It is well established that the primary function of voltage-gated Ca_v Ca²⁺ channels within axon terminals is to serve as a conduit for Ca²⁺ ions that trigger the fusion of neurotransmitter-laden vesicles with the presynaptic membrane (reviewed in Dolphin and Lee, 2020). Mutations in genes encoding presynaptic Ca_v channels cause neurological disorders such as epilepsy, migraine, and ataxia (Pietrobon, 2010). When studied in animal models, these mutations lead to aberrant synaptic transmission and alterations in network excitability (Tottene et al., 2009; Eikermann-Haerter et al., 2011). Due to the steep Ca²⁺-dependence of neurotransmitter release (Dodge and Rahamimoff, 1967), factors that modulate Ca_v channel function can dramatically alter synaptic output. Thus, efforts to understand the pathophysiological consequences of disease-causing mutations have largely centered on how they affect biophysical parameters of channel opening, closing, or inactivation (Pietrobon, 2010).

However, Ca_v channels are known to engage in pathways that do not involve their Ca²⁺ conductance. For example, voltage-dependent changes in the conformation of Ca_v1.1 channels, rather than the incoming Ca²⁺ ions, initiate skeletal muscle contraction (Adams et al., 1990). Despite its importance for understanding fundamental cell biological processes in health and disease, the prevalence of non-conducting roles of Ca_v channels in neurons is poorly understood. Analyses of Ca_v knockout (KO) mice have helped illuminate the physiological functions of Ca_v channels (Pietrobon, 2005), however, this approach does not distinguish between the Ca²⁺-dependent and Ca²⁺-independent contributions of these channels. Moreover, complex double and triple-KO approaches are needed given that most synapses in the nervous system utilize more than one Ca_v subtype (Dolphin and Lee, 2020).

To overcome these hurdles, we focused on the Ca_v1.4 L-type channel, which is the predominant Ca_v subtype in rod and cone photoreceptors in the retina (Mansergh et al., 2005; Chang et al., 2006; Liu et al., 2013). More than 140 mutations in the human CACNA1F gene encoding Ca_v1.4 have been identified, many of which cause heterogeneous forms of vision impairment (Waldner et al., 2018). The absence of photoreceptor synaptic responses in Ca_v1.4 KO mice has been attributed to a requirement for Ca_v1.4 in the presynaptic release of glutamate (Mansergh et al., 2005; Chang et al., 2006; Liu et al., 2013). However, an additional contributing factor is that photoreceptor synapses do not form in Ca_v1.4 KO mice (Liu et al., 2013; Zabouri and Haverkamp, 2013; Regus-Leidig et al., 2014). Does this developmental failure reflect a requirement for Ca_v1.4-mediated Ca²⁺ signals or an alternative, non-conducting role for the Ca_v1.4 protein in organizing the photoreceptor synapse? Here, we generated a mouse strain that expresses a non-conducting mutant form of Ca_v1.4 to answer this question.

Rod and cone photoreceptors communicate visual information via a triadic ‘ribbon’ synapse containing postsynaptic neurites of a depolarizing (ON) bipolar cell and two horizontal cells (HCs) (Masland, 2012; Figure 1A). The ribbon, an organelle that critically regulates the properties of glutamate release, is a morphological hallmark of this and other sensory synapses (Matthews and Fuchs, 2010). During the first postnatal week in mice, ribbons develop from spherical precursors and are concentrated in the outer plexiform layer (OPL) where mature rod and cone synapses are localized (Blanks et al., 1974; Regus-Leidig et al., 2009). In Ca_v1.4 KO mice, the development of these synapses appears stunted in that spheres, rather than ribbons, are present at all developmental ages and often found in the outer nuclear layer (ONL) that normally contains only photoreceptor somas. The terminals of rods and cones (i.e., spherules and pedicles, respectively) (Figure 1A) of Ca_v1.4 KO mice are misshapen, and a number of proteins typical of mature synapses are absent or aberrantly localized (Raven et al., 2008; Liu et al., 2013; Zabouri and Haverkamp, 2013; Regus-Leidig et al., 2014). If Ca_v1.4 Ca²⁺ signals are needed for the assembly of photoreceptor synapses, then ribbons and key synaptic proteins should be lacking from photoreceptors of a mouse strain expressing a non-conducting mutant form of Ca_v1.4, just as in Ca_v1.4 KO mice.

Figure 1 with 2 supplements see all

Download asset Open asset

Figure 1—source data 1

Traces corresponding to I_Ca that were used for analyses of rod peak I_Ca in Figure 1D are shown in the ‘.PDF’ file.

Individual values obtained for peak I_Ca in different cells are listed in the ‘.xlsx’ file.

https://cdn.elifesciences.org/articles/62184/elife-62184-fig1-data1-v2.xlsx

Download elife-62184-fig1-data1-v2.xlsx

To test this prediction, we deployed a strategy based on a disease-causing mutation in the Ca_v1.3 L-type channel. The mutation results in the insertion of a glycine residue into transmembrane segment 6 of domain I (G369i, Figure 1B) and prevents Ca²⁺ influx through the channel during membrane depolarizations (Baig et al., 2011). We determined if this mutation has a similar effect in Ca_v1.4 in electrophysiological recordings of transfected human embryonic kidney cells (HEK293T). The wild-type (WT) and G369i mutant channels were co-expressed with β₂ and α₂δ−4 subunits which are the major Ca_v auxiliary subunits forming Ca_v1.4 complexes in photoreceptor synaptic terminals (Lee et al., 2015). G369i mutant channels were associated with the plasma membrane but did not mediate significant inward Ba²⁺ currents (I_Ba) like WT channels (Figure 1—figure supplement 1). To functionally verify that G369i mutant channels reached the cell-surface, we analyzed ON gating currents (I_gating), which result from the movement of charged voltage-sensing domains prior to channel opening. For this purpose, we utilized the α₂δ−1 subunit which we find to produce larger current densities and better resolution of gating currents than with α₂δ−4. Compared to robust peak I_Ba densities mediated by WT channels (−20.70 ± 5.31 pA/pF), those in cells transfected with G369i mutant channels were negligible (−1.14 ± 0.11 pA/pF, p=0.03) and not significantly different from that in cells transfected only with β₂ and α₂δ−1 as negative controls (−0.56 ± 0.15 pA/pF, p=0.72, by Kruskal-Wallis test and post-hoc Dunn’s test; Figure 1C). I_gating was evident for both WT and G369i mutant channels, but not in the negative control cells. To estimate the number of channels with functional voltage-sensors in the membrane, we analyzed the time integral of I_gating evoked at the reversal potential (Q_max, [Wei et al., 1994]). When normalized to cell size, Q_max did not differ for WT (4.22 ± 1.34 fC/pF) and G369i mutant channels (3.22 ± 0.48 fC/pF, p=0.57 by unpaired t-test; Figure 1C). Thus, G369i prevents Ca²⁺ conductance but not cell-surface trafficking or voltage-sensor movements in Ca_v1.4.

Armed with this knowledge, we incorporated the G369i mutation into Ca_v1.4 channels in vivo by genome editing in mice (Figure 1—figure supplement 2). In agreement with our findings in the heterologous expression system, inward currents such as those measured in rods of WT mice, were not evident in those of the knock-in mouse strain (G369i KI). The absence of depolarization-evoked currents was similar in G369i KI mice and a mouse strain completely lacking Ca_v1.4 expression (Ca_v1.4 KO, Figure 1D). However, Ca_v1.4 protein was detected in retinal lysates of G369i KI mice by Western blot (Figure 1—figure supplement 2). Moreover, immunofluorescence corresponding to G369i mutant channels was abundant in the OPL, where photoreceptor synapses normally form, and juxtaposed near the ribbon as in WT mouse retina (Figure 1E). Therefore, while it does not mediate voltage-dependent Ca²⁺ currents, the G369i mutant channel is expressed and targeted appropriately in photoreceptors of G369i KI mice. Electroretinograms (ERGs) of dark-adapted G369i KI mice lacked b-waves at all but the highest light intensities, consistent with a disruption of rod synaptic transmission (Figure 1I). A-waves, which represent light-dependent hyperpolarization of photoreceptors, were not significantly different in WT and G369i KI mice (Figure 1H). Thus, G369i KI mice show defects in photoreceptor function that are largely restricted to synaptic terminals.

