A non-conducting role of the Cav1.4 Ca2+ channel drives homeostatic plasticity at the cone photoreceptor synapse

  1. J Wesley Maddox
  2. Gregory J Ordemann
  3. Juan de la Rosa Vázquez
  4. Angie Huang
  5. Christof Gault
  6. Serena R Wisner
  7. Kate Randall
  8. Daiki Futagi
  9. Nihal A Salem
  10. R Dayne Mayfield
  11. Boris V Zemelman
  12. Steven H DeVries
  13. Mrinalini Hoon
  14. Amy Lee

  • Curated by eLife

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    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 valuable 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. The mechanism of compensation of Cav1.4 loss by Cav3 remains unclear but appears to involve post-transcriptional processes. 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.

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Abstract

In congenital stationary night blindness type 2 (CSNB2)—a disorder involving the Ca v 1.4 (L-type) Ca 2+ channel—visual impairment is mild considering that Ca v 1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Ca v 1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Ca v 1.4. Surprisingly, Ca v 3 (T-type) Ca 2+ currents were detected in cones of G369i KI mice and Ca v 1.4 KO mice but not in cones of wild-type mouse, ground squirrel, and macaque retina. Whereas Ca v 1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e., cone-mediated) visual behavior. Cone synapses, which fail to form in Ca v 1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Ca v 1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Ca v 3 channels maintain cone synaptic output provided that the nonconducting role of Ca v 1.4 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.

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  1. eLife

    eLife assessment

    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 valuable 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. The mechanism of compensation of Cav1.4 loss by Cav3 remains unclear but appears to involve post-transcriptional processes. 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.

  2. eLife

    Public Review (Joint Version of all Reviewers)

    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 fact that the mechanism by which Cav3 channels might partially compensate for the loss of Cav1.4 calcium currents remains unclear.

  3. eLife

    Author response:

    The following is the authors’ response to the previous reviews.

    Intro.

    47-48 rewrite sentence

    This sentence has been rewritten as: Photoreceptor synapses are specialized with a vesicle-associated ribbon organelle and postsynaptic neurites of horizontal and bipolar cells that invaginate deep within the terminal

    Results

    Major comment. Lines 100-103

    The new rod data presented here looks like an n = 1. Neither the Results section nor Supp Fig S1, describe the number of cells used. Nor do the authors offer a statistical description with averages, etc.. In addition, the single traces are much improved over their previous study (Maddox et al eLife 2020), but the authors have not described any new approach or trick that improved their rod Ica. Neither Methods section nor Supp section describes the procedure for patching rods (solutions, or Vh which is critical for assessing T-type currents).

    Suggestion, if more data exists, then present it. Otherwise, drop the argument.

    The recording methodology for recording rods was like that for cones and this has been clarified in the Methods section (lines 725-752). Averaged data (n= at least 5 per group) and statistical analyses have been added to Fig.S1 (renamed Figure 2-Figure Supplement 1), and clearly show that no Ca2+ currents are present in the KI rods.

    Supp Fig S2. The legend needs to be fixed. Conversion to PDF file may have created these formatting errors.

    This has been corrected (renamed Figure 3-Figure Supplement 2).

    Fig 8 a. The position of the light stimulus bar in the KO panel appears to be out of place, shifted too far to the left.

    This has been corrected.

    Major comments. 219-221

    The use of Fluo3-AM is not properly stated here. The text reads "cone pedicles filled with the Ca2+ indicator Fluo3". The wording used could be wrongly interpreted as: whole-cell filling of the cones via patch electrode. However, the Methods section describes bathing the retina in Fluo3-AM, which presumably fills PRs, HCs tips, Mueller glia and bpc dendrites. The Results section should acknowledge that the retina was loaded with Fluo3-AM.

