate (1 mg/kg S.Q.). The animal was intubated and maintained with inhaled isoflurane (0.75-2.0%) and propofol (7-8 mg/kg/hr I.V.). Crystalloid fluids (5 mL/kg/hr) were administered I.V., and phenylephrine (5-10 mcg/kg/hr I.V.) was used for blood pressure support. The animal was under anesthesia for approximately 3 hours prior to perfusion. Transcardial perfusion was approached through a midline thoracotomy. Immediately prior to perfusion, 5 mL of Euthasol® (sodium pentobarbital 390 mg/mL/phenytoin sodium 50 mg/mL) was administered I.V. The descending thoracic aorta was clamped, the pericardium was opened, and the right atrium was cut. The apex of the left ventricle was sharply incised, and a large bore cannula (Yankauer suction handle, 5 mm internal diameter) was inserted through the left ventricle until it could be palpated in the ascending aorta. The cannula was clamped in place at the apex of the left ventricle. The animal was perfused with 4000 mL of cold phosphate-buffered saline at ∼500 mL/min. Eyes were removed approximately 1 hour following perfusion. Eyes were dissected, and eye cups were allowed to dark adapt for ∼30 min and stored in bicarbonate buffered Ames’ media at 32°C equilibrated with 95% O2/5% CO2 prior to slice preparations. Vertical sections of central retina were prepared from 5 mm retina punches as described for mice.
Serial block-face scanning electron microscopy and 3D reconstructions
Eye cups were prepared from P42 WT and G369i KI littermates and fixed using 4% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4, for 4 hours at room temperature followed by additional fixation overnight at 4°C. Glutaraldehyde-fixed eye cups were then washed 3 times in 0.1M cacodylate buffer. Retinas were thereafter isolated and embedded in Durcupan resin after staining, dehydration, and embedding as described previously (Della Santina et al., 2016). A Thermo Scientific VolumeScope serial block face scanning electron micrscope was used to image embedded retinas. Retinal regions comprising a 2x2 montage of 40.96 μm tiles were imaged at a resolution of 5nm/pixel and section thickness of 50 nm. Image stacks were aligned, and cone photoreceptor terminals reconstructed using TrakEM2 (NIH). Postsynaptic partners at cone ribbons were followed to the inner nuclear layer to determine their identity. Amira software was used for 3D visualization of reconstructed profiles.
Electroretinography
Retinal function was assessed using the Celeris system (Diagnosys, Inc.) paired with the Espion software (Diagnosys, Inc.). Mice were anaesthetized under red light (660 nm) via intraperitoneal (I.P.) injection with ketamine/xylazine mixture (100 mg/kg ketamine, 10 mg/kg xylazine). Tropicamide ophthalmic solution (1 %) and hypromellose lubricant eye gel (0.3 %) were administered topically to both eyes before the mouse was secured to a heated (37 °C) platform to maintain body temperature. Ag/AgCl corneal stimulators were placed on each eye. After collecting data from individual mice, atipamezole (Antisedan, 1-2 mg/kg) was I.P. injection administered to reverse the effects of the ketamine and xylazine. For photopic ERGs, eyes were light adapted using a background green light at 20 cd·s/m2 for 10 min. Following light adaptation, eight different light intensity pulses (-0.5, 0, 0.5, 1, 1.5, 2, 2.3 and 2.6 log cd·s/m2) were delivered on top of the background green light. ERG a-waves were measured from baseline to the peak of the negative potential. ERG b-waves were measured from the peak of the a-wave to the peak of the positive potential. For flickering ERGs, mice were dark adapted (>12 hrs). White light pulses from -4 to 2.5 log cd·s/m2 were delivered in 0.5 log unit steps at 10 Hz. Response amplitudes were measured from the trough to the peak of each response at all light intensities. Each intensity stimulus was delivered 10 times, with a 3 s interval between each stimulus, and averaged. The mean response amplitudes recorded in the right and left eye of each mouse are reported for all quantified ERG data.
