Function of cone and cone-related pathways in CaV1.4 IT mice

CaV1.4 L-type calcium channels are predominantly expressed in photoreceptor terminals playing a crucial role for synaptic transmission and, consequently, for vision. Human mutations in the encoding gene are associated with congenital stationary night blindness type-2. Besides rod-driven scotopic vision also cone-driven photopic responses are severely affected in patients. The present study therefore examined functional and morphological changes in cones and cone-related pathways in mice carrying the CaV1.4 gain-of function mutation I756T (CaV1.4-IT) using multielectrode array, patch-clamp and immunohistochemical analyses. CaV1.4-IT ganglion cell responses to photopic stimuli were seen only in a small fraction of cells indicative of a major impairment in the cone pathway. Though cone photoreceptors underwent morphological rearrangements, they retained their ability to release glutamate. Our functional data suggested a postsynaptic cone bipolar cell defect, supported by the fact that the majority of cone bipolar cells showed sprouting, while horizontal cells maintained contacts with cones and cone-to-horizontal cell input was preserved. Furthermore a reduction of basal Ca2+ influx by a calcium channel blocker was not sufficient to rescue synaptic transmission deficits caused by the CaV1.4-IT mutation. Long term treatments with low-dose Ca2+ channel blockers might however be beneficial reducing Ca2+ toxicity without major effects on ganglion cells responses.

www.nature.com/scientificreports/ only expressed in rod and cone photoreceptors but has also been shown in bipolar cells its particular role and contribution to cellular Ca 2+ influx there is still elusive 1,19,30 . While sprouting of second order rod bipolar and horizontal cells has been found in Ca V 1.4-IT retinas 4,21,23 , and literature previously focused on rod pathway connections morphological data about cone bipolar cells in CSNB2 models are scarce 25,31 and the degree of synaptic remodelling is unknown. Therefore we focused on cone signalling pathways of Ca V 1.4-IT retinas. Measuring light induced ganglion cell activity while isolating specific signalling pathways (through different light levels as well as pharmacologically) we showed that the cone pathway was severely affected albeit the tonic vesicle release from cones was comparable to wild type. This was remarkable because our immunohistochemical analyses revealed that cone pedicles lost their regular mosaic arrangement in the outer plexiform layer (OPL) and cone bipolar cell dendrites extended into the ONL. Because the rod pathway was still responsive in Ca V 1.4-IT retinas we also tested whether the functional changes seen in synaptic transmission could be reversed by L-type calcium channel specific drugs.

Materials and methods
Animals. Animals were housed in groups of 2-6 per cage under standard laboratory conditions (12:12 light/ dark, lights on at 07:00 h, 22 ± 2 °C, 50-60% humidity) with food and water available ad libitum. Experimental procedures were designed to minimize animal suffering and the number of used animals and approved by the national ethical committee on animal care and use (Austrian Federal Ministry for Science and Research). All methods were performed in accordance with the relevant guidelines and regulations.