To define the contribution of Ca_v1.4 Ca²⁺ signals to the organization of the photoreceptor synapse, we compared the molecular composition and structure of photoreceptor synapses in the retinas of WT, G369i KI, and Ca_v1.4 KO mice. We focused on rod terminals due to their simpler synaptic organization (i.e. one large, arc-shaped ribbon and a single type of postsynaptic bipolar cell) as compared to cones (i.e. multiple short ribbons and types of postsynaptic bipolar cells). Furthermore, rod synapses are more severely disrupted than cone synapses when the presynaptic abundance of Ca_v1.4 is compromised (Ball et al., 2002; Katiyar et al., 2015; Wang et al., 2017; Kerov et al., 2018). We first compared the prevalence of ribbons and spheres using antibody labeling for CtBP2, a component of the major ribbon protein Ribeye (Schmitz et al., 2000; Figure 2A). We restricted analyses to CtBP2-labeled structures in rod spherules by excluding those in cone arrestin-labeled cone pedicles. The minimum ribbon length was arbitrarily set at 0.95 μm based on the absence of values higher than this in Ca_v1.4 KO retina. While shorter on average than in WT retina, arc-shaped ribbons were plentiful in the OPL of G369i retina (Figure 2B–D) and co-clustered with proteins characteristic of mature rod synapses (Figure 2E) such as PSD-95 and β-dystroglycan (Koulen et al., 1998; Blank et al., 1997), which were undetectable in the OPL of Ca_v1.4 KO retina (Figure 2F,G). Bassoon, a protein involved in ribbon morphogenesis (tom Dieck et al., 2005), and RIM2, a protein implicated in regulating Ca_v1.4 at rod synapses (Grabner et al., 2015), were tightly clustered with ribbons in G369i KI as in WT retina (Figure 2H,I). Thus, although leading to shorter ribbons, the presynaptic assembly of rod synapses can proceed without Ca_v1.4 Ca²⁺ signals.

Figure 2

Download asset Open asset

Figure 2—source data 1

The image archive contains all images used in quantitative analyses in Figure 2B–D.

The data were exported as 8-bit ‘tif’ files (1600 × 1600 pixels). Values obtained for individual data points (and outlier analysis) for summary graphs in Figure 2B–D are contained in ‘.xlsx’ files.

https://cdn.elifesciences.org/articles/62184/elife-62184-fig2-data1-v2.zip

Download elife-62184-fig2-data1-v2.zip

We next determined if postsynaptic elements were also intact at G369i KI rod synapses. At the depolarized voltage of photoreceptors in darkness, Ca_v1.4-dependent glutamate release activates a metabotropic glutamate receptor (mGluR6), which suppresses the activity of TRPM1-dependent channels (Shiells et al., 1981; Shen et al., 2009). Light-dependent hyperpolarization of photoreceptors reduces Ca_v1.4 Ca²⁺ signals, which slows the rate of glutamate release, thus disinhibiting the postsynaptic conductance and enabling excitatory postsynaptic currents (EPSCs) that communicate light signals via the ON pathway (Shiells et al., 1981; Figure 3A). In agreement with previous work (Koike et al., 2010), mGluR6 was clustered postsynaptic to rod ribbons, and TRPM1 was situated at the tips of RBC dendrites in WT retina (Figure 3B–D). Although the subcellular distribution of the postsynaptic proteins was partially obscured by the diffuse pattern of TRPM1 labeling, arc-shaped ribbons in apposition to colocalized TRPM1 and mGluR6 could clearly be distinguished in G369i KI retina but not in Ca_v1.4 KO retina (Figure 3B–D). To test if this signaling pathway was functional in RBCs of G369i KI mice, we used an agonist and antagonist of mGluR6 (L-AP4 and CPPG, respectively) to simulate light responses in RBCs (Shen et al., 2009; Figure 3A). As expected (Morgans et al., 2009; Shen et al., 2009), the CPPG-evoked EPSC was robust and outwardly rectifying in WT RBCs (Figure 3E,F). Although significantly lower in amplitude compared to WT RBCs, this EPSC was measurable in all RBCs that were recorded in G369i KI mice but was never detected in any of the RBC recordings in Ca_v1.4 KO mice (Figure 3E,F). These results indicate that the molecular components of the postsynaptic signaling complex in RBCs can be assembled in the absence of Ca_v1.4 Ca²⁺ signals, albeit less efficiently than in their presence.

Figure 3

Download asset Open asset

Figure 3—source data 1

Traces corresponding to EPSCs that were used for analyses of EPSC amplitudes in Figure 3F are included in the ‘.PDF’ file; individual values obtained for EPSC amplitudes in different cells are listed in the ‘.xlsx’ file.

https://cdn.elifesciences.org/articles/62184/elife-62184-fig3-data1-v2.zip

Download elife-62184-fig3-data1-v2.zip

Sprouting of HC and RBC neurites out of the OPL and into the ONL, and the appearance of ectopic ribbons and other presynaptic proteins in the ONL, occurs as a result of presynaptic functional impairments in rods and cones (McCall and Gregg, 2008). Thus, the prevailing view has been that synaptic transmission is needed to maintain the laminar organization of photoreceptor synapses in the OPL. Consistent with this notion, HC and RBC sprouting (Figure 4A) are particularly prominent in Ca_v1.4 KO mice, which lack evidence of photoreceptor transmission (Chang et al., 2006; Raven et al., 2008; Liu et al., 2013; Zabouri and Haverkamp, 2013). If these defects arise solely from the absence of Ca_v1.4 Ca²⁺ signals that trigger glutamate release, the severity of neurite sprouting should be similar in G369i KI mice as in Ca_v1.4 KO mice. Contrary to this prediction, HC and RBC neurite sprouting was less prominent in G369i KI mice than in Ca_v1.4 KO mice (Figure 4A,B) with most of the synapses situated normally in the OPL (Figure 4C). These results indicate a supporting, but non-essential, role for Ca_v1.4 Ca²⁺ signals and/or glutamatergic transmission for the proper lamination of rod synapses within the OPL.

Figure 4 with 2 supplements see all

Download asset Open asset

Figure 4—source data 1

The image archive contains all images used in quantitative analyses in Figure 4B.