    The cell types, and their processes (Muellers, HCs, bpc, PRs), present in a cone pedicle ROI will likely contribute to the Fluo3 readout of Ca2+ in the OPL, because 1) the EM images in Fig 7 highlight how interdigitated the processes are with the presynapse, 2) all express Cav channels, and many if not all express L-Type Cavs in their processes (glia, HC, on-bcs and PRs), and 3) all are depolarized with the addition of high extracellular KCl. The inclusion of Isradipine will inhibit L-type Cavs on pre- and post-synaptic targets, failing to specifically isolate PR Ca2+. Furthermore, Glu Receptor blockers are used here, which would be a great idea if the cones were stimulated with light; however, KCl bypasses the excitatory synaptic pathway and depolarizes all processes within the ROI. Hence, all cellular parts in the ROI will potentially contribute to Fluo3-Ca2+ signals.

    Suggestions for presentation of these findings. Ultimately your conclusion is suitable " 233 to 234...... Taken together, our results suggest that Cav3 channels nominally support Ca2+ signals and synaptic transmission in cones of G369i KI mice". The dramatic reduction in Fluo3-Ca2+ signals in the OPL G369i retinas (Fig 9) is a valuable finding for the following reasons: 1) the results do not show a clear compensation from intracellular stores that could potentially supersede the T-type currents in the G369i (which is an argument you make), and 2) there is a massive loss of Ca2+ influx in the OPL of G369i retinas. Since G369i is specific to the PRs, and only cones are present in the mutant G369i, the loss of Fluo3-Ca2+ signal in the mutant ROI reflects in large part loss of cone Fluo3-Ca2+ signals. Your findings illustrate the severity of the mutation, which has also been addressed in the various electro-physio sections of the MS.

    Figure 9 also needs to be more clear about 1) the loading of the cells with AM-dye, and 2) the presence of glia, HCs and bc dendrites in the PNA demarcated ROIs.

    We regret that we did not make this more clear, but our Fluo 3 loading protocol of whole retina followed by vertical slicing allowed for loading primarily of photoreceptors in the portion of the outer retina that we imaged. We clarified this with the following edit to the text (lines 220-226):

    “To test if the diminished HC light responses correlated with lower presynaptic Ca2+ signals in G369i KI cones, we performed 2-photon imaging of vertical slices prepared from whole retina that was incubated with the Ca2+ indicator Fluo3-AM and Alexa-568-conjugated peanut agglutinin (PNA) to demarcate regions of interest (ROIs) corresponding to cone pedicles. With this approach Fluo3 fluorescence was detected only in photoreceptors and ganglion cells and not inner retinal cell-types (e.g., horizontal cells, bipolar cells, Mueller cell soma). Thus, Ca2+ signals reported by Fluo3 fluorescence near PNA-labeling originated primarily from cones.”

    We also note that given the considerably larger volume of the cone pedicle relative to the postsynaptic neurites of horizontal and bipolar cells, as well as neighboring glia, it seems unlikely that the latter would contribute significantly to the isradipine-sensitive Ca2+ signal measured in the ROI above the PNA labeling. Moreover, to our knowledge the contribution of Cav1 L-type channels to postsynaptic Ca2+ signals in the dendritic tips of horizontal cells and bipolar cells has not been demonstrated.

  4. Version published to 10.7554/elife.94908.3 on eLife
  5. Version published to 10.7554/elife.94908 on eLife
  6. Version published to 10.7554/elife.94908.2 on eLife
  7. eLife

    eLife assessment

    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 valuable 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. The mechanism of compensation of Cav1.4 loss by Cav3 remains unclear but appears to involve post-transcriptional processes. 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.

  8. eLife

    Joint Public Review

    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 mechanism by which Cav3 channels might partially compensate for the loss of Cav1.4 calcium currents.

  9. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Reviewer #1 (Recommendations For The Authors):

    The authors should perform experiments to answer this question: does Cav3 transcription increase in the G369i-KI, or is there instead some post-transcriptional modulation that permits surface expression of functional Cav3-containing channels in the absence of typical HVA Ca conductances? Also, the authors should determine whether G369i-KI can mediate Ca2+ release from intracellular stores and whether release from stores is upregulated as Cav3-containing channel expression (or function) is increased.