Ca V 1.4 mouse lines. We used two mouse models, Ca V 1.4-IT and Ca V 1.4-KO (Cacna1fΔEx14-17), previously described in 21,22 . Both male and female mice were investigated. Genotyping was performed as described in 21 . Immunohistochemistry. 10 to 15 weeks old mice were anesthetized with isoflurane (Vetflurane®k, Virbac) and killed by cervical dislocation. Vertical sections: The following steps were conducted at room temperature if not stated otherwise. Eyes were quickly removed from the eye socket, opened at the scleral-corneal rim and immersed for 10 min with 4% paraformaldehyde (PFA) in 1X phosphate-buffered saline (1X PBS, pH 7.4). Cornea, lens and vitreous were removed. Eye cups were then fixed with 4%PFA/1X PBS for 20 min, washed four times with 1X PBS and cryoprotected by a graded sucrose series: 10% sucrose in 1X PBS for 1 h, 20% sucrose in 1X PBS for 1 h and 30% sucrose in 1X PBS overnight at 4 °C. Eyecups were orientated along the dorsoventral axis, embedded in OCT Medium (Tissue-Tek O.C.T Compound; Sakura Finetek, Tokyo, Japan) and frozen in liquid nitrogen. Vertical sections (16 µm) were cut on a cryostat (Leica Microsystems, Wetzlar, Germany), mounted on gelatine coated slides and stored at − 20 °C. For immunofluorescence experiments, sections were washed three times in 1X PBS-T (1X PBS + 0.1% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA), blocked for 1 h in 1X PBS-T containing 1% bovine serum albumin (BSA, Sigma-Aldrich, A7030) and incubated overnight at 4 °C with primary antibodies diluted in 1X PBS-T at concentrations listed in Supplementary table 1. After washing three times with 1X PBS-T, sections were incubated with the secondary antibodies (Supplementary table 2) for one. Additional washes preceded the counterstaining with DAPI (1:10,000; Sigma, D-9542) and eventually the sections were mounted using Poly/Mount (Polysciences, Inc., Warrington, PA, USA).
For whole-mounts eyecups were fixed for 30 min in 4% PFA/1X PBS. The retina was dissected by removing the sclera and incubated in blocking solution (1% BSA in 1X PBS-T with 0.02% sodium azide) for 1 h. Primary antibodies were diluted in antibody solution (1% BSA in 1X PBS + 1% Triton X-100 with 0.02% sodium azide) and incubated for 1 week on a shaker. Afterwards, whole-mounts were washed three times in 1X PBS for 30 min. Secondary antibodies were diluted in 1X PBS-T with 0.02% sodium azide and incubated overnight at room temperature on a shaker. After washing again three times in 1X PBS-T for 30 min, whole-mounts were flattened by cutting 4 times ("clover-leaf " cuts) and mounted using Aqua Poly/Mount (Polysciences, Inc.).
Sections and whole-mounts were imaged with a confocal laser scanning microscope (Leica TCS SP5-II; Leica Microsystems, Wetzlar, Germany) at 40× magnification (NA 1.30). Series of micrographs were taken at 0.25 and 0.42 µm intervals and collapsed to a z-projection with maximum intensities in ImageJ (National Institutes of Health, Bethesda, Maryland, USA). The analysis of retinal layer thickness, soma and pedicle size and the quantification of cell numbers was conducted using ImageJ. Images were adjusted for contrast, brightness using ImageJ and assembled in Adobe Photoshop CS5.
Microelectrode array recordings. Mice were dark-adapted for at least 2 h before the experiment, and sacrificed by cervical dislocation after isoflurane anaesthesia between circadian zeitgeber time ZT5.5 and ZT8.5. Animals were 10 to 15 weeks old at the time of the experiment. The ventral position on each eye was marked with a soldering tool (BP645CEU 6W, Weller, Apex, NC, USA) before excision, and the eyes were put in bath solution (in [mM]: 110 NaCl, 2.5 KCl, 1 CaCl 2 , 1.6 MgCl 2 , 10 D-Glucose, and 22 NaHCO 3 ; bubbled with 5% CO 2 /95% O 2 ) for dissection. After cornea and lens removal, the retina was isolated and mounted on a dark grey nitrocellulose filter (13006-50-ACN, Sartorius Stedim, Göttingen, Germany) with a central 3 × 3 mm aperture, with the dorsal part of the retina placed as centred as possible. All operations were performed under dim red light conditions.