The data were exported as 8-bit ‘tif’ files (800 × 600 pixels). Values obtained for individual data points (and outlier analysis) for summary graphs in are contained in ‘.xlsx’ files.

https://cdn.elifesciences.org/articles/62184/elife-62184-fig4-data1-v2.zip

Download elife-62184-fig4-data1-v2.zip

Compared to the spherical appearance of ribeye-labeled structures in the OPL/ONL of Ca_v1.4 KO and other mouse strains with presynaptic dysfunction (Chang et al., 2006; McCall and Gregg, 2008; Raven et al., 2008; Liu et al., 2013; Zabouri and Haverkamp, 2013), those in the ONL of G369i KI mice exhibited mature ribbon morphology and were associated with synaptic proteins as in the WT OPL (Figure 4C,D). To further analyze the seemingly normal organization of these synapses in the OPL and ONL, we performed 3-dimensional (3D) reconstructions of spherules in G369i KI retina via serial block-face scanning electron microscopy (Figure 4E–G). Each of the four rod terminals sampled from G369i KI retina had a single ribbon that was anchored at the presynaptic membrane and was apposed to a triad of postsynaptic partners (Figure 4F–G). However, several peculiarities were observed. First, three of the four spherules that were analyzed contained more than one ribbon, rather than the single ribbon characteristic of rod terminals in the WT mouse retina (Blanks et al., 1974; Figure 4E–G). Second, some ribbons appeared club-shaped (Figure 4—figure supplement 1). Third, at least one of the extraneous ribbons was ‘floating’ rather than anchored near the presynaptic membrane. These alterations of ribbon architecture in G369i KI rod spherules resemble the types of ribbon plasticity caused by reductions in presynaptic Ca²⁺ in mouse rods due to light or Ca²⁺ chelators (Spiwoks-Becker et al., 2004; Dembla et al., 2020). Thus, the lack of Ca_v1.4 Ca²⁺ signals in G369i KI rods could have caused fragmentation of the anchored ribbon, leading to the appearance of additional short, floating ribbons.

A particularly prominent anomaly of the reconstructed spherules was an alteration in postsynaptic architecture. Normally, within the invagination of mouse rod spherules are two HC neurites and an intervening RBC neurite (Figure 4E). Compared to HCs, the RBC neurite tip and the resident mGluR6 receptors are maintained relatively distant—hundreds of nanometers—from the release site (Rao-Mirotznik et al., 1995). This complex organization is thought to be critical for controlling glutamate release volume and spillover dynamics, allowing for differential activation of distinct glutamate receptor populations (Petralia et al., 2018). For spherules in the OPL as well as in the ONL of G369i KI retina, branching neurites of HCs and RBCs formed non-invaginating triadic contacts close to the anchored presynaptic ribbon (Figure 4F,G), rather than invading rod spherules as in WT retina (Figure 4E). Nevertheless, immunolabeling for mGluR6 remained clustered near ribbons in G369i KI retina (Figure 3B; Figure 4—figure supplement 2), suggesting that pre and postsynaptic apposition is maintained even in the absence of the normal spacing between RBC tips and the release sites. In agreement with these findings, mGluR6 labeling was significantly closer to that for ribbons in G369i KI than in WT retina as revealed by nearest-neighbor analyses (Figure 4—figure supplement 2). Thus, while dispensable for postsynaptic partner selection at rod synapses, Ca_v1.4 Ca²⁺ signals are required for the invaginating arrangement of the corresponding neurites within the rod spherule and their proximity to release sites.

Like other presynaptic Ca_v channels, Ca_v1.4 is expected to interact directly or indirectly with a diverse array of proteins (Dolphin and Lee, 2020), some of which are required for ribbon formation in photoreceptors (tom Dieck et al., 2005; Kiyonaka et al., 2012). Thus, the Ca_v1.4 complex could act as a central scaffold for protein interactions that drive the assembly of the ribbon and associated components of the active zone. Alternatively, but not mutually exclusively, voltage-dependent conformational changes in the Ca_v1.4 protein, which are intact in G369i mutant channels (Figure 1C), could be involved. For example, Ca_v1.4 could conformationally couple to intracellular Ca²⁺ release channels, as Ca_v1.1 does in skeletal muscle (Adams et al., 1990), which could represent a Ca²⁺ source that aids presynaptic development.

A limitation of our study is that the mutant G369i channel was expressed in all retinal cell-types that normally express Ca_v1.4. In addition to photoreceptors, Ca_v1.4 appears to be expressed in bipolar cells and at much lower levels in other cell-types in mouse retina (Yan et al., 2020). Because we find specific immunoreactivity for Ca_v1.4 in the outer retina to be limited to photoreceptor terminals (Liu et al., 2013), we favor the interpretation that Ca_v1.4 protein is not present to any large extent within bipolar cell soma or dendrites and that the postsynaptic structural defects of G369i KI rod synapses result primarily from the absence of presynaptic Ca_v1.4 Ca²⁺ signals in rods. In addition to supporting glutamatergic transmission, presynaptic Ca_v1.4 Ca²⁺ signals could strengthen trans-synaptic signaling via cell-adhesion molecules such as NGL-2 in HCs (Soto et al., 2013) and ELFN1 and pikachurin in RBCs (Sato et al., 2008; Wang et al., 2017), which collectively enable the invagination of the corresponding neurites into the rod terminal. Considering that rod ribbons are shorter in G369i KI than in WT retina (Figure 2B), Ca_v1.4 Ca²⁺ signals may also regulate the activities of Ca²⁺ sensitive proteins such as GCAP2, which binds to Ribeye and controls ribbon stability in a Ca²⁺-dependent manner (Schmitz, 2014).

There are notable parallels and distinctions in the rod phenotypes of G369i KI mice and those lacking expression of either of the auxiliary subunits of Ca_v1.4, β₂ or α₂δ−4. Ca_vβ and α₂δ subunits are known to facilitate the forward trafficking and maintenance of Ca_v channels in the plasma membrane (Dolphin and Lee, 2020). Thus, it is not surprising that β₂ KO and α₂δ−4 KO mice exhibit severely diminished Ca_v1.4 Ca²⁺ signals, and similar to G369i KI mice (Figures 1G–I and 4A–C), there is evidence for a loss of rod synaptic transmission in ERGs, and significant sprouting of postsynaptic RBCs and HCs into the ONL in these mouse strains (Ball et al., 2002; Katiyar et al., 2015; Wang et al., 2017; Kerov et al., 2018). However, a striking difference is that ribbons and other aspects of the presynaptic complex are preserved in G369i KI but not in β₂ KO or α₂δ−4 KO mice (Ball et al., 2002; Katiyar et al., 2015; Wang et al., 2017; Kerov et al., 2018). Because we find significant levels of the mutant Ca_v1.4 protein within rod terminals of G369i KI mice (Figure 1E,F), which is not the case in β₂ KO or α₂δ−4 KO mice (Ball et al., 2002; Katiyar et al., 2015; Wang et al., 2017; Kerov et al., 2018), we propose that a minimal complement of Ca_v1.4 channels is needed to enable ribbon morphogenesis.

How synapses achieve their diverse forms and functions remains a fundamental question in neuroscience. Current evidence indicates that most synapses are assembled by intrinsic programs involving a variety of scaffolding proteins and cell-adhesion molecules (Kurshan and Shen, 2019), with ensuing patterns of neurotransmitter release needed primarily to instruct further refinements in synapse morphology and connectivity (Sando et al., 2017; Sigler et al., 2017). Our study identifies Ca_v1.4 as a nexus for both activity-dependent and independent mechanisms that drive the maturation of the rod synapse. Elucidating the signaling pathways that underlie the dual functions of Ca_v1.4 is an important challenge for future studies.

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Adams BA
   2. Tanabe T
   3. Mikami A
   4. Numa S
   5. Beam KG

   (1990) Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs

   Nature 346:569–572.

   https://doi.org/10.1038/346569a0

   • PubMed
   • Google Scholar
2. 1. Baig SM
   2. Koschak A
   3. Lieb A
   4. Gebhart M
   5. Dafinger C
   6. Nürnberg G
   7. Ali A
   8. Ahmad I
   9. Sinnegger-Brauns MJ
   10. Brandt N
   11. Engel J
   12. Mangoni ME
   13. Farooq M
   14. Khan HU
   15. Nürnberg P
   16. Striessnig J
   17. Bolz HJ

   (2011) Loss of ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness

   Nature Neuroscience 14:77–84.

   https://doi.org/10.1038/nn.2694

   • PubMed
   • Google Scholar
3. 1. Ball SL
   2. Powers PA
   3. Shin HS
   4. Morgans CW
   5. Peachey NS
   6. Gregg RG

   (2002)

   Role of the β₂ subunit of voltage-dependent calcium channels in the retinal outer plexiform layer

   Investigative Ophthalmology & Visual Science 43:1595–1603.