    We performed transcriptomic (drop-seq) analysis to test whether a Cav3 subtype is upregulated in cones of G369i KI mice. These experiments show that, consistent with previous studies (PMID 35803735, 26000488), Cacna1h appears to be the primary Cav3 subtype expressed mouse cones. However, as shown in new Supp.Fig.S3, there was no significant difference in the levels of Cacna1h transcripts in WT and G369i KI cones. Therefore, we propose that there may be some post-transcriptional modification, or alteration in a pathway that regulates channel availability, that enables the contribution Cav3 channels to the whole-cell Ca2+ current in the absence of functional Cav1.4 channels cones.

    We also performed Ca2+ imaging experiments in WT vs G369i KI cone terminals to assess whether the diminutive Cav3 current in G369i KI cone terminals may be compensated by upregulation of a Ca2+ signal such as from intracellular stores. Arguing against this possibility, depolarization-evoked Ca2+ signals in G369i KI cones were dramatically reduced compared to WT cones (new Fig.9).

    Reviewer #2 (Recommendations For The Authors):

    Major points-

    (1) It is stated in too many places that cone features in the Cav1.4 knock-in are "intact", preserved, or spared, but this representation is not accurate. There are two instances in this study that qualify as intact when comparing KI to WT: 1) the photopic a-waves in the Cav1.4 knock-in (also demonstrated in Maddox et al 2020) and 2) latency to the platform (current MS, Figure 7f). However, in the numerous instances listed below, the authors compared the Cav1.4 knock-in to the Cav1.4 knock-out, and then referred to the KI as exhibiting intact responses. The reference point for intactness needs to be wildtype, as appropriately done for Figures 2 and 3, and when comparing the KI to the KO the phrasing should be altered; for example: "the KI was spared from the extensive degeneration witnessed in the KO....".

    In most cases, we clearly note that there are key differences in the WT and the G369i KI cone synapses, which highlight the importance of Cav1.4-specific Ca2+ signals for certain aspects of the cone synapse. We disagree with the reviewer on the point that we did not often use the WT as a reference since most of our experiments involved comparisons of only WT and G369i KI (Figs. 3-6) or WT, G369i KI, and Cav1.4 KO (Figs.1,7—and in these cases comparisons specifically between WT and G369i KI mice were included). We used “intact” as a descriptor for G369i KI cone synapses since these are actually present, albeit abnormal in the G369i KI retina, whereas cone synapses are completely absent in the Cav1.4 KO retina. To avoid confusion, we modified our use of “intact” and “preserved” where appropriate.

    A. Abstract, line 34 to 35: ".......preserved in KI but not in KO.".

    Abstract was rewritten and this line was removed.

    B. Line 36: "....synaptogenesis remains intact". The MS documents many differences in the morphology of KI and WT cones (immunofluorescence and electron microscopy data), which is counter to an intact phenotype.

    The sentence was: “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.”

    Here the meaning of “intact” refers to the Ca2+ -independent role of Cav1.4, not synapses. Thus, we have left the sentence unchanged.

    C. This strikes the right balance, lines 67 to 68: "....although greatly impaired.....".

    D. Line 149, "Cone signaling to a postsynaptic partner is intact in G369i KI mice". This description is inaccurate. Here there is only WT and KI, and the text reads as follows in line 162: "terminals (Figure 6b). The ON and OFF components of EPSCs in G369i KI HCs were measurable, although lower in amplitude than in WT (Figure 6a,b)." Neither "measurable" nor "lower in amplitude" meet the definition of "intact", and actual numerical values are lacking in the text.

    We have added results showing that there are no light responses in the Cav1.4 KO horizontal cells and have modified the sentence to: “Cone synaptic responses are present in horizontal cells of G369i KI but not Cav1.4 KO mice”.

    We have modified discussion of these results as (line 210-213): “Consistent with the lack of mature ribbons and abnormal cone pedicles (Fig.1), HC light responses were negligible in Cav1.4 KO mice (Fig.8a,b). In contrast, the ON and OFF responses were present in G369i KI HCs although significantly lower in amplitude than in WT HCs (Fig. 8a,b).”