Visible platform swim test
The visible platform swim test was performed as has been previously described 30. WT, G369i KI, and Cav1.4 KO male and female mice (6-9 weeks old) were adapted to the procedure room for at least 1 h prior to beginning the experiments. A water-filled, 4-foot diameter galvanized steel tank and a visible white platform with a diameter of 10 cm was used for the swim test. Mice were subjected to 6 swim trials per day. Assays conducted under photopic (55 lux) and scotopic conditions (0 lux) were performed on days 1 and 2, respectively. Light intensity for photopic and scotopic conditions was measured at the platform using an Extech HD450 light meter (FLIR Systems, Nashua, New Hampshire). Mice were given 90 s to find the platform before being removed. After one trial was performed on all mice, the platform was moved to one of three different locations, top left, top center, and top right in relation to the initial site of mouse placement in tank. Each trial was recorded using an infrared camera (Basler AG, Ahrensburg, Germany) and EthoVision XT16 software (Noldus Information Technology). Locating the platform was considered successful when mice contacted the platform with a head-on approach, even if mice failed to escape onto the platform. Mice were allowed to rest on the platform for 15 s at the end of each trial before being returned to a pre-warmed cage. Latency to find the platform was manually recorded and confirmed using recorded videos. Male and female mice were tested separately, and no sex differences in performance were identified post-hoc using two-way RM-ANOVA (Supplementary Table 1). Male and female data were combined into a single group for each genotype. Latency to platform was calculated by averaging the final 3 trials under scotopic and photopic conditions for each mouse and compared using Kruskal-Wallis one-way ANOVA with Dunn’s posthoc multiple comparison test.
Data analysis
Electrophysiological data were analyzed by custom routines written in IgorPro software (Wavemetrics) and statistical analysis was performed using Prism software (GraphPad). Data were analyzed for normality by Shapiro Wilk test followed by parametric (t-test) or non-parametric methods (Kruskal Wallis or Mann-Whitney).
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Elife9 (2020).Care, R.A., et al.Mature Retina Compensates Functionally for Partial Loss of Rod Photoreceptors. Cell rep31, 107730 (2020).Magupalli, V.G., et al.Multiple RIBEYE-RIBEYE interactions create a dynamic scaffold for the formation of synaptic ribbons. J. Neurosci. 28, 7954–7967 (2008).Ingram, N.T., Sampath, A.P. & Fain, G.L. Voltage-clamp recordings of light responses from wild-type and mutant mouse cone photoreceptors. J. Gen. Physiol. 151, 1287–1299 (2019).Spiwoks-Becker, I., Glas, M., Lasarzik, I. & Vollrath, L. Mouse photoreceptor synaptic ribbons lose and regain material in response to illumination changes. Eur J Neurosci19, 1559–1571 (2004).Regus-Leidig, H., Specht, D., Tom Dieck, S. & Brandstatter, J.H. Stability of active zone components at the photoreceptor ribbon complex. Mol Vis16, 2690–2700 (2010).Sheets, L., Kindt, K.S. & Nicolson, T. Presynaptic CaV1.3 channels regulate synaptic ribbon size and are required for synaptic maintenance in sensory hair cells. J. Neurosci. 32, 17273–17286 (2012).Wong, H.C., et al.Synaptic mitochondria regulate hair-cell synapse size and function. Elife8 (2019).Goel, P., et al.Homeostatic scaling of active zone scaffolds maintains global synaptic strength. J Cell Biol218, 1706–1724 (2019).Hong, H., et al.Structural Remodeling of Active Zones Is Associated with Synaptic Homeostasis. J. Neurosci. 40, 2817–2827 (2020).Williams, B., Maddox, J.W. & Lee, A. Calcium Channels in Retinal Function and Disease. Annu Rev Vis Sci (2022).Macosko, E.Z., et al.Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell161, 1202–1214 (2015).Tringham, E., et al.T-type calcium channel blockers that attenuate thalamic burst firing and suppress absence seizures. Sci Transl Med 4, 121ra119 (2012).Perez-Reyes, E., Van Deusen, A.L. & Vitko, I. Molecular pharmacology of human Cav3.2 T-type Ca2+ channels: block by antihypertensives, antiarrhythmics, and their analogs. The Journal of pharmacology and experimental therapeutics328, 621–627 (2009).Choi, S.Y., Jackman, S., Thoreson, W.B. & Kramer, R.H. Light regulation of Ca2+ in the cone photoreceptor synaptic terminal. Vis Neurosci25, 693–700 (2008).Bijveld, M.M., et al.Assessment of night vision problems in patients with congenital stationary night blindness. PLoS One8, e62927 (2013).Peloquin, J.B., Rehak, R., Doering, C.J. & McRory, J.E. Functional analysis of congenital stationary night blindness type-2 CACNA1F mutations F742C, G1007R, and R1049W. Neuroscience 150, 335-345 (2007).Hoda, J.C., Zaghetto, F., Koschak, A. & Striessnig, J. Congenital stationary night blindness type 2 mutations S229P, G369D, L1068P, and W1440X alter channel gating or functional expression of Cav1.4 L-type Ca2+ channels. J. Neurosci. 25, 252-259 (2005).Kerov, V., et al.alpha2delta-4 Is Required for the Molecular and Structural Organization of Rod and Cone Photoreceptor Synapses. J. Neurosci. 38, 6145–6160 (2018).Lee, A., et al.Characterization of Cav1.4 complexes (α11.4, β2, and α2δ−4) in HEK293T cells and in the retina. J. Biol. Chem. 290, 1505-1521 (2015).Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using microManager. Curr Protoc Mol Biol Chapter14, Unit14 20 (2010).Edelstein, A.D., et al.Advanced methods of microscope control using muManager software. J Biol Methods1 (2014).DeVries, S.H. & Schwartz, E.A. Kainate receptors mediate synaptic transmission between cones and ’Off’ bipolar cells in a mammalian retina. Nature397, 157–160 (1999).
Effect of ML218 on HEK293T cells transfected with Ca<sub>v</sub>1.4, β<sub>2x13</sub>, and α<sub>2</sub>δ−4 or Ca<sub>v</sub>3.2.
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 Ca<sub>v</sub>3-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 I<sub>Ca</sub> 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.
10.7554/eLife.94908.1.sa4eLife AssessmentBroseNilsReviewing EditorMax Planck Institute of Experimental MedicineGöttingenGermanyConvincingIncompleteUseful
Based on analyses of retinae from genetically modified mice, and from wild-type ground squirrel and macaque employing microscopic imaging, electrophysiology, and pharmacological manipulations, this useful study on the role of Cav1.4 calcium channels in cone photoreceptor cells (i) shows that the expression of a Cav1.4 variant lacking calcium conductivity supports the development of cone synapses beyond what is observed in the complete absence of Cav1.4, and (ii) indicates that the cone pathway can partially operate even without calcium flux through Cav1.4 channels, thus preserving behavioral responses under bright light. The evidence for the function of Cav1.4 protein in synapse development is convincing, and in agreement with a closely related earlier study by the same authors on rod photoreceptors, but the evidential support for the notion of a homeostatic compensation of Cav1.4 loss by Cav3 is incomplete. As congenital Cav1.4 dysfunction can cause stationary night blindness, this work relates to a wide range of neuroscience topics, from synapse biology to neuro-ophthalmology.
Cav1.4 calcium channels control voltage-dependent calcium influx at photoreceptor synapses, and congenital loss of Cav1.4 function causes stationary night blindness CSNB2. Based on a broad portfolio of methodological approaches - genetic mouse models, immunolabeling and microscopic imaging, serial block-face-SEM, ERGs, and electrophysiology - the authors show that cone photoreceptor synapse development is strongly perturbed in the absence of Cav1.4 protein, and that expression of a nonconducting Cav1.4 channel mitigates these perturbations. Further data indicate that Cav3 channels are present, which, according to the authors, may compensate for the loss of Cav1.4 calcium currents and thus maintain cone synaptic transmission. These data, which are in agreement with a similar study by the same authors on rod photoreceptor synapses, help to explain what functional defects exactly cause CSNB2 and why it is accompanied by only mild visual impairment.