   • PubMed
   • Google Scholar
4. 1. Blank M
   2. Koulen P
   3. Kröger S

   (1997) Subcellular concentration of beta-dystroglycan in photoreceptors and glial cells of the chick retina

   The Journal of Comparative Neurology 389:668–678.

   https://doi.org/10.1002/(SICI)1096-9861(19971229)389:4<668::AID-CNE9>3.0.CO;2-Z

   • PubMed
   • Google Scholar
5. 1. Blanks JC
   2. Adinolfi AM
   3. Lolley RN

   (1974) Synaptogenesis in the photoreceptor terminal of the mouse retina

   The Journal of Comparative Neurology 156:81–93.

   https://doi.org/10.1002/cne.901560107

   • PubMed
   • Google Scholar
6. 1. Chang B
   2. Heckenlively JR
   3. Bayley PR
   4. Brecha NC
   5. Davisson MT
   6. Hawes NL
   7. Hirano AA
   8. Hurd RE
   9. Ikeda A
   10. Johnson BA
   11. McCall MA
   12. Morgans CW
   13. Nusinowitz S
   14. Peachey NS
   15. Rice DS
   16. Vessey KA
   17. Gregg RG

   (2006) The nob2 mouse, a null mutation in Cacna1f: anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses

   Visual Neuroscience 23:11–24.

   https://doi.org/10.1017/S095252380623102X

   • PubMed
   • Google Scholar
7. 1. Della Santina L
   2. Kuo SP
   3. Yoshimatsu T
   4. Okawa H
   5. Suzuki SC
   6. Hoon M
   7. Tsuboyama K
   8. Rieke F
   9. Wong ROL

   (2016) Glutamatergic monopolar interneurons provide a novel pathway of excitation in the mouse retina

   Current Biology 26:2070–2077.

   https://doi.org/10.1016/j.cub.2016.06.016

   • PubMed
   • Google Scholar
8. 1. Dembla E
   2. Dembla M
   3. Maxeiner S
   4. Schmitz F

   (2020) Synaptic ribbons foster active zone stability and illumination-dependent active zone enrichment of RIM2 and Cav1.4 in photoreceptor synapses

   Scientific Reports 10:5957.

   https://doi.org/10.1038/s41598-020-62734-0

   • PubMed
   • Google Scholar
9. 1. Dodge FA
   2. Rahamimoff R

   (1967) Co-operative action a calcium ions in transmitter release at the neuromuscular junction

   The Journal of Physiology 193:419–432.

   https://doi.org/10.1113/jphysiol.1967.sp008367

   • PubMed
   • Google Scholar
10. 1. Dolphin AC
    2. Lee A

    (2020) Presynaptic calcium channels: specialized control of synaptic neurotransmitter release

    Nature Reviews Neuroscience 21:213–229.

    https://doi.org/10.1038/s41583-020-0278-2

    • PubMed
    • Google Scholar
11. 1. Eikermann-Haerter K
    2. Yuzawa I
    3. Qin T
    4. Wang Y
    5. Baek K
    6. Kim YR
    7. Hoffmann U
    8. Dilekoz E
    9. Waeber C
    10. Ferrari MD
    11. van den Maagdenberg AM
    12. Moskowitz MA
    13. Ayata C

    (2011) Enhanced subcortical spreading depression in familial hemiplegic migraine type 1 mutant mice

    Journal of Neuroscience 31:5755–5763.

    https://doi.org/10.1523/JNEUROSCI.5346-10.2011

    • PubMed
    • Google Scholar
12. 1. Grabner CP
    2. Gandini MA
    3. Rehak R
    4. Le Y
    5. Zamponi GW
    6. Schmitz F

    (2015) RIM1/2-Mediated facilitation of Cav1.4 Channel Opening Is Required for Ca2+-Stimulated Release in Mouse Rod Photoreceptors

    Journal of Neuroscience 35:13133–13147.

    https://doi.org/10.1523/JNEUROSCI.0658-15.2015

    • PubMed
    • Google Scholar
13. 1. Katiyar R
    2. Weissgerber P
    3. Roth E
    4. Dörr J
    5. Sothilingam V
    6. Garcia Garrido M
    7. Beck SC
    8. Seeliger MW
    9. Beck A
    10. Schmitz F
    11. Flockerzi V

    (2015) Influence of the β2-Subunit of L-Type Voltage-Gated cav channels on the structural and functional development of photoreceptor ribbon synapses

    Investigative Opthalmology & Visual Science 56:2312–2324.

    https://doi.org/10.1167/iovs.15-16654

    • PubMed
    • Google Scholar
14. 1. Kerov V
    2. Laird JG
    3. Joiner ML
    4. Knecht S
    5. Soh D
    6. Hagen J
    7. Gardner SH
    8. Gutierrez W
    9. Yoshimatsu T
    10. Bhattarai S
    11. Puthussery T
    12. Artemyev NO
    13. Drack AV
    14. Wong RO
    15. Baker SA
    16. Lee A

    (2018) α₂δ-4 Is Required for the Molecular and Structural Organization of Rod and Cone Photoreceptor Synapses

    The Journal of Neuroscience 38:6145–6160.

    https://doi.org/10.1523/JNEUROSCI.3818-16.2018

    • PubMed
    • Google Scholar
15. 1. Kiyonaka S
    2. Nakajima H
    3. Takada Y
    4. Hida Y
    5. Yoshioka T
    6. Hagiwara A
    7. Kitajima I
    8. Mori Y
    9. Ohtsuka T

    (2012) Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent ca(2+) channel

    Journal of Biochemistry 152:149–159.

    https://doi.org/10.1093/jb/mvs054

    • PubMed
    • Google Scholar
16. 1. Koike C
    2. Obara T
    3. Uriu Y
    4. Numata T
    5. Sanuki R
    6. Miyata K
    7. Koyasu T
    8. Ueno S
    9. Funabiki K
    10. Tani A
    11. Ueda H
    12. Kondo M
    13. Mori Y
    14. Tachibana M
    15. Furukawa T

    (2010) TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade

    PNAS 107:332–337.

    https://doi.org/10.1073/pnas.0912730107

    • PubMed
    • Google Scholar
17. 1. Koulen P
    2. Fletcher EL
    3. Craven SE
    4. Bredt DS
    5. Wässle H

    (1998) Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina

    The Journal of Neuroscience 18:10136–10149.

    https://doi.org/10.1523/JNEUROSCI.18-23-10136.1998

    • PubMed
    • Google Scholar
18. 1. Kurshan PT
    2. Shen K

    (2019) Synaptogenic pathways

    Current Opinion in Neurobiology 57:156–162.

    https://doi.org/10.1016/j.conb.2019.03.005

    • PubMed
    • Google Scholar
19. 1. Lee A
    2. Wang S
    3. Williams B
    4. Hagen J
    5. Scheetz TE
    6. Haeseleer F

    (2015) Characterization of Cav1.4 complexes (α11.4, β2, and α2δ4) in HEK293T cells and in the retina

    The Journal of Biological Chemistry 290:1505–1521.

    https://doi.org/10.1074/jbc.M114.607465

    • PubMed
    • Google Scholar
20. 1. Liu X
    2. Kerov V
    3. Haeseleer F
    4. Majumder A
    5. Artemyev N
    6. Baker SA
    7. Lee A

    (2013) Dysregulation of _ca(v)1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2

    Channels 7:514–523.