Recordings were carried out with perforated 120-electrode micro-electrode arrays (MEA; 120pMEA100/30iR-Ti-pr, Multichannel Systems, Reutlingen, Germany). Experiments were performed as described 32 . Briefly, the dorsal retina was placed ganglion cell-side down in the recording chamber and continuously perfused with fresh bath solution at 30 °C. Dorsal part was chosen based on cone opsin spectral distribution and visual stimulation Whole-cell patch clamp recordings from horizontal cells. Slice preparation. Horizontal slice preparation was performed as described 34 . Briefly, the retina was cut in four pieces which were embedded into 1.8% low-melting agarose dissolved in Ames´ Medium (US Biological, Salem, MA). Approximately 150 µm thick slices were cut with a vibratome (Leica Microsystems, Wetzlar, Germany) at room light condition. Slice were kept in Ames´ Medium at 37 °C (pH 7.2) in an incubator containing 5% CO 2 and 55% O 2 . Whole-cell recordings were carried at room temperature. 63X water immersion objective (Zeiss, Jena, Germany) was mounted on a fixedstage microscope (Zeiss Axio Examiner) equipped with Dodt contrast. Currents were recorded with an EPC-10 patch-clamp amplifier (Heka Elektronk, Lambrecht, Germany) low-pass filtered at 2.9 kHz using a built-in Bessel filter, and digitized at 10 kHz with Patchmaster software (Heka Elektronik). Patch pipettes were pulled from borosilicate glass (Sutter Instruments, Novato, CA, USA) to a final resistance of 4-7 MΩ. Series resistance was compensated up to 50%.

Results
In this study we provide further insight in rod and cone synaptic dysfunction in a mouse line that harbours the Ca V 1.4 gain-of-function mutation Ile-to-Thr at residue 756 (Ca V 1.4-IT; 4,21-23 ) reported in human patients 28,29 . Because also photopic responses are severely affected in patients with CSNB2 8,35 , yet previous analyses emphasized on the rod system, we focused primarily on the cone pathway. To this end, we characterized rod and cone pathways at the ganglion cell output using multielectrode array (MEA) recordings and correlated them with morphological analyses. Ca V 1.4-IT ganglion cell responses are more affected under photopic light stimulation. First, we dissected ganglion cell responses triggered by rod and cone activation using scotopic and photopic full-field flash stimuli in MEA recordings. We subdivide them into OFF and ON ganglion cells using dark and bright flashes (Fig. 1). OFF ganglion cells increase their firing rate upon dark flashes (negative contrast, Fig. 1a), while ON ganglion cells increase firing rate to bright flashes (positive contrast, Fig. 1b). We found a higher baseline firing frequency under both light conditions ([Hz]: scotopic: WT = 1.2 ± 0.1, Ca V 1.4-IT = 2.2 ± 0.2 ***; photopic: WT: 1.6 ± 0.12, Ca V 1.4-IT: 3.2 ± 0.2 ***p < 0.01; WT: N = 5, Ca V 1.4-IT: N = 4; mean ± SEM; Statistics: ***p < 0.01, Kruskal-Wallis test with Dunn's multiple comparison test; for mesopic light see Knoflach et al. 22 ) together with a significantly increased latency in OFF and ON ganglion cell responses (Fig. 1a,b; compare mesopic light: Knoflach et al. 22 ). More strikingly, in the Ca V 1.4-IT retina less than one third of the ganglion cells responding to scotopic stimulation also showed a photopic response (21.9% of ON and 33.4% of OFF ganglion cells), whereas in wild type the majority of ganglion cells responded under both light conditions (Fig. 1c). Immunohistochemical analyses with the ganglion cell marker RNA-binding protein with multiple splicing showed comparable ganglion cell numbers in central and peripheral wild type and Ca V 1.4-IT retinas (Fig. 1d,e). Our data indicated that roddriven responses are largely preserved whereas cone-driven pathways are strongly impaired. The impairment in the cone pathway could either be explained by a defect at the cone-to-cone bipolar cell synapse or further downstream at the cone bipolar-to-ganglion cell synapse.  Morphology of cone photoreceptor synaptic terminals in Ca V 1.4-IT retinas. We and others previously reported a reduction in the size of the ONL in the Ca V 1.4-IT retinas 21,23 indicating a loss of (rod) photoreceptors. To now also quantify the number of cones, we stained whole-mount retinas with an anti-cone arrestin antibody. The number of cones was comparable in wild type and Ca V 1.4-IT (wild type: 458.8 ± 43.6, Ca V 1.4-IT:   22 . Re-visiting cone terminal morphology revealed that what seemed like enlarged pedicles was a cluster of cone terminals and individual pedicles were significantly smaller than wild type (Fig. 2a,c,d). We tracked cone axons through different layers of a z-stack and observed clusters of pedicles which either derived from a single or different cones (Fig. 2d), displaying axonal branching. The branches occurred in different levels of the ONL (see Fig. 2a, arrows). In the central retina of a few mice (1 wild type and 2 Ca V 1.4-IT) we were able to count cone somata (in the ONL) and pedicles (in the OPL) in the same scanned image. While in the wild type the pedicles to soma ratio was almost 1:1 (only ~ 2% more pedicles than somas), we found 45% more synaptic terminals than cone somas in Ca V 1.4-IT, suggesting that axonal branching is a common feature.