The strengths of the present study are its conceptual and experimental soundness, the broad spectrum of cutting-edge methodological approaches pursued, and the convincing differential analysis of mutant phenotypes.
Weaknesses mainly concern the experiments and arguments leading to the authors' notion that Cav3 channels may partially compensate for the loss of Cav1.4 calcium currents in cone synapses. It is possible that the non-conducting Cav1.4 variant supports synapse development and the Cav3 channel then provides the calcium influx. However, in its current state, the study does not unequivocally assess Cav3 expression in wild-type cones, it lacks direct evidence of Cav3 expression and upregulation, e.g. via single cell transcriptomics, immunolabeling, or an elaboration on electrophysiology, and it does not test the authors' earlier idea that Cav1.4 might couple to intracellular calcium stores at photoreceptor synapses
This paper by Maddox et al. presents the results of a study of Ca channel function in mouse cone photoreceptor synaptic terminals. It builds on earlier work by the same authors (Maddox et al. 2020 in eLife) which demonstrated that a non-conducting but voltage-sensing variant of Cav1.4 (G369i knock-in, or KI) could substitute for WT Cav1.4 to promote relatively normal rod synapse development despite an inability to support Ca2+-dependent glutamatergic transmission to postsynaptic bipolar cells. Cav1.4 knock-out (KO) rod synapses, however, were completely disorganized, indicating that the presence of Cav1.4 protein is critical for synaptic organization. Here, the authors extend their study of the G369i-KI retina to demonstrate that G369i-KI cones develop working (though disrupted and sometimes aberrant) synapses that support some visual function owing to compensatory expression of Cav3-containing Ca channels that can mediate some Ca2+-dependent transmission from cones to postsynaptic cells. This compensatory expression of a low voltage-activated Ca conductance was not noted previously (Maddox et al. 2020) in G369i-KI rods.
Strengths:
In all, this is a scientifically sound study that shows obvious differences between synaptic terminal morphology and organization, macroscopic Ca currents, transmission to postsynaptic horizontal and bipolar cells (with whole-cell recording and ERG, respectively), and visually-guided behavior in experimental groups.
Weaknesses:
The major criticism that I have of the study is that it infers Ca channel molecular composition based solely on pharmacological analysis, which, as the authors note, is confounded by the cross-reactivity of many of the "specific" channel-type antagonists. The authors note that Cav3 mRNAs have been found in cones, but here, they do not perform any analysis to examine Cav3 transcript expression after G369i-KI nor do they examine Ca channel transcript expression in monkey or squirrel cones, which serve as controls of sorts for the G369i-KI (i.e. like WT mouse cones, cones of these other species do not seem to exhibit LVA Ca currents).
Secondarily, in Maddox et al. 2020, the authors raise the possibility that G369i-KI, by virtue of having a functional voltage-sensing domain-might couple to intracellular Ca2+ stores, and it seems appropriate that this possibility be considered experimentally here.
As a minor point: the authors might wish to note - in comparison to another retinal ribbon synapse-that Zhang et al. 2022 (in J. Neuroscience) performed a study of mouse rod bipolar cells found a number of LVA and HVA Ca conductances in addition to the typical L-type conductance mediated by Cav1-containing channels.