    E. Please add a legend to Figure 6a to indicate the intensities. The shape of the KI responses is different from the control which is worthy of discussion: i) there is no clear cessation of HC EPSCs in the KI during the light ON period (when release stops, Im fluctuations should be minimal), and ii) the "peaked" appearances of the initial 500ms of the On and Off periods are very similar in shape for the KI (hard to interpret in the same fashion as a control response). How were the On and Off amplitudes analyzed? Furthermore, the OFF current is not summarized in Figure 6D, but should not this be when Cav3 should be opening and triggering release: Off response-EPSC? Lastly, Figure 6b,d shows a ~70% reduction in On-current in the KI, and the KI example of 6b an 80% reduction in Off current compared to WT. Yet, the only place asterisks are used to indicate sig diff is the DNQX data within each genotype in Fig 6d. These data cannot be described as showing "intact" KI responses, and the absence of numerical and statistical values needs to be addressed.

    New Fig.8a depicting the horizontal cell light responses has been modified to include the legend indicating light intensities. The ON and OFF amplitudes were analyzed as the peak current amplitudes. This information has been added to the legend.

    The reviewer is correct in that the OFF response represents the EPSC whereas the ON response represents the decrease in the EPSC with light. To avoid confusion, we changed the y axis label for the averaged data to read ON or OFF “response” rather than “current” in new Fig.8b.

    As the reviewer suggests, the more transient nature of the KI response during the light ON period could result from aberrant continuation of vesicular release during the light-induced hyperpolarization of cones in the KI mice, in contrast to the prolonged suppression of release by light which is evident in the WT responses. We speculated on this difference as follows (lines 237-241):

    “In addition to its smaller amplitude, the transient nature of the ON response in G369i KI HCs suggested inadequate cessation of cone glutamate release by light (Fig.8b). Slow deactivation of Cav3 channels and/or their activation at negative voltages20 could give rise to Ca2+ signals that support release following light-induced hyperpolarization of G369i KI cones.”

    We added astericks to new Fig.8b,d indicating statistical differences and description of the tests in the legend.

    F. line 168 the section titled "Light responses of bipolar cells and visual behavior is spared in G369i KI but not Cav1.4 KO mice".

    Changed to: “Light responses of bipolar cells and visual behavior are present in G369i KI but not Cav1.4 KO mice”

    Last sentence of erg results, 189-190: "These results suggest that cone-to-CBC signaling is intact in G369i KI mice.". "Spared and intact" are not accurate descriptions. The ERG data presented here shows massive differences between WT and the KI, except in the instance of awaves.

    This sentence was removed.

    As for Figure 6, the results text related to Figure 7a-d does not present real numbers for ERG responses, and there is no indication of significant differences there or in the Figure panels. For instance, in Figure 7b, b-waves are KI are comparable to KO, except at the two highest-intensity flashes that show KI responses ~20% the amplitude of WT. Presentation of KI and KO data on a 6- to 10-fold expanded scale higher than WT can be misleading: a quick read of these Figure panels might make one incorrectly conclude that the KI is intact while the KO is impaired when compared to WT. The Methods section needs more details on the ERG analysis (e.g. any filtering out of oscillatory potentials when measuring b-wave, and what was the allowable range of time-to-peak for b-wave amplitude, etc..).

    The vertical scaling of the ERG results in new Fig.10c,d has been changed so as to reflect clearly diminished responses of the KO and KI vs the WT. Further details regarding the ERG analysis was added to the Methods section.

    G. Can you point to other studies that have used the "visible platform swim test" used in Figure 7e, f, and specify further how mice were dark/light adapted prior to the recordings?

    As referenced in the Methods, original line 674, the methods we used for the swim test were described in our previous study (PMID 29875267). Other studies that have used this assay include PMIDs: 28262416, 26402607.