Cones couple to surrounding photoreceptors at the tip of fine processes, so-called telodendria 37-39 which emerge from their pedicles. Our cone arrestin staining suggested irregular telodendrial contacts between photoreceptors (Fig. 2a,c) in Ca V 1.4-IT retinas which might be due to rod terminal retraction ( Supplementary  Fig. 1, 4,22,23 ). Together our analyses elicited that cones changed more profoundly than previously appreciated, however retained their synaptic terminals in the OPL (Fig. 2a). We therefore set out to investigate their synaptic function.

Functionality of the cone pathway in Ca V 1.4-IT retinas.
To explore the glutamate release from cones, we performed whole-cell patch-clamp recordings in postsynaptic horizontal cells as a read-out for transmitter release from cone presynaptic terminals. In horizontal cells the constant release of glutamate from cone photoreceptors results in a persistent inward current accompanied by tonic activity composed of high-frequency excitatory postsynaptic currents (EPSCs, Fig. 3a, see also Feigenspan & Babai 40 ). Under laboratory light conditions, the cumulative distribution of Ca V 1.4-IT EPSC amplitudes was significantly shifted to the left (Fig. 3c), indicating more events with smaller current amplitude. However, we saw no difference in the mean amplitude of the EPSCs (Fig. 3b) between wild type and mutant mice. Moreover, neither the inter-event interval nor the overall event frequency showed any statistical difference (Fig. 3d,e). Ca V 1.4-IT cones therefore released glutamate at a comparable rate as in wild type meaning the cone terminal per se is functional. Interestingly, we found a reduction in cell membrane capacitance as a measure for horizontal cell size (Fig. 3g). This finding is in good agreement with our morphological data which showed a reduction in the soma size of calbindin-stained horizontal cells in Ca V 1.4-IT retinas compared to wild type (Fig. 3f).
Since we detected release from cones, we investigated the dysfunction of pathways involving the cone-to cone bipolar cell synapse. We addressed this hypothesis of a cone-to-cone bipolar cell transmission failure by pharmacologically isolating the OFF cone bipolar cell pathway as this is feasible without blocking synaptic transmission of other glutamatergic synapses in inner retina. We made use of L-AP4, a group III mGluR agonist which binds to mGluR6 receptors expressed in depolarizing ON-bipolar cells, thereby eliminating rod-and cone-driven ON responses in ganglion cells as well as rod-driven OFF responses. The cone-to-OFF cone bipolar cell pathway ( Supplementary Fig. 2), however, remained available and OFF ganglion cells would respond to a dark flash provided that OFF cone bipolar cells received input. As expected, the application of 50 µM L-AP4 abolished all ON responses in wild type (Fig. 4a,d) and Ca V 1.4-IT retinas (Fig. 4d). Wild type OFF responses were still present under both scotopic (with reduced strength due to the block of the primary rod pathway) and photopic conditions (Fig. 4a-d). By contrast, Ca V 1.4-IT retinas exhibited only baseline ganglion cell activity and light-induced OFF responses were strongly reduced (Fig. 4b,c). In Ca V 1.4-IT, merely 1% of the OFF ganglion cells kept responding during L-AP4 perfusion under scotopic condition (compared to 76.9% in wild type) and 3.9% were L-AP4 resistant under photopic illumination (vs. 81.9% in wild type) (Fig. 4d). These data indicated a defect in the secondary rod and in the cone pathway of Ca V 1.4-IT retinas both involving the cone-to-OFF cone bipolar cell synapse.