    https://doi.org/10.4161/chan.26376

    • PubMed
    • Google Scholar
21. 1. Mansergh F
    2. Orton NC
    3. Vessey JP
    4. Lalonde MR
    5. Stell WK
    6. Tremblay F
    7. Barnes S
    8. Rancourt DE
    9. Bech-Hansen NT

    (2005) Mutation of the calcium channel gene Cacna1f disrupts calcium signaling, synaptic transmission and cellular organization in mouse retina

    Human Molecular Genetics 14:3035–3046.

    https://doi.org/10.1093/hmg/ddi336

    • PubMed
    • Google Scholar
22. 1. Masland RH

    (2012) The neuronal organization of the retina

    Neuron 76:266–280.

    https://doi.org/10.1016/j.neuron.2012.10.002

    • PubMed
    • Google Scholar
23. 1. Matthews G
    2. Fuchs P

    (2010) The diverse roles of ribbon synapses in sensory neurotransmission

    Nature Reviews Neuroscience 11:812–822.

    https://doi.org/10.1038/nrn2924

    • PubMed
    • Google Scholar
24. 1. McCall MA
    2. Gregg RG

    (2008) Comparisons of structural and functional abnormalities in mouse b-wave mutants

    The Journal of Physiology 586:4385–4392.

    https://doi.org/10.1113/jphysiol.2008.159327

    • PubMed
    • Google Scholar
25. 1. Morgans CW
    2. Zhang J
    3. Jeffrey BG
    4. Nelson SM
    5. Burke NS
    6. Duvoisin RM
    7. Brown RL

    (2009) TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells

    PNAS 106:19174–19178.

    https://doi.org/10.1073/pnas.0908711106

    • PubMed
    • Google Scholar
26. 1. Petralia RS
    2. Wang YX
    3. Mattson MP
    4. Yao PJ

    (2018) Invaginating structures in mammalian synapses

    Frontiers in Synaptic Neuroscience 10:4.

    https://doi.org/10.3389/fnsyn.2018.00004

    • PubMed
    • Google Scholar
27. 1. Pietrobon D

    (2005) Function and dysfunction of synaptic calcium channels: insights from mouse models

    Current Opinion in Neurobiology 15:257–265.

    https://doi.org/10.1016/j.conb.2005.05.010

    • PubMed
    • Google Scholar
28. 1. Pietrobon D

    (2010) CaV2.1 channelopathies

    Pflügers Archiv - European Journal of Physiology 460:375–393.

    https://doi.org/10.1007/s00424-010-0802-8

    • PubMed
    • Google Scholar
29. 1. Rao-Mirotznik R
    2. Harkins AB
    3. Buchsbaum G
    4. Sterling P

    (1995) Mammalian rod terminal: architecture of a binary synapse

    Neuron 14:561–569.

    https://doi.org/10.1016/0896-6273(95)90312-7

    • PubMed
    • Google Scholar
30. 1. Raven MA
    2. Orton NC
    3. Nassar H
    4. Williams GA
    5. Stell WK
    6. Jacobs GH
    7. Bech-Hansen NT
    8. Reese BE

    (2008) Early afferent signaling in the outer plexiform layer regulates development of horizontal cell morphology

    The Journal of Comparative Neurology 506:745–758.

    https://doi.org/10.1002/cne.21526

    • PubMed
    • Google Scholar
31. 1. Regus-Leidig H
    2. Tom Dieck S
    3. Specht D
    4. Meyer L
    5. Brandstätter JH

    (2009) Early steps in the assembly of photoreceptor ribbon synapses in the mouse retina: the involvement of precursor spheres

    The Journal of Comparative Neurology 512:814–824.

    https://doi.org/10.1002/cne.21915

    • PubMed
    • Google Scholar
32. 1. Regus-Leidig H
    2. Atorf J
    3. Feigenspan A
    4. Kremers J
    5. Maw MA
    6. Brandstätter JH

    (2014) Photoreceptor degeneration in two mouse models for congenital stationary night blindness type 2

    PLOS ONE 9:e86769.

    https://doi.org/10.1371/journal.pone.0086769

    • PubMed
    • Google Scholar
33. 1. Sando R
    2. Bushong E
    3. Zhu Y
    4. Huang M
    5. Considine C
    6. Phan S
    7. Ju S
    8. Uytiepo M
    9. Ellisman M
    10. Maximov A

    (2017) Assembly of excitatory synapses in the absence of glutamatergic neurotransmission

    Neuron 94:312–321.

    https://doi.org/10.1016/j.neuron.2017.03.047

    • PubMed
    • Google Scholar
34. 1. Sato S
    2. Omori Y
    3. Katoh K
    4. Kondo M
    5. Kanagawa M
    6. Miyata K
    7. Funabiki K
    8. Koyasu T
    9. Kajimura N
    10. Miyoshi T
    11. Sawai H
    12. Kobayashi K
    13. Tani A
    14. Toda T
    15. Usukura J
    16. Tano Y
    17. Fujikado T
    18. Furukawa T

    (2008) Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation

    Nature Neuroscience 11:923–931.

    https://doi.org/10.1038/nn.2160

    • PubMed
    • Google Scholar
35. 1. Schmitz F
    2. Königstorfer A
    3. Südhof TC

    (2000) RIBEYE, a component of synaptic ribbons: a protein's journey through evolution provides insight into synaptic ribbon function

    Neuron 28:857–872.

    https://doi.org/10.1016/s0896-6273(00)00159-8

    • PubMed
    • Google Scholar
36. 1. Schmitz F

    (2014) Presynaptic [Ca(2+)] and GCAPs: aspects on the structure and function of photoreceptor ribbon synapses

    Frontiers in Molecular Neuroscience 7:3.

    https://doi.org/10.3389/fnmol.2014.00003

    • PubMed
    • Google Scholar
37. 1. Shen Y
    2. Heimel JA
    3. Kamermans M
    4. Peachey NS
    5. Gregg RG
    6. Nawy S

    (2009) A transient receptor potential-like channel mediates synaptic transmission in rod bipolar cells

    Journal of Neuroscience 29:6088–6093.

    https://doi.org/10.1523/JNEUROSCI.0132-09.2009

    • PubMed
    • Google Scholar
38. 1. Shiells RA
    2. Falk G
    3. Naghshineh S

    (1981) Action of glutamate and aspartate analogues on rod horizontal and bipolar cells

    Nature 294:592–594.

    https://doi.org/10.1038/294592a0

    • PubMed
    • Google Scholar
39. 1. Sigler A
    2. Oh WC
    3. Imig C
    4. Altas B
    5. Kawabe H
    6. Cooper BH
    7. Kwon HB
    8. Rhee JS
    9. Brose N

    (2017) Formation and maintenance of functional spines in the absence of presynaptic glutamate release

    Neuron 94:304–311.

    https://doi.org/10.1016/j.neuron.2017.03.029

    • PubMed
    • Google Scholar
40. 1. Soto F
    2. Watkins KL
    3. Johnson RE
    4. Schottler F
    5. Kerschensteiner D

    (2013) NGL-2 regulates pathway-specific neurite growth and lamination, synapse formation, and signal transmission in the retina

    Journal of Neuroscience 33:11949–11959.

    https://doi.org/10.1523/JNEUROSCI.1521-13.2013

    • PubMed
    • Google Scholar
41. 1. Spiwoks-Becker I
    2. Glas M
    3. Lasarzik I
    4. Vollrath L

    (2004) Mouse photoreceptor synaptic ribbons lose and regain material in response to illumination changes

    European Journal of Neuroscience 19:1559–1571.

    https://doi.org/10.1111/j.1460-9568.2004.03198.x

    • PubMed
    • Google Scholar
42. 1. tom Dieck S
    2. Altrock WD
    3. Kessels MM
    4. Qualmann B
    5. Regus H
    6. Brauner D
    7. Fejtová A
    8. Bracko O
    9. Gundelfinger ED
    10. Brandstätter JH