    (2) The Maddox et al 2020 study does not safely address whether rods have a residual T-type Ca2+ current in the Cav 1.4 KO or KI. The study showed that membrane currents measured from rods in the KI and KO retina were distinct from WT, supporting their claim that L-type Ca2+ current is absent in the KI and KO. However, the recordings had shortcomings that challenge the analysis of Ca2+ currents: i) collected at room temp (22-24{degree sign}C), ii) at an unknown distance from the terminal (uncertain voltage clamp), iii) with a very slow voltage ramp rate that is not suitable for probing T-type currents (Figure 1d Maddox 2020, 140 mV over 1 sec: 7msec/1mV), and iv) at a signal-to-noise that does not allow to resolve a membrane current under 1 pA (avg wt rod Ca2+ current was -3.5 pA, and line noise ~1pA peak-to-peak in Maddox 2020). Suggestion: say T-type currents were not probed in Maddox et al 2020, but Davison et al 2022 did not find PCR signal for Cav3.2 in rods.

    We disagree that recordings in the Maddox 2020 study were not sufficient to uncover a T-type current. The voltage ramps in that study were not much slower than that of the Davison et al. 2022 study (they used 0.19 mV/ms). Moreover, in new Supp. Fig.S1, we show that like the slower voltage ramp (0.15 mV/ms) used in the prior study of G369i KI rods, the voltage ramps we used in the present study (0.5 mV/ms), which clearly evoke currents with T-type properties in G369i KI cones (Fig.2a,b, Fig.3a,b) do not evoke currents in WT or G369i KI rods.

    Minor comments.

    (1) Suggestion: add an overview panel to Figure 1 that shows the rod terminals in the KI. The problem is that cropping out the ribbon and active zone signals from rods, to highlight cones, can give the impression that the cones are partially spared in the KI, and the rods are not spared at all. (yet you nicely clarify this in Figure 4 and in the legend and text, etc.).

    We chose to modify the legend with this information as in Fig.4 rather than modify the figure.

    (2) Mouse wt cone Ca2+ currents look like L-type currents, as do your monkey and squirrel cone recordings, and also much like those of mouse rods (see Figure S5, Hagiwara et al., 2018 or Grabner and Moser 2021). Your pharm data from mice and squirrels further supports your conclusion, and certainly took much effort. Davison et al 2022 J Neurosci showed PCR results that support their claim that a Cav3 current exists in wt cones. Questions: 1) have you tried PCR? 2) Can you offer more details on what Cav3 KO you tried and what antibodies failed to confirm the KO? As the authors know, one complication is that the deletion of one Cav can be compensated for by the expression of a new Cav. There are 3 types of Cav3s and removal of one type may be compensated for by another Cav3.

    We have included drop-seq data (new Supp.Fig.S3) implicating Cav3.2 as the main Cav3 subtype in cones and have modified our discussion of these results accordingly. These experiments did not reveal any changes in Cav3 subtype expression in G369i KI vs WT cones.

    (3) Lines 95/96- onward, spend more time telling the story. When working out the biophysical and pharmacological behavior of the Ca2+ currents, you might want to initially refer to the membrane current as a membrane current, and then state how your voltage protocols, intra- and extra-cell solutions, and drugs helped you verify 1) L-type and 2) T-type Ca2+ currents.

    We have modified the text with more detail.

    (4) If data is in hand, add a ramp I-V to Figure S2, which shows the response of the ground squirrel cone. The steps in S2a are excellent for making your point that a transient current is missing, and the bipolar is a great control to illustrate ML218 works. However, a comparison of a squirrel cone ramp to a bipolar ramp response could complete the figure.

    See Reponse to #5 below.

    (5) Consider moving Supplementary Figures S2 and S3 to the main text; these are highly relevant to the story, novel, and well-executed.

    Fig.S2 and S3 were added as new Figs.4,5. The new Fig.4 includes voltage ramps in ground squirrel cones (panel a) to compare with the bipolar data (panel f).

    (6) The nice electron microscopy reconstructions are not elaborated on in any detail, and there is no mention of ribbon size. Is the resolution sufficient to estimate ribbon size, the number of synaptic vesicles around the ribbon and in the adjacent cytosol? The images indicate major changes in the morphology of the terminals. Is the glial envelope similar in WT and KI?