Because group III mGluRs are also expressed in other retinal neurons 41 , we did further experiments in which we perfused the whole-mounts with 2 µM strychnine. As a competitive antagonist of glycine-gated Cl − channels 42 , strychnine (yellow in Supplementary Fig. 2) blocks the input to OFF cone bipolar cells by the AII amacrine cell, hence affects ganglion cell OFF responses driven via the primary rod pathway. The secondary and tertiary (rod → (cone →) OFF cone bipolar cell) rod pathways would not be affected by this manipulation. We expected similar defects in the OFF pathway under scotopic conditions as seen under L-AP4 perfusion in Ca V 1.4-IT retinas if the secondary rod pathway is indeed dysfunctional.
In fact, only 28.3% (scotopic) and 7.2% (photopic) of the Ca V 1.4-IT OFF ganglion cells continued responding during 2 µM strychnine perfusion compared to 81.2% (scotopic) and 87.9% (photopic) of wild type still showing an OFF response ( Supplementary Fig. 3a-d). The strychnine-resistant Ca V 1.4-IT OFF ganglion cells showed a "delayed" light-induced response, a behaviour that was never observed in wild type retinas. These "delayed" responses accounted for all responding Ca V 1.4-IT OFF ganglion cells observed during strychnine perfusion. We concluded that the scotopic and photopic OFF responses in Ca V 1.4 IT ganglion cells were driven by the primary rod pathway. As photopic ON responses in Ca V 1.4-IT ganglion cells were even less preserved than OFF responses it seems likely that also transmission to ON cone bipolar cells is similarly affected, even though we could not test this directly. Together these findings supported the notion of a cone-to-cone bipolar cell transmission defect, thus we analysed the morphology of different second-order neurons to elucidate the effect on the postsynaptic side. www.nature.com/scientificreports/ www.nature.com/scientificreports/

Morphology of cone bipolar and horizontal cells neurons in Ca V 1.4-IT retinas. Dendritic sprout-
ing of postsynaptic cells as a hallmark of outer retina synaptic dysfunction was evident in rod (RBCs labelled with PKCα, Fig. 5a) and cone bipolar cells (CBCs labelled with secretagogin, SCGN; labelling OFF types 2, 3, 4, 5 and ON types 6 and 8) of Ca V 1.4-IT retinas. We examined dendritic sprouting more specifically in both OFF cone (types 3a, 3b and type 4 stained by HCN4, PKARIIβ and calsenilin, respectively) and ON bipolar cells (Goα) (Fig. 5a). Since anti-Goα labels all ON-type bipolar cells, we identified ON cone bipolar cell processes by costaining rod bipolar cell dendrites with PKCα. We compared Ca V 1.4-IT with wild type and Ca V 1.4-KO retinas, using the same markers. While secretagogin, PKARIIβ, calsenilin and Go α stainings were similar in Ca V 1.4-KO and Ca V 1.4-IT, the HCN4 staining showed no sprouting of type 3a OFF cone bipolar cells in Ca V 1.4-IT retinas. The elongated dendrites of cone bipolar cells did not contact mislocated rod terminals in the ONL (rod terminals labelled with PSD-95, Fig. 5b), in contrast 72.5 ± 7.1% of the rod bipolar cell sprouts contacted a spherule (Ca V 1.4-IT: N = 3, n = 126). Conversely, 51.5 ± 5.2% of displaced rod spherules were contacted by a rod bipolar cell sprout (N = 3, n = 179, example Fig. 5b, top). Like type 3a cone bipolar cell dendrites, also horizontal cell dendrites were maintained in the OPL presumably connecting to cone terminals 43 , while their axonal arbors rewired with displaced rod terminals. We showed this using Neurofilament 200 (NF200), a marker for horizontal cell axons 44,45 , co-labelling all horizontal cell processes with Calbindin to distinguish dendritic (calbindin-only) from axonal processes (NF200 and calbindin doublelabelled). In Ca V 1.4-IT retinas, most sprouting processes were co-labelled with both markers and therefore likely of axonal origin (Fig. 6a) with few exceptions (Fig. 6a, arrows), indicating that horizontal cell dendritic sprouting was rare.