This is an important study that tests the hypothesis that Cav1.4 calcium channels do more than provide a voltage-dependent influx of Ca2+ into photoreceptors. The relevant background can be divided into two tranches. First, deletion of Cav1.4 channels (Cav1.4 knock-out) disrupts rod and cone photoreceptors and their synapses in the outer plexiform layer. Second, knock-in of a non-conducting Cav1.4 channel (Cav1.4 knock-in) partially spares the organization of the outer plexiform layer and photoreceptor synapses (Maddox et al., eLife 2020), which is remarkable considering the disruption of the outer plexiform layer in the Cav1.4 knock-out. In addition, phototransduction, assessed by scotopic and phototopic electroretinography (a-wave amplitude) in the Cav1.4 knock-in retina was partially spared for rods and only slightly impaired for cones. However, the non-conducting Cav1.4 channel of the Cav1.4 knock-in failed to rescue synaptic transmission across the outer retina (electroretinography: b-wave amplitude, Maddox et al., eLife 2020). The 2020 Maddox et al. (eLife) focused more on the rod pathway, while the current work addressed the cone pathway.
Strengths
The study addresses the important question of how disruption of Cav1.4 function in both rod and cone photoreceptors leads to impairment primarily of the rod pathway for scotopic vision. This is clinically relevant as human mutations lead to stationary night blindness rather than blindness. The work relevance provides excellent single-cell electrophysiological recordings of Ca2+ currents from cones of wild-type, Cav1.4 knock-out, and Cav1.4 knock-in mice and, in addition, from ground squirrel and monkey cones. To make these recordings successfully in the various species and the compromised retinas (Cav1.4 knock-out and Cav1.4 knock-in) is very impressive. The findings clearly advance our understanding of Ca2+ channel function in cones. In addition, the study presents high-quality electron microscopy reconstructions of cones and further physiological and behavioral data related to the cone pathway.
Weaknesses
The major critiques are related to the description of the Cav1.4 knock-in mouse as "sparing" function, which can be remedied in part by a simple rewrite, and in certain places, the data may need to be examined more critically. In particular, the authors should address features in the data presented in Figures 6 and 7 that seem to indicate that the retina of the Cav1.4 knock-in is not intact, but the interpretation given by the authors as "intact" is not appropriate and made without rigorous statistical testing.
Cav1.4 voltage-gated calcium channels play an important role in neurotransmission at mammalian photoreceptor synapses. Mutations in the CACNA1f gene lead to congenital stationary night blindness that particularly affects the rod pathway. Mouse Cav1.4 knockout and Cav1.4 knockin models suggest that Cav1.4 is also important for the cone pathway. Deletion of Cav1.4 in the knockout models leads to signaling malfunctions and to abundant morphological re-arrangements of the synapse suggesting that the channel not only has a role in the influx of Ca2+ but also in the morphological organization of the photoreceptor synapse. Of note, also additional Cav-channels have been previously detected in cone synapses by different groups, including L-type Cav1.3 (Wu et al., 2007; pmid; Kersten et al., 2020; pmid), and also T-type Cav3.2 (Davison et al., 2021; pmid 35803735).
In order to study a conductivity-independent role of Cav1.4 in the morphological organization of photoreceptor synapses, the authors generated the knockin (KI) mouse Cav1.4 G369i in a previous study (Maddox et al., eLife 2020; pmid 32940604). The Cav1.4 G369i KI channel no longer works as a Ca2+-conducting channel due to the insertion of a glycine in the pore-forming unit (Madox et al. elife 2020; pmid 32940604). In this previous study (Madox et al. elife 2020; pmid 32940604), the authors analyzed Cav1.4 G369i in rod photoreceptor synapses. In the present study, the authors analyzed cone synapses in this KI mouse.
For this purpose, the authors performed a comprehensive set of experimental methods including immunohistochemistry with antibodies (also with quantitative analyses), electrophysiological measurements of presynaptic Ca2+ currents from cone photoreceptors in the presence/absence of inhibitors of L-type- and T-type- calcium channels, electron microscopy (FIB-SEM), ERG recordings and visual behavior tests of the Cav G369i KI in comparison to the Cav1.4 knockout and wild-type control mice.