    Since ribbons were quantified extensively in the confocal analyses in Fig.6, we felt it unnecessary to add this to the EM analysis which focused mainly on aspects of 3D structure (i.e., arrangement of ribbons, postsynaptic wiring, cone pedicle morphology). We added further discussion of the change in morphology of the G369i KI cone pedicle (lines 200-203): “Compared to WT, ribbons in G369i KI pedicles appeared disorganized and were often parallel rather than perpendicular to the presynaptic membrane (Fig.7a-c). Consistent with our confocal analyses (Fig.1), G369i KI cone pedicles extended telodendria in multiple directions rather than just apically (Fig. 7a).”

    While we did not opt to characterize the glial envelope in WT cones, we did add an analysis of synaptic vesicles around ribbons to Table 2.

    (7) Discussion line 250: "we found no evidence for a functional contribution of Cav3 in our recordings of cones in WT mice (Figures. 2,3), ground squirrels, or macaque (Supplementary Figures S2 and S3).". I would not use "functional" in this context because when comparing your work to Davison et al 2022, they defined functional as a separate response component driven by Cav3. For instance, they examined the influence of their T-type current on exocytosis (by membrane capacitance) and other features like spiking Ca2+ transients. Suggestion: substitute functional with "detectable", and say "we found no detectable Cav currents". Or if you had Ttype staining, but not T-type Ca2+ currents, then say "no functional current even though there is staining...".

    We have modified the text as (lines 336-338): “However, in contrast to recordings of WT mouse cone pedicles in a previous study21, we found no evidence for Cav3-mediated currents in somatic recordings of cones in WT mice (Figs.2,3).”

    We propose an alternative interpretation of the results in the Davison et al study concerning the conclusion that Cav3 channels contribute to Ca2+ spikes and exocytosis. That study used 100 µM Ni2+ to block a “T-type” contribution to spike activity in cones. In their Figs.4,5, the spikes are suppressed by 100 µM Ni2+ and 10 µM nifedipine, a Cav1 antagonist, and spared by the T-type selective drug Z944. This is problematic for several reasons. First, as shown by the authors

    (their Fig.2A1,A2) and others (PMID: 15541900), 100 µM Ni2+ inhibits Cav1-type currents in photoreceptors. Second, Z944 potentiates Cav1 current in their mouse cones (their Fig.2C1,C2). Thus, both reagents are suboptimal for dissecting the contribution of either Cav subtype to spiking activity. With respect to Cav3 channels and exocytosis, these authors interpreted a reduction in exocytosis upon holding at -39 mV compared to at -69 mV as indicating a loss of a T-type driven component of release. However, Cav1 channel inactivation (PMID: 12473074) could lead to the observed reduction in exocytosis at -30 mV.

    (8) Additional literature related to your Intro and Discussion. Regarding CSNB2, related mutations of active zone proteins, and what happens to Ca2+ currents when ribbons are deleted, you might want to consider the following studies that measure Ca2+ currents from rods: conditional KO of RIM1/2 (Grabner et al 2015 JN), KO of ELKS1/2 (Hagiwara et al, 2018 JCB), and KO of Ribeye (Grabner and Moser eLife 2021). In these studies, the Cav currents were absent in rods of the ELKS1/2 DKO, strongly reduced (80%) in the RIM1/2DKO, but altered in more subtle ways (activation-inactivation) without significantly changing steady-state Ca2+ current in the Ribeye KO. This does not seem to support some of the arguments you have made in the Introduction and Discussion regarding ribbon size and Ca2+ currents, yet the suggested literature is related to the topic at hand.

    A description of these synaptic proteins as potential mediators of the effect of Cav1.4 on ribbon morphogenesis was added to the Discussion, lines 325-327.

    (9) Line 129: "Along with the major constituents of the ribbon, CtBP2, and RIBEYE", for clarity Ribeye has two domains, one that is identical to CtBP2 (B-domain) and the unique Ribeye domain (A-domain) that is only expressed at ribbon synapses. And, Piccolino is also embedded in the ribbon (Brandstaetter lab, Wichmann/Moser labs). In other words, Ribeye and Piccolino are the major constituents of the ribbon.

    To avoid confusion, we simply mention Ctbp2 and RIBEYE in the context of the corresponding antibodies that were used to label ribbons.