Furthermore, our analyses showed that the majority of Ca V 1.4-IT horizontal cell sprouts contacted displaced rod spherules, and that most displaced spherules were approached by horizontal cell processes (Fig. 6b, asterisk), with few exceptions (Fig. 6b, arrows). These findings are consistent with a preservation of horizontal cell-cone contacts.

Modulation of ganglion cell responses by dihydropyridines. While an increased intracellular Ca 2+
concentration was proposed at the ribbon synapse, reported in Ca 2+ imaging experiments 23 mainly from rod terminals (in line with the pronounced hyperpolarizing shift seen in the voltage-dependence of Ca V 1.4-IT channels 29 ) cone photoreceptors retained the ability of releasing glutamate (Fig. 3). However, the cone-to-cone bipolar cell synapses were not transmitting modulations of light intensity (as in our flash stimuli) efficiently, revealed by the cone and secondary rod pathway dysfunction proven in our L-AP4 and strychnine experiments. We hypothesized that, if an increased baseline Ca 2+ level in photoreceptor terminals was mainly responsible for the dysfunction we observed (response delays and failures to respond), then we should be able to reverse some of the effects by reducing the basal Ca 2+ load. Hence, we tested whether we could ameliorate the ganglion cell phenotype, using the response delay as a robust read out parameter, by blocking retinal LTCCs in Ca V 1.4-IT retinas. We had to conduct these experiments in scotopic light level because only there did we get enough responding www.nature.com/scientificreports/ ganglion cells for a meaningful analysis, thus involving a potential rescue of the secondary rod pathway in addition to potential improvements on rod release as a proxy for cone terminal function. The dihydropyridine LTCC blocker nilvadipine has previously been reported in different studies to decrease LTCC mediated currents in photoreceptors [46][47][48][49] but the effective concentration to block Ca V 1.4 channels was unknown. Therefore we first determined the nilvadipine sensitivity of Ca V 1.4 and Ca V 1.4-IT channels in a heterologous expression system. Remarkably, the Ca V 1.4-IT mutation increased the nilvadipine sensitivity almost tenfold (Fig. 7). In a second step, we evaluated the inhibition of two Ca V 1.3 splice isoforms 50 by nilvadipine: full-length Ca V 1.3 (Ca V 1.3 L ) and a variant with shorter C-terminus (Ca V 1.3 42a ). Nilvadipine blocked the calcium channels with different IC 50 with the highest affinity for Ca V 1.4-IT (Supplementary table 3; Fig. 7: www.nature.com/scientificreports/ The prerequisite for further functional analyses in the retina was, that the Ca V 1.4-IT ganglion cells were still able to respond at scotopic dark (OFF) flashes (Fig. 4b, control condition), suggesting that not all calcium channels were open at background light levels. Thus, further depolarization induced by the negative contrast could indeed trigger an additional light-dependent glutamate release.
We then recorded wild type and Ca V 1.4-IT ganglion cell responses at scotopic light level in the presence of different concentrations of nilvadipine (30 nM to 3 µM; Fig. 8). In wild type, we also expected a decrease in the ganglion cell response latency upon blocking LTCCs, at least with some concentrations, because a reduction of Ca 2+ influx would mimic a more hyperpolarized state of the photoreceptors, akin to light adaptation. Indeed, in the presence of 30 nM nilvadipine a trend towards a faster response was observed compared to the control ringer solution (Fig. 8a). But we found no statistically significant differences in any concentration compared to control. In Ca V 1.4-IT retinas, other than an increased variability, we did not observe any change in the light-induced ganglion cell response delay compared to control.