The authors found that the non-conducting Cav channel is properly localized in cone synapses and demonstrated that there are no gross morphological alterations (e.g., sprouting of postsynaptic components that are typically observed in the Cav1.4 knockout). These findings demonstrate that cone synaptogenesis relies on the presence Cav1.4 protein but not on its Ca2+ conductivity. This result, obtained at cone synapses in the present study, is similar to the previously reported results observed for rod synapses (Maddox et al., eLife 2020, pmid 32940604). No further mechanistic insights or molecular mechanisms were provided that demonstrated how the presence of the Cav channels could orchestrate the building of the cone synapse.
Strengths:
The study has been expertly performed. A comprehensive set of experimental methods including immunohistochemistry with antibodies (also with quantitative analyses), electrophysiological measurements of presynaptic Ca2+ currents from cone photoreceptors in the presence/absence of inhibitors of L-type- and T-type- calcium channels, electron microscopy (FIB-SEM), ERG recordings and visual behavior tests of the Cav G369i KI in comparison to the Cav1.4 knockout and wild-type control mice.
Weaknesses:
The study has been expertly performed but remains descriptive without deciphering the underlying molecular mechanisms of the observed phenomena, including the proposed homeostatic switch of synaptic calcium channels. Furthermore, a relevant part of the data in the present paper (presence of T-type calcium channels in cone photoreceptors) has already been identified/presented by previous studies of different groups (Macosko et al., 2015; pmid 26000488; Davison et al., 2021; pmid 35803735; Williams et al., 2022; pmid 35650675). The degree of novelty of the present paper thus appears limited.
10.7554/eLife.94908.1.sa5Author Response:MaddoxJ. WesleyAuthorOrdemannGregory J.AuthorVázquezJuan de la RosaAuthorHuangAngieAuthorGaultChristofAuthorWisnerSerena R.AuthorRandallKateAuthorFutagiDaikiAuthorDeVriesSteven H.AuthorHoonMrinaliniAuthorLeeAmyAuthorhttp://orcid.org/0000-0001-8021-0443
We thank the reviewers and editor for their careful analysis of our manuscript and their appreciation of its strengths. Our plans to address the reviewers’ concerns regarding the weaknesses of the study are outlined below.
Reviewing Editor (Public Review):
“Weaknesses mainly concern the experiments and arguments leading to the authors' notion that Cav3 channels may partially compensate for the loss of Cav1.4 calcium currents in cone synapses. It is possible that the non-conducting Cav1.4 variant supports synapse development and the Cav3 channel then provides the calcium influx. However, in its current state, the study does not unequivocally assess Cav3 expression in wild-type cones, it lacks direct evidence of Cav3 expression and upregulation, e.g. via single cell transcriptomics, immunolabeling, or an elaboration on electrophysiology, and it does not test the authors' earlier idea that Cav1.4 might couple to intracellular calcium stores at photoreceptor synapses.”
Current transcriptomic studies indicate that Cav3 transcripts are present at extremely low levels compared to that for Cav1.4 in cones of young mice (PMID 26000488, summarized in PMID 35650675), adult mice (PMID: 36807640), macaque (PMID 30712875), and human (PMID 31075224). Thus, it was somewhat surprising that Davison et al reported the presence of low voltage activated (LVA) Cav3-like currents with amplitudes that were ~50% of that for the Cav1 current in mouse cones at -40 mV (PMID 35803735). Using similar pharmacological criteria as Davison et al, we did not find functional evidence for a LVA current in cones of wild-type (WT) mouse retina: the Ca2+ current in our recordings was suppressed by the Cav1 antagonist isradipine (Fig 3a) but minimally affected in the expected voltage range by the Cav3 antagonist ML218 (Fig 3b). In WT mouse, voltage clamp steps from -90 mV to more depolarized voltages failed to show a transient inward current at onset (Fig 2e), which is a hallmark of LVA calcium currents. In addition, by standard physiological and pharmacological critera, we could not identify LVA currents in cones of ground squirrel (Fig.3c,d) and macaque retina (Supp. Fig.S3). Our results argue against a significant role for LVA currents in mammalian cones.