    (2005) Molecular dissection of the photoreceptor ribbon synapse: physical interaction of bassoon and RIBEYE is essential for the assembly of the ribbon complex

    The Journal of Cell Biology 168:825–836.

    https://doi.org/10.1083/jcb.200408157

    • PubMed
    • Google Scholar
43. 1. Tottene A
    2. Conti R
    3. Fabbro A
    4. Vecchia D
    5. Shapovalova M
    6. Santello M
    7. van den Maagdenberg AM
    8. Ferrari MD
    9. Pietrobon D

    (2009) Enhanced excitatory transmission at cortical synapses as the basis for facilitated spreading depression in ca(v)2.1 knockin migraine mice

    Neuron 61:762–773.

    https://doi.org/10.1016/j.neuron.2009.01.027

    • PubMed
    • Google Scholar
44. 1. Waldner DM
    2. Bech-Hansen NT
    3. Stell WK

    (2018) Channeling vision: cav1.4-a critical link in retinal signal transmission

    BioMed Research International 2018:1–14.

    https://doi.org/10.1155/2018/7272630

    • Google Scholar
45. 1. Wang Y
    2. Fehlhaber KE
    3. Sarria I
    4. Cao Y
    5. Ingram NT
    6. Guerrero-Given D
    7. Throesch B
    8. Baldwin K
    9. Kamasawa N
    10. Ohtsuka T
    11. Sampath AP
    12. Martemyanov KA

    (2017) The auxiliary calcium channel subunit α2δ4 is required for axonal elaboration, synaptic transmission, and wiring of rod photoreceptors

    Neuron 93:1359–1374.

    https://doi.org/10.1016/j.neuron.2017.02.021

    • PubMed
    • Google Scholar
46. 1. Wei X
    2. Neely A
    3. Lacerda AE
    4. Olcese R
    5. Stefani E
    6. Perez-Reyes E
    7. Birnbaumer L

    (1994)

    Modification of Ca2+ channel activity by deletions at the carboxyl terminus of the cardiac alpha 1 subunit

    The Journal of Biological Chemistry 269:1635–1640.

    • PubMed
    • Google Scholar
47. 1. Yan W
    2. Laboulaye MA
    3. Tran NM
    4. Whitney IE
    5. Benhar I
    6. Sanes JR

    (2020) Mouse retinal cell atlas: molecular identification of over sixty amacrine cell types

    The Journal of Neuroscience 40:5177–5195.

    https://doi.org/10.1523/JNEUROSCI.0471-20.2020

    • PubMed
    • Google Scholar
48. 1. Zabouri N
    2. Haverkamp S

    (2013) Calcium channel-dependent molecular maturation of photoreceptor synapses

    PLOS ONE 8:e63853.

    https://doi.org/10.1371/journal.pone.0063853

    • PubMed
    • Google Scholar

Acceptance summary:

This paper provides convincing evidence that presynaptic calcium channel proteins play an organizing role during synapse development, additionally to calcium influx and activity. This work provides insights into the cellular elements involved in synaptic structure and organization of rod ribbon synapses, with relevant implications in the study of development at other synapses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "A dual role for Ca_v1.4 Ca²⁺ channels in the molecular and structural organization of the rod photoreceptor synapse" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Joshua H Singer (Reviewer #2); Wallace B Thoreson (Reviewer #3). Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work cannot be considered for publication in eLife in its current form.

The main reason for our decision is related to issues that critically affect the main conclusions of the manuscript. Reviewer #2 raised a serious concern about the lack of an essential control experiment needed to demonstrate that the KI rods do generate light responses and that there is a deficit in transmission at the rod synapse. This issue was discussed during the consultation process, and reviewer #3, the Senior Editor and myself agreed with reviewer #2 that this experiment is indispensable in order for this work to be considered for publication in eLife. However, we would be willing to consider a new manuscript including this experiment and addressing all the other concerns expressed by the reviewers. Please keep in mind that this would be considered a new submission.

Reviewer #1:

The Ca_v1.4 calcium channel is the major calcium channel in the OPL of the retina, which drive neurotransmitter release from both rods and cones. Ca_V1.4 knockout mice have presynaptic and postsynaptic morphological abnormalities in the retina that had previously been described. This manuscript reports the use of a knock in mouse model to disambiguate the effects of the Ca_v1.4 Ca²⁺ channel from those of calcium itself on synapse structure in the OPL. To do so, the authors make use of a KI mouse where the WT Ca_v1.4 channel is replaced by a channel with a point mutation that renders it unable to conduct calcium. Interestingly, the authors find a small change in the size of and relatively moderate disorganization of presynaptic structures in the KI mouse model, more significant disruptions were observed in postsynaptic cells. The authors conclude that the Ca²⁺ channel protein is needed for proper formation and maintenance of the pre-synaptic structure, but calcium entry through channels are necessary for proper recruitment of post-synaptic partners. Overall, the results are novel and the conclusion is well supported by the data.

Specific comments:

1) The experiments in this paper focused on the synapses in rods. In WT, rod ribbons are easily distinguishable from cone ribbons based on size and shape. In the KI model, rod ribbons become smaller and one cannot assume that they will maintain their normal shape. Indeed, the authors describe multiple ribbons (some misshapen) in some rods in EM. Is the assumption that all spots are rod ribbons in the KO and KI, but only the crescent shaped ones are rod ribbons in the WT? If some are mis-assigned, how might this affect the quantification of the effects on ribbon size. If they can't be convincingly distinguished, then wouldn't it be fairer comparison to analyze all ribbons in the OPL regardless of genotype?

2) If there is a reliable way to distinguish rod ribbons from cone ribbons, there seems to be a missed opportunity here to look at effects on cone ribbons and their post-synaptic partners, at least at the level of IHC.

Reviewer #2:

This manuscript by Maddox et al. presents a study of Ca_v1.4 in retinal synapse development. The central issue addressed is differentiating the signaling / organizational role(s) of the channel protein from those of Ca²⁺ influx through the channel during retinal rod photoreceptor synapse development. The authors generated a non-conducting channel (G369i KI) that appears to be trafficked normally to synapses and assessed differences between G369i mutants, Ca_v1.4 KO, and wild-type animals. They found substantial differences between G369i mutants and Ca_v1.4 KO retinas, with the G369i mutant more closely resembling the wild-type. The authors suggest, then, that the channel protein serves an organizing role during synapse development.

One critical control experiment has not been performed, and this must be included in any revision of the manuscript: although the authors show that rods themselves lack Ca²⁺ currents (Figure 1D), the authors do not provide evidence that the G369i KI rods respond to light and that postsynaptic rod bipolar cells do not (indicating a true deficit of synaptic transmission at the rod to rod bipolar cell synapse). As illustrated in Figure 3, the postsynaptic rod bipolar cells do appear to have developed mGluR6-mediated signaling cascades, but the authors' interpretation of the result, that the postsynaptic signaling can develop in the complete absence of presynaptic glutamate release, is not supported by the data presented. As well, the efficiency of Ca²⁺-exocytosis coupling at the rod presynapse is incredibly high, and it is worth noting that Ca²⁺ from sources other than voltage-gated Ca²⁺ channels could give rise to enough glutamate release to modulate synapse development.

With regard to the pre- and postsynaptic organization of the synapse, as illustrated in Figure 4: it seems a bit counterintuitive to me that the absence of an invaginating synapse would result in a closer apposition of ribeye and mGluR6 proteins, given that mGluR6 normally is located at the dendritic tips inserted in the very center of the invagination. The authors should clarify their explanation of this observation. As well, it is worth commenting that the authors addressed the integrity of the presynaptic active zone by visualizing ribeye and bassoon proteins using fluorescence immunohistochemistry, but neither protein-to my knowledge-interacts directly with Cavs. Is there a reason that the authors did not examine Rim / RimBP or CAST/ELKS expression as an assay of active zone organization? These proteins interact more directly with the Ca²⁺ channels.