However, the ganglion cell baseline firing frequency, measured during constant mean background grey levels, increased upon nilvadipine perfusion compared to control conditions in both wild type and mutant retinas. The baseline frequencies decreased again with increasing nilvadipine concentrations, always in a comparable fashion in Ca V 1.4-IT and wild type retinas, and at 3 μM were lower than in the control condition (Fig. 8b). Of note, when we perfused wild type retinas with 1 μM nilvadipine more than 60% of the ganglion cells showed light responses, while only in two out of four Ca V 1.4-IT retinas a few cells were still responding. During 3 µM nilvadipine perfusion only one wild type retina was still active (Fig. 8a), while Ca V 1.4-IT retinas showed no responses. These data fit with the nilvadipine sensitivity for wild type and mutant Ca V 1.4-IT channels seen in heterologous expression systems. In conclusion, a reduction of basal Ca 2+ influx by a dihydropyridine LTCC blocker is not sufficient to rescue deficits in photoreceptor synaptic transmission caused by the Ca V 1.4-IT mutation. www.nature.com/scientificreports/

Discussion
The variability of symptoms in human CSNB2 patients ranges from night blindness to light sensitivity indicating changes in both rod and cone pathways. The majority of literature, however, focused on rod pathway components; only few abnormalities have been reported in neurons downstream of cones 25,31 . Our results suggested that the Ca V 1.4-IT mutation does not prevent neurotransmitter release from photoreceptors, but rather induces morphological rearrangement of the retinal network resulting in a dysfunction of the cone pathway. We reported sprouting of ON and OFF cone bipolar cells dendrites that did not contact any displaced rod photoreceptor terminals. This finding is in contrast to rod bipolar cells which have been shown to make ectopic synapses 4    www.nature.com/scientificreports/ retraction seems to be a trigger for rod bipolar cell elongation, however, the trigger for cone bipolar cell sprouting is elusive. Of note, type 3a OFF cone bipolar cells were the only subtype not showing dendritic elongation. The underlying reason for this cellular subtype specificity is yet unresolved. A key difference might be found in the receptor composition, as type 3a and type 2 OFF bipolar cells (not investigated here) express only kainatetype glutamate receptors 51 that induce a longer desensitized state compared to AMPA-type glutamate receptors expressed in other OFF bipolar cell types 52 . However, we showed that cone-contacting horizontal cell dendrites, which largely express AMPA-type glutamate receptors 53 , showed little sprouting and even maintained functional contacts with cones. Therefore different mechanisms might underlie the resilience of type 3a cone bipolar cells and horizontal cells towards perturbances at cone terminals and the ensuing remodelling of the cone contacts. Although Ca V 1.4 channels are expressed in both cones and rods [1][2][3][4] , the gain-of-function mutation affected cones in a different way than rods. While rods retract their axonal terminals into the ONL and show cell death 4,21,23 , we did not find evidence for cell death in cones. A possible explanation might be related to the photoreceptor specific internal Ca 2+ modulation. Cone pedicles possess the machinery to remove intra-terminal free Ca 2+ more rapidly during light adaptation compared to rods spherules 54 . Therefore cones might also be less susceptible to Ca 2+ -induced toxicity. Phenotypic differences in rod and cone phenotype have been found in different mouse models carrying mutations in the Ca V 1.4 channel complex. While some found that cones are spared 13,17,31,55,56 , other reported drastic changes compared to rods 14,27 . Still, a recent study showed that the Ca V 1.4.IT mutation can exert different functional phenotypes depending on splice variant and subunit composition 57 . Deeper knowledge about the channel composition in rods and cones will therefore be essential to elucidate the CSNB2 phenotype.
