A non-conducting role of the Ca_v1.4 Ca²⁺ channel drives homeostatic plasticity at the cone photoreceptor synapse

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

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.

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.

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.

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.