Presumably, the G369i KI mutation should not affect the voltage-dependent gating of the channel. Has gating in the mutant been characterized? Is the voltage-sensing role of the channel important in development? These are interesting-to me, at least-questions that might be addressed experimentally or, at a minimum, in a revised discussion.

Reviewer #3:

This well-written, carefully done study shows that structural features, not calcium influx, of Ca_V1.4 L-type calcium channels are sufficient for proper organization of many key molecules at rod ribbon synapses. However, formation of the synaptic invagination and proper arrangement of post-synaptic partners requires calcium influx. To perform this study, the authors created knockin mice that express a non-conducting form of Ca_V1.4. The study was technically proficient and clearly described, with helpful illustrations accompanying each figure. Further work will be needed to understand which particular structural elements are important and how calcium influx regulates formation of the invaginating synapse. Testing the ability of rods to respond to light and transmit those signals to rod bipolar cells is important for interpreting their results. One relatively straightforward way to test this would be to record the A- and B-waves of the electroretinogram (ERG) in their mice.

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

The main reason for our decision is related to issues that critically affect the main conclusions of the manuscript. Reviewer #2 raised a serious concern about the lack of an essential control experiment needed to demonstrate that the KI rods do generate light responses and that there is a deficit in transmission at the rod synapse. This issue was discussed during the consultation process, and reviewer #3, the Senior Editor and myself agreed with reviewer #2 that this experiment is indispensable in order for this work to be considered for publication in eLife. However, we would be willing to consider a new manuscript including this experiment and addressing all the other concerns expressed by the reviewers. Please keep in mind that this would be considered a new submission.

We thank the reviewers for their careful assessment of our manuscript. We have addressed the primary concern that we perform a critical experiment to demonstrate that the KI rods do generate light responses and that there is a deficit in transmission at the rod synapse. We have addressed this concern as suggested by Reviewer 3 via electroretinograms (ERGs). These experiments show that while KI rods exhibit normal responses to light (i.e., normal a-waves), they show no evidence of b-waves that reflect transmission from rods to rod bipolar cells (RBCs). We have addressed these and other concerns raised by the reviewers as described below.

Reviewer #1:

The Ca_v1.4 calcium channel is the major calcium channel in the OPL of the retina, which drive neurotransmitter release from both rods and cones. Ca_V1.4 knockout mice have presynaptic and postsynaptic morphological abnormalities in the retina that had previously been described. This manuscript reports the use of a knock in mouse model to disambiguate the effects of the Ca_v1.4 Ca²⁺ channel from those of calcium itself on synapse structure in the OPL. To do so, the authors make use of a KI mouse where the WT Ca_v1.4 channel is replaced by a channel with a point mutation that renders it unable to conduct calcium. Interestingly, the authors find a small change in the size of and relatively moderate disorganization of presynaptic structures in the KI mouse model, more significant disruptions were observed in postsynaptic cells. The authors conclude that the Ca²⁺ channel protein is needed for proper formation and maintenance of the pre-synaptic structure, but calcium entry through channels are necessary for proper recruitment of post-synaptic partners. Overall, the results are novel and the conclusion is well supported by the data.

Specific comments:

1) The experiments in this paper focused on the synapses in rods. In WT, rod ribbons are easily distinguishable from cone ribbons based on size and shape. In the KI model, rod ribbons become smaller and one cannot assume that they will maintain their normal shape. Indeed, the authors describe multiple ribbons (some misshapen) in some rods in EM. Is the assumption that all spots are rod ribbons in the KO and KI, but only the crescent shaped ones are rod ribbons in the WT? If some are mis-assigned, how might this affect the quantification of the effects on ribbon size. If they can't be convincingly distinguished, then wouldn't it be fairer comparison to analyze all ribbons in the OPL regardless of genotype?

We regret that we did not include sufficient details regarding our quantitative analyses of ribbons in the Materials and methods section. A key detail relevant to the reviewer’s concern is that we restricted our analyses to rod ribbons using double-labeling with cone-arrestin antibodies to generate 3-D binary masks in order to eliminate the confounding influence of the shorter ribbons that characterize cone terminals. We then used a Matlab-based routine (ObjectFinder) to determine the lengths along the major axis all Ctbp2-labeled structures in the portion of the OPL. Because this was done in confocal z-stacks with more than 30 optical sections (voxel size of 0.09 𝜇𝑚 × 0.09 𝜇𝑚 × 0.2 𝜇𝑚 (𝑋 × 𝑌 × 𝑍)), we were able to capture the full-dimensions of each Ctbp2-labeled structure. Thus, we are confident that we are not mistaking cone ribbons for rod ribbons in the KO or KI, and that our approach allowed us to quantitate differences in the distribution of ribbons and spheres specifically in rod terminals of the 3 genotypes. We have added more details regarding ribbon analysis to the Materials and methods section and Results and Discussion.

2) If there is a reliable way to distinguish rod ribbons from cone ribbons, there seems to be a missed opportunity here to look at effects on cone ribbons and their post-synaptic partners, at least at the level of IHC.

We agree that this is an important future direction, but a bit more complicated to address given the diversity of cone bipolar cells and types of synaptic contacts that cones make with them. To fit with the focus of this short report format, we opted to limit our analyses to the rod phenotype of the KI.

Reviewer #2:

This manuscript by Maddox et al. presents a study of Ca_v1.4 in retinal synapse development. The central issue addressed is differentiating the signaling / organizational role(s) of the channel protein from those of Ca²⁺ influx through the channel during retinal rod photoreceptor synapse development. The authors generated a non-conducting channel (G369i KI) that appears to be trafficked normally to synapses and assessed differences between G369i mutants, Ca_v1.4 KO, and wild-type animals. They found substantial differences between G369i mutants and Ca_v1.4 KO retinas, with the G369i mutant more closely resembling the wild-type. The authors suggest, then, that the channel protein serves an organizing role during synapse development.

One critical control experiment has not been performed, and this must be included in any revision of the manuscript: although the authors show that rods themselves lack Ca²⁺ currents (Figure 1D), the authors do not provide evidence that the G369i KI rods respond to light and that postsynaptic rod bipolar cells do not (indicating a true deficit of synaptic transmission at the rod to rod bipolar cell synapse). As illustrated in Figure 3, the postsynaptic rod bipolar cells do appear to have developed mGluR6-mediated signaling cascades, but the authors' interpretation of the result, that the postsynaptic signaling can develop in the complete absence of presynaptic glutamate release, is not supported by the data presented.

We agree this is an important control experiment. Thus, we have added ERG analyses in Figure 1GI, which show that while KI rods exhibit normal responses to light (i.e., normal a-waves), they show no evidence of b-waves that reflect transmission from rods to rod bipolar cells (RBCs).

As well, the efficiency of Ca²⁺-exocytosis coupling at the rod presynapse is incredibly high, and it is worth noting that Ca²⁺ from sources other than voltage-gated Ca²⁺ channels could give rise to enough glutamate release to modulate synapse development.