Although in Ca V 1.4-IT retinas cone terminals remained in the OPL the characteristic mosaic of cone terminals was lost and clusters of smaller pedicles, often branching from the same axon appeared. Cone axonal branching might be related to changes in the Ca 2+ level in the presynaptic terminal because this phenotype has been observed in two different Ca V 1.4 KO models 14,25 and one carrying a Ca V 1.4 mutation ( 21 , this study). It might even be a common feature that cone pedicles cluster while they are still releasing but have lost most of their downstream partners as suggested previously also for horizontal cell ablated mouse retinas 58 . Nevertheless, we would have expected a more pronounced difference in wild type and Ca V 1.4-IT EPSCs. Still, our horizontal cell recordings were performed on under ambient room light conditions and we cannot exclude a more pronounced defect on light-induced vesicle release not revealed during steady-state release. While the regulation of vesicle release would be an important question to address, we cannot exclude that the strong impairment in the photopic response is also due to the postsynaptic receptors inability to respond to the changes (leading to e.g. cone bipolar cell sprouting).
Under the hypothesis of a higher Ca 2+ influx in photoreceptor terminals, the remaining rod-driven signal seen in our MEA experiments allowed us to test whether we can modulate the Ca V 1.4-IT-mediated Ca 2+ influx pharmacologically. Yet, we did not see a faster signal transmission in scotopic light as a consequence of the nilvadipine-induced reduction of Ca 2+ influx. This finding that does not support the notion of a similarly increased intracellular Ca 2+ load in cone terminals at steady state or a consequentially higher glutamate level in the synaptic cleft which might lead to postsynaptic glutamate receptor desensitisation. Therefore, experiments with a Ca V 1.4 channel gating modulator would be required to optimize activation and inactivation properties of the mutated channels and thus extend the dynamic range of Ca 2+ signalling in Ca V 1.4-IT retinal neurons.
Nevertheless, in the adult mutant retina, molecular and/or structural rearrangements of the inner plexiform layer might contribute to the transmission delay. In fact, in the presence of strychnine, we observed a "delayed" ganglion cell response also in the Ca V 1.4-IT retina. Further investigations would have exceeded the scope of the present study, but the phenotype was reminiscent of a complex, non-physiological, amacrine cell influence 59 .
We can exclude that our approach failed due to a lack of nilvaldipine block of Ca V 1.4-IT channels because the mutant channels even showed a tenfold higher sensitivity compared to wild type Ca V 1.4 channels in a heterologous expression system. Such behaviour was previously shown also in gain-of-function variants of Ca V 1.3 L-type calcium channels 60,61 possibly explained by the state-dependent action of dihydropyridines 50,62 . Ca V 1.3 which has been suggested to be the second major L-type calcium channel in photoreceptor terminals 30 might have been unaffected by the treatment based on the lower nilvadipine sensitivity that we observed. Still, other L-type calcium channel expressed in the IPL 43,45,63 might account for the increased baseline firing rate in ganglion cells that we observed, elicited by disinhibition effects (e.g. Ca V 1.2 has a markedly higher dihydropyridine sensitivity 50,[64][65][66]. Altogether our data indicated that the rod pathway is still active whereas pathways involving cone-to-cone bipolar cell transmission including the secondary pathway, are strongly impaired. In human CSNB2 patients that show a congenital rod-cone dysfunction 8 this difference might also be reflected and should be considered in the ERG interpretation (e.g. influence of secondary rod pathway in the scotopic ERG 67,68 ) and when treatment options are discussed. The fact that the gain of channel function in adult Ca V 1.4-IT mice cannot be reversed by simply decreasing the Ca 2+ levels will have to be taken into account for pharmaco-therapeutic approaches which aim at restoring channel function. It might nevertheless prove beneficial to use low-dose Ca 2+ channel blockers for long term treatments aimed to reduce Ca 2+ overload and toxicity in the retina, as we've seen only moderate acute effects on ganglion cells responses.

Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary  Information files).