A problem that we discovered (as did Davison et al, their Fig.2C) was that Cav3 blockers (e.g., ML218 and Z944) have non-specific actions on the high voltage activated (HVA) Ca2+ current (presumably mediated by Cav1.4) in WT mouse cones. This is clearly shown in our Supp. figure S1a-b where ML218 causes a dose-dependent negative shift in the I-V relationship but also inhibition of current density in HEK293T cells transfected with Cav1.4. We are planning a second study to thoroughly characterize these actions of ML218 and Z944 on Cav1 channels as the results are important for understanding the actions of these drugs in cell-types with mixed populations of Cav1 and Cav3 channels.
A second problem is that dihydropyridines (DHP) used in both our study and that of Davison et al (e.g., isradipine, nifedipine) incompletely and slowly block Cav1 channels at negative membrane potentials (PMID: 12853422). Due to the slow kinetics of DHP block, Cav1 currents in the presence of such blockers can appear to inactivate rapidly (see Fig.6A in PMID 11487617). Thus, the Cav current recorded in the presence of DHP blockers in WT mouse cones may represent unblocked Cav1.4-mediated currents that appear rapidly inactivating, and therefore misconstrued as being mediated by Cav3 channels.
Given the caveats of the pharmacological approach, we agree that stronger evidence is needed to rule out a small contribution of Cav3 channels in WT mouse cones. As mentioned in our text, we have found that currently available Cav3 antibodies produce similar patterns of immunofluorescence in WT and corresponding Cav3 KO retina so analysis at the level of Cav proteins is not possible. Thus, we are planning to compare the relative expression of Cav channel genes in cones using drop-seq experiments of G369i KI and WT mouse retina. We also plan to elaborate on our electrophysiological dissection of the HVA and LVA currents.
Among the 3 Cav3 subtypes, Cav3.2 was the only one detected in mouse cones by Davison et al using nested RT-PCR (PMID 35803735). Thus, we obtained the Cav3.2 mouse strain from JAX (B6;129-Cacna1htm1Kcam/J) and generated a Cav3.2 KO/G369i KI double mutant mouse strain. If the Cav3 current that appears in the G369i KI cones is mediated by Cav3.2, then it should be undetectable in cones of the double mutant mice. Moreover, if these Cav3.2 channels contribute to the residual cone synaptic responses in G369i KI mice, then the double mutant mice should be deficient in this regard. We will test these predictions in patch clamp recordings and ERGs.
Finally, we will conduct Ca2+ imaging experiments in cone terminals of the WT vs G369i KI mice to test whether increased coupling of Cav channels to intracellular Ca2+ release may be involved in cone synaptic responses of the G369i KI mice.
Reviewer #1 (Public Review):
Weaknesses:
“The major criticism that I have of the study is that it infers Ca channel molecular composition based solely on pharmacological analysis, which, as the authors note, is confounded by the cross-reactivity of many of the "specific" channel-type antagonists. The authors note that Cav3 mRNAs have been found in cones, but here, they do not perform any analysis to examine Cav3 transcript expression after G369i-KI nor do they examine Ca channel transcript expression in monkey or squirrel cones, which serve as controls of sorts for the G369i-KI (i.e. like WT mouse cones, cones of these other species do not seem to exhibit LVA Ca currents).”
Actually, we also used non-pharmacological (i.e., electrophysiological) criteria to back up our interpretation that Cav3 channels contribute to the Cav current in cones primarily in the absence of functional Cav1.4 channels. For example, in Fig.2, we show that the Ca2+ current in G369i KI and Cav1.4 KO mice exhibit the hallmarks of the Cav3 channel (negative activation and inactivation voltages and window current, rapid inactivation), which are quite distinct from the Ca2+ currents in WT cones. In recordings of ground squirrel and macaque cones (Supp.Figs.S2-3), negative holding voltages do not unmask a LVA current according to various criteria. In addition to the transcriptomic approaches described above, we plan to elaborate on the electrophysiological evidence for the absence of a LVA current in WT mouse cones as part of the revision.