    (10) Abstract: consider to rephrase "Ca2+-independent role of Cav1.4" by "Ca2+-permeationindependent role of Cav1.4" or alike

    Sentence changed to: “In CSNB2, we propose that Cav3 channels maintain cone synaptic output provided that the nonconducting role of Cav1.4 in cone synaptogenesis remains intact.”

    Reviewer #3 (Recommendations For The Authors):

    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 of 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.

    We respectfully disagree regarding the mechanistic advance of our study. 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. Our study provides an important contrast to the Maddox et al 2020 study in showing that rods and cones respond differentially to loss of Cav1.4 function, which is also relevant to the visual phenotypes of CSNB2. How the presence of Cav1.4 orchestrates cone synaptogenesis is an important topic that is outside the scope of our present study.

    In the present study, the authors also propose a homeostatic switch from L-type to (newly occurring) T-type calcium channels in the Cav1.4 G369i KI mouse as a consequence of the deficient calcium channel conductivity in the Cav1.4 G369i Cav1.4 KI mouse. In cones of the Cav1.4 G369i, the high-voltage activated, L-type Ca2+-entry was abolished, in agreement with their previous paper (Maddox et al., eLife 2020, pmid 32940604). The authors found a lowvoltage activated Ca2+ current instead that they assigned to T-type Ca2+-currents based on pharmacological inhibitor experiments. T-type Ca2+-currents/channels were already previously identified in other studies by independent groups and independent techniques

    (electrophysiology, RT-PCR, single-cell sequencing) in cones of wild-type mice (Davison et al.,

    2021, pmid 35803735; Macosko et al., 2015, pmid 26000488; Williams et al., 2022, pmid 35650675). In the present manuscript (Figures 3a/b), the authors also observed a low-voltage activated, T-type like current in cones of wild-type mice, that is isradipine-resistant and affected by the T-type inhibitor ML218. This finding appears compatible with a T-type-like current in wildtype cones and is consistent with the published data mentioned above, although the authors interpret this data in a different way in the discussion.

    Due to the noise inherent in whole cell voltage clamp measurements and some crossover effects in the pharmacology, we cannot completely exclude the presence of a T-type current in WT mouse cones. However, our results very clearly support a conclusion opposite to that stated by the reviewer. Namely, if WT mouse cones have T-type Ca currents, then they are far smaller than those in the Cav1.4 G369i KI and KO cones. In particular, while we identified message for Cav3.2 in WT mouse cones, we were unable to identify a functional T-type current by either voltage clamp measurements or pharmacology. See below for a detailed rebuttal.

    This proposal of a homeostatic switch is not convincingly supported in this reviewer's opinion

    (for further details, please see below). Furthermore, no data on possible molecular mechanisms were provided that would support such a proposal of a homeostatic switch of calcium channels. No mechanistic/molecular insights were provided for a proposed homeostatic switch between Ltype to T-type channels that the authors propose to occur between wild-type and Cav1.4 G369i as a consequence of conduction-deficient Cav1.4 G369i channels. Is this e.g. based on posttranslational modifications that switch on T-type channels or regulation at the transcriptional level inducing expression of T-type calcium channel or on other mechanisms? The authors remain descriptive with their central hypotheses. No molecular mechanisms/signaling pathways were provided that would support the idea of such a homeostatic switch.

    Homeostatic plasticity refers to the maintenance of neuronal function in response to some perturbation in neuronal activity and can result from changes in the expression of ion channel genes (PMID: 36377048, 32747440, 19778903) or regulatory pathways that modulate ion channels (PMID: 15051886, 32492405). We present multiple lines of evidence showing that Cav3 currents appear in cones upon genetically induced Cav1.4 loss of function and can support cone synaptic responses and visual behavior if cone synapse structure is maintained. Our new transcriptomic studies show no difference between levels of Cav3 channel transcripts in WT and G369i KI cones, suggesting that the appearance of the Cav3 currents in G369i KI cones does not result from an increase in Cav3 gene expression. We are currently investigating our transcriptomic dataset to determine if Cav3 regulatory pathways are upregulated in G369i KI cones and will present this in a follow-up study.