This is an excellent point. We added this text to the Discussion:

“Like other presynaptic Ca_v channels, Ca_v1.4 is expected to interact directly or indirectly with a diverse array of proteins (Dolphin and Lee, 2020), some of which are required for ribbon formation in photoreceptors (tom Dieck et al., 2005; Kiyonaka et al., 2012). Thus, the Ca_v1.4 complex could act as a central scaffold for protein interactions that drive the assembly of the ribbon and associated components of the active zone. Alternatively, but not mutually exclusively, voltagedependent conformational changes in the Ca_v1.4 protein, which are intact in G369i mutant channels (Figure 1C), could trigger signaling pathways that promote synapse maturation. For example, Ca_v1.4 could conformationally couple to intracellular Ca²⁺ release channels, as Ca_v1.1 does in skeletal muscle (Adams et al., 1990), which could represent a Ca²⁺ source needed for synapse development.”

With regard to the pre- and postsynaptic organization of the synapse, as illustrated in Figure 4: it seems a bit counterintuitive to me that the absence of an invaginating synapse would result in a closer apposition of ribeye and mGluR6 proteins, given that mGluR6 normally is located at the dendritic tips inserted in the very center of the invagination. The authors should clarify their explanation of this observation.

We have modified the text accordingly:

“A particularly prominent anomaly of the reconstructed spherules was an alteration in postsynaptic architecture. Normally, within the invagination of mouse rod spherules are two HC neurites and an intervening RBC neurite (Figure 4E). Compared to HCs, the RBC neurite tip and the resident mGluR6 receptors are maintained relatively distant—hundreds of nanometers—from the release site (Rao-Mirotznik et al., 1995). This complex organization is thought to be critical for controlling glutamate release volume and spillover dynamics, allowing for differential activation of distinct glutamate receptor populations (Petralia et al., 2018). For spherules in the OPL as well as in the ONL of G369i KI retina, branching neurites of HCs and RBCs formed non-invaginating triadic contacts close to the anchored presynaptic ribbon (Figure 4F,G), rather than invading rod spherules as in WT retina (Figure 4E). Nevertheless, immunolabeling for mGluR6 remained clustered near ribbons in G369i KI retina (Figure 4C), suggesting that pre- and post-synaptic apposition is maintained even in the absence of the normal spacing between RBC tips and the release sites. In agreement with these findings, mGluR6 labeling was significantly closer to that for ribbons in G369i KI than in WT retina as revealed by nearest-neighbor analyses (Figure 4—figure supplement 2). Thus, while dispensable for postsynaptic partner selection at rod synapses, Ca_v1.4 Ca²⁺ signals are required for the invaginating arrangement of the corresponding neurites within the rod spherule and their proximity to release sites.”

As well, it is worth commenting that the authors addressed the integrity of the presynaptic active zone by visualizing ribeye and bassoon proteins using fluorescence immunohistochemistry, but neither protein-to my knowledge-interacts directly with Cavs. Is there a reason that the authors did not examine Rim / RimBP or CAST/ELKS expression as an assay of active zone organization? These proteins interact more directly with the Ca²⁺ channels.

The goal of the experiments shown in Figure 2F-G was to show that some major constituents of the presynaptic active zone are intact in G369i KI terminals, rather than to assess the localization of proteins known to interact with Ca_v1.4. However, we have added labeling for RIM2 to Figure 2I.

Presumably, the G369i KI mutation should not affect the voltage-dependent gating of the channel. Has gating in the mutant been characterized? Is the voltage-sensing role of the channel important in development? These are interesting-to me, at least-questions that might be addressed experimentally or, at a minimum, in a revised discussion.

The biophysical consequences of the G369i mutation have been characterized in the context of Ca_v1.3 (Baig et al., 2011). Similar detailed studies of this mutation in Ca_v1.4 are complicated by the notoriously low current amplitudes produced upon transfection of this channel, relative to other Ca_v channels, in HEK293T cells. However, we have added experiments showing that gating currents, which represent voltage-sensor movements, are relatively normal in G369i mutant channels (Figure 1C). We have also discussed the potential role of voltage-sensing in the Discussion (see point 2 response, above).

Reviewer #3:

This well-written, carefully done study shows that structural features, not calcium influx, of Ca_V1.4 L-type calcium channels are sufficient for proper organization of many key molecules at rod ribbon synapses. However, formation of the synaptic invagination and proper arrangement of post-synaptic partners requires calcium influx. To perform this study, the authors created knockin mice that express a non-conducting form of Ca_V1.4. The study was technically proficient and clearly described, with helpful illustrations accompanying each figure. Further work will be needed to understand which particular structural elements are important and how calcium influx regulates formation of the invaginating synapse. Testing the ability of rods to respond to light and transmit those signals to rod bipolar cells is important for interpreting their results. One relatively straightforward way to test this would be to record the A- and B-waves of the electroretinogram (ERG) in their mice.

Done.

Author details

1. J Wesley Maddox

   1. Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
   2. Iowa Neuroscience Institute, University of Iowa, Iowa City, United States
   3. Pappajohn Biomedical Institute, University of Iowa, Iowa City, United States

   Present address

   Department of Neuroscience, University of Texas-Austin, Austin, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1630-2746

2. Kate L Randall

   1. Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
   2. Iowa Neuroscience Institute, University of Iowa, Iowa City, United States
   3. Pappajohn Biomedical Institute, University of Iowa, Iowa City, United States

   Present address

   Department of Neuroscience, University of Texas-Austin, Austin, United States

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7199-9806

3. Ravi P Yadav

   Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Brittany Williams

   1. Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
   2. Iowa Neuroscience Institute, University of Iowa, Iowa City, United States
   3. Pappajohn Biomedical Institute, University of Iowa, Iowa City, United States

   Present address

   Department of Cell Biology & Physiology, Carolina Institute for Developmental Disabilities, and Neuroscience Center, University of North Carolina, Chapel Hill, United States

   Contribution

   Formal analysis, Funding acquisition, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. Jussara Hagen

   1. Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
   2. Iowa Neuroscience Institute, University of Iowa, Iowa City, United States
   3. Pappajohn Biomedical Institute, University of Iowa, Iowa City, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

6. Paul J Derr

   Department of Neuroscience, University of Wisconsin-Madison, Madison, United States

   Contribution

   Investigation, Gave final approval of the version

   Competing interests

   No competing interests declared

7. Vasily Kerov

   1. Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
   2. Iowa Neuroscience Institute, University of Iowa, Iowa City, United States

   Contribution

   Investigation, Gave final approval of the version

   Competing interests

   No competing interests declared

8. Luca Della Santina

   Department of Ophthalmology, University of California, San Francisco, San Francisco, United States

   Contribution

   Software, Writing - review and editing

   Competing interests

   No competing interests declared

9. Sheila A Baker

   1. Iowa Neuroscience Institute, University of Iowa, Iowa City, United States
   2. Department of Biochemistry, University of Iowa, Iowa City, United States
   3. Department of Ophthalmology, Iowa City, United States

   Contribution

   Resources, Writing - review and editing

   Competing interests

   No competing interests declared

10. Nikolai Artemyev

    1. Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    2. Iowa Neuroscience Institute, University of Iowa, Iowa City, United States
    3. Department of Ophthalmology, Iowa City, United States

    Contribution

    Formal analysis, Investigation, Writing - review and editing

    Competing interests

    No competing interests declared

11. Mrinalini Hoon

    1. Department of Neuroscience, University of Wisconsin-Madison, Madison, United States
    2. Department of Ophthalmology and Visual Science, University of Wisconsin-Madison, Madison, United States

    Contribution

    Formal analysis, Funding acquisition, Visualization, Writing - review and editing

    Competing interests

    No competing interests declared

12. Amy Lee

    1. Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, United States
    2. Iowa Neuroscience Institute, University of Iowa, Iowa City, United States
    3. Pappajohn Biomedical Institute, University of Iowa, Iowa City, United States

    Present address

    Department of Neuroscience, University of Texas-Austin, Austin, United States

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    amy.lee1@austin.utexas.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8021-0443

• 1,766

  Page views

• 310

  Downloads

• 13

  Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.