“Secondarily, in Maddox et al. 2020, the authors raise the possibility that G369i-KI, by virtue of having a functional voltage-sensing domain-might couple to intracellular Ca2+ stores, and it seems appropriate that this possibility be considered experimentally here.”
We will conduct Ca2+ imaging experiments in cone terminals of the WT vs G369i KI mice to test whether increased coupling of Cav channels to intracellular Ca2+ release may be involved in cone synaptic responses of the G369i KI mice.
“As a minor point: the authors might wish to note - in comparison to another retinal ribbon synapse-that Zhang et al. 2022 (in J. Neuroscience) performed a study of mouse rod bipolar cells found a number of LVA and HVA Ca conductances in addition to the typical L-type conductance mediated by Cav1-containing channels.”
We are aware of the extensive evidence for the expression of Cav3 channels in retinal bipolar cells (PMID 11604141, 22909426, 19275782, 35896423) and our recordings of cone bipolar cells in ground squirrel confirm this (Supp. Fig.S2D). We could add reference to this work in our revision.
Reviewer #2 (Public Review):
Weaknesses:
“The major critiques are related to the description of the Cav1.4 knock-in mouse as "sparing" function, which can be remedied in part by a simple rewrite, and in certain places, the data may need to be examined more critically. In particular, the authors should address features in the data presented in Figures 6 and 7 that seem to indicate that the retina of the Cav1.4 knock-in is not intact, but the interpretation given by the authors as "intact" is not appropriate and made without rigorous statistical testing.”
We intended to use “sparing” and “intact” to indicate that cone synapses are present and to some extent functional, in contrast to their complete absence in the Cav1.4 KO mouse. However, we recognize this may be misinterpreted as “normal”. As suggested by the reviewer, we will revise our statistical analyses and text to clarify that cone synaptic responses do indeed differ significantly in G369i KI as compared to WT mice. We feel that this will be a strong addition to the study and will emphasize the key point that Cav3 cannot fully compensate for loss of Cav1.4 with respect to cone synapse structure and function.
Reviewer #3 (Public Review):
Weaknesses:
“The study has been expertly performed but remains descriptive without deciphering the underlying molecular mechanisms of the observed phenomena, including the proposed homeostatic switch of synaptic calcium channels. Furthermore, a relevant part of the data in the present paper (presence of T-type calcium channels in cone photoreceptors) has already been identified/presented by previous studies of different groups (Macosko et al., 2015; pmid 26000488; Davison et al., 2021; pmid 35803735; Williams et al., 2022; pmid 35650675). The degree of novelty of the present paper thus appears limited.”
We respectfully disagree that our paper lacks novelty. As indicated by Reviewer 2, a major advance of our study is in providing a mechanism that can explain the longstanding conundrum that congenital stationary night blindness type 2 mutations that would be expected to severely compromise Cav1.4 function do not produce complete blindness. We also disagree that the presence of T-type channels in cone photoreceptors has been unequivocally demonstrated, as the non-biased transcriptomic approaches show very little Cav3 transcript expression in mouse cones (PMIDs 26000488, 35650675, 36807640), macaque cones (PMID 30712875), and human cones (PMID 31075224). Transcription may not equate to translation, particularly at low expression levels. We also note that the one study to date that suggests a functional contribution of Cav3 channels in mouse cones (Davison et al., 2021; pmid 35803735) used a DHP to isolate the “LVA” current, which is problematic as described above. Our demonstration of minimal or undetectable Cav3-type currents in mammalian cones using physiological and pharmacological approaches, while a negative result, adds important context to the recent literature. As described in our response to the editor’s review, our planned revisions include testing whether Cav3 transcripts are upregulated in G369i KI cones and whether the Cav3.2 subtype suggested to be present in cones (PMID 35803735) contributes to Cav currents in these cells using Cav3.2 KO and Cav3.2 KO/G369i KI double mutant mice.