    The authors show residual photopic signaling in the non-conducting Cav1.4 G369i KI mouse as judged by the recording of postsynaptic currents, ERG recordings and visual behavior tests though in a reduced manner. The residual cone-based signaling could be based on the nonaffected T-type Ca2+ channel conductivity in cone synapses. Given that the L-type current through Cav1.4 is gone in the Cav1.4 G369i KI as previously shown (Maddox et al., 2020, pmid 32940604), the T-type calcium current will remain. However as discussed above, this does not necessarily support the idea of a homeostatic switch.

    A major point which we highlighted with new results is that despite the expression of Cav3 transcripts in WT mouse cones, Cav3 channels do not contribute to the cone Ca2+ current. This is at odds with the Davison et al study (PMID: 35803735, see our response to Reviewer 2, pt 7 for caveats of this study), but our results convincingly show that the Cav3 current appears only when Cav1.4 is genetically inactivated. Pharmacological or electrophysiological methods that should reveal the presence of Cav3 currents do not change the properties of the Ca2+ current in cones of WT mice, ground squirrel, or macaque:

    • Figs.2-4: Voltage steps to -40 mV (Fig 2e) that activate a sizeable T-current in G369i KI mouse cones produce a negligible transient at pulse onset in WT mouse cones. Similarly, transient currents that are obvious in G369i KI mouse cones during the final step to -30 mV are absent in WT cones. When we block Cav1.4 with isradipine either in cones of WT mice or ground squirrel, the current that remains does not resemble a Cav3 current but rather a scaled down version of the L-type current. ML218, which readily blocks Cav3 channels in HEK293T cells and in G369i KI cones, has only minor effects in cones of WT mice and ground squirrel; these effects of ML218 can be attributed to non-specific actions on Cav1.4 (new Supp.Fig.S2). New Fig.4 (moved from the supplementary data to the main article) clearly shows that the ML218-sensitive current in ground squirrel cones exhibits properties of Cav1.4 not Cav3 channels.

    • Figs.2,5: Holding voltages that inactivate Cav3 channels have no effect on the Ca2+ current in cones of WT mice or macaque (recordings of macaque cones were moved from the supplement to the main article as new Fig.5).

    In Figure 4 the authors measured an increase in the size of the active zone (as judged by the size of the bassoon cluster) and of the synaptic ribbons in the Cav1.4 G369i. A mechanistic explanation for this phenomenon was not provided and the underlying molecular mechanisms were not unraveled.

    The FIB-SEM data uncover some ultrastructural alteration/misalignments of the synaptic ribbons and misalignments of the regular arrangement of the postsynaptic dendrites in the G369i KI mice. Also concerning this observation, the study remains descriptive and does not reveal the underlying mechanisms as it would be expected for eLife.

    We respectfully disagree on the descriptive nature of our study and the need for a full characterization of the molecular mechanism underlying the cone synaptic defects in the G369i KI mouse.

    An important study in the field (Zanetti et al., Sci. Rep. 2021; pmid 33526839) should be also cited that used a gain-of-function mutation of Cav1.4 to analyze its functional and structural role in the cone pathway.

    We have added citation of this paper to the Discussion (lines 354-356).

    In conclusion, 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. I think that the study might be better suited in a more specialized journal than eLife.

    We thank the reviewer for acknowledging the rigor of our study but disagree with their evaluation regarding the novelty of our work as outlined in our responses above.

  10. Version published to 10.1101/2023.12.05.570129v3 on bioRxiv
  11. Version published to 10.1101/2023.12.05.570129v2 on bioRxiv
  12. eLife

    Author Response:

    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.

  13. Version published to 10.7554/elife.94908.1 on eLife
  14. eLife

    eLife assessment

    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.

  15. eLife

    Reviewer #1 (Public Review)

    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

  16. eLife

    Reviewer #2 (Public Review)

    Summary:
    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.

  17. eLife

    Reviewer #3 (Public Review)

    Summary
    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.

  18. eLife

    Reviewer #4 (Public Review)

    Summary:
    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.

  19. Version published to 10.1101/2023.12.05.570129v1 on bioRxiv