April 2015
Volume 56, Issue 4
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Physiology and Pharmacology  |   April 2015
Influence of the β2-Subunit of L-Type Voltage-Gated Cav Channels on the Structural and Functional Development of Photoreceptor Ribbon Synapses
Author Affiliations & Notes
  • Rashmi Katiyar
    Institute of Anatomy and Cell Biology, Department of Neuroanatomy, Saarland University School of Medicine, Homburg, Germany
  • Petra Weissgerber
    Department of Pharmacology and Toxicology, Saarland University School of Medicine, Homburg, Germany
  • Elisabeth Roth
    Department of Pharmacology and Toxicology, Saarland University School of Medicine, Homburg, Germany
  • Janka Dörr
    Department of Pharmacology and Toxicology, Saarland University School of Medicine, Homburg, Germany
  • Vithiyanjali Sothilingam
    Division of Ocular Neurodegeneration, Institute for Ophthalmic Research, Centre for Ophthalmology, Eberhard Karls University, Tübingen, Germany
  • Marina Garcia Garrido
    Division of Ocular Neurodegeneration, Institute for Ophthalmic Research, Centre for Ophthalmology, Eberhard Karls University, Tübingen, Germany
  • Susanne C. Beck
    Division of Ocular Neurodegeneration, Institute for Ophthalmic Research, Centre for Ophthalmology, Eberhard Karls University, Tübingen, Germany
  • Mathias W. Seeliger
    Division of Ocular Neurodegeneration, Institute for Ophthalmic Research, Centre for Ophthalmology, Eberhard Karls University, Tübingen, Germany
  • Andreas Beck
    Department of Pharmacology and Toxicology, Saarland University School of Medicine, Homburg, Germany
  • Frank Schmitz
    Institute of Anatomy and Cell Biology, Department of Neuroanatomy, Saarland University School of Medicine, Homburg, Germany
  • Veit Flockerzi
    Department of Pharmacology and Toxicology, Saarland University School of Medicine, Homburg, Germany
  • Correspondence: Frank Schmitz, Institute of Anatomy and Cell Biology, Department of Neuroanatomy, Saarland University School of Medicine, 66421 Homburg, Germany; frank.schmitz@uks.eu
  • Veit Flockerzi, Department of Pharmacology and Toxicology, Saarland University School of Medicine, 66421 Homburg, Germany; veit.flockerzi@uks.eu
  • Footnotes
     RK, PW, and ER contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science April 2015, Vol.56, 2312-2324. doi:10.1167/iovs.15-16654
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      Rashmi Katiyar, Petra Weissgerber, Elisabeth Roth, Janka Dörr, Vithiyanjali Sothilingam, Marina Garcia Garrido, Susanne C. Beck, Mathias W. Seeliger, Andreas Beck, Frank Schmitz, Veit Flockerzi; Influence of the β2-Subunit of L-Type Voltage-Gated Cav Channels on the Structural and Functional Development of Photoreceptor Ribbon Synapses. Invest. Ophthalmol. Vis. Sci. 2015;56(4):2312-2324. doi: 10.1167/iovs.15-16654.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The cacnb2 gene encodes the β2 subunit (Cavβ2) of voltage-gated Ca2+ channels in photoreceptors, and its targeted deletion in mice has previously been shown to cause altered retinal morphology and synaptic transmission. The purpose of this study was to provide a detailed morphologic study combined with experiments on the altered functions of photoreceptor ribbon synapses lacking Cavβ2.

Methods.: A cacnb2-deficient mouse strain was generated and deletion of the Cavβ2 in the retina documented by biochemical and immunhistochemical approaches. Ultrastructural changes of photoreceptor ribbon synapses were examined by electronmicroscopy and functional implications of the lack of Cavβ2 studied by depolarization-induced Ca2+ influx into isolated photoreceptor cells and electroretinography.

Results.: Voltage-gated Ca2+ influx into rod photoreceptors lacking Cavβ2 was abolished and the typical rod ribbon-type active zones were absent in Cavβ2-deficient retinas. The active zone and the architecture of the presynaptic terminals were severely altered in rod synapses. Cone photoreceptor and the bipolar cell ribbon synapses were largely spared from ultrastructural changes although peanut agglutinin (PNA) labelling and photopic ERG analyses demonstrated that also cone pathways were disturbed in Cavβ2-deficient retinas.

Conclusions.: The presence of the Cavβ2 is essential for the structural integrity and function of the rod photoreceptor synapse. The Cavβ2 is less essential for the morphology of cone and bipolar cell ribbon synapses, although the impaired photopic electroretinogram suggests a functional alteration also of the cone-mediated signaling in Cavβ2-deficient retinas.

Photoreceptors, like retinal bipolar cells, communicate with their secondary neurons via ribbon synapses, specialized chemical synapses that support tonic and phasic exocytosis. In photoreceptors the ribbon synapses translate light-triggered graded changes of membrane potential into modulations of a continuous synaptic vesicle exocytosis.1,2 Photoreceptors form large presynaptic terminals with a large synaptic ribbon attached to a single active zone. This active zone is characterized by an electron-dense projection, the “arciform density.” The protein RIBEYE is a major component of the synaptic ribbon,3–5 which is associated with large numbers of release-ready synaptic vesicles. Exocytosis in rod photoreceptor terminals preferentially occurs at the active zone, at the base of the synaptic ribbon, where voltage-gated Ca2+ channels are clustered. These channels are essential for synaptic vesicle exocytosis and retinal signaling.1,2,6–8 In photoreceptor synapses, the entire presynaptic plasma membrane is invaginated by a postsynaptic dendritic complex that contains the dendritic tips of horizontal and bipolar cells as well as presynaptic, dystroglycan-containing fingerlike-extensions that contact the postsynaptic dendrites. Cone terminals, like retinal bipolar cells, contain several ribbon-type active zones. 
The major Ca2+ channels at photoreceptor synapses consist of the pore-forming Cav1.4 α1-, a Cavβ-, and a Cavα2δ4-subunit.9–14 Mutations in CACNA1F, the gene encoding Cav1.4, cause incomplete X-linked congenital stationary night blindness type 2 (CSNB2),8,15–17 a human retinal disorder with abnormal electrophysiological response and visual impairments. Targeted disruption7 and spontaneous mutations6 of the mouse cacna1f revealed similar retinal dysfunction. Mutation in the Cavα2δ4 gene (CACNA2D4) causes autosomal recessive cone dystrophy in humans and cone–rod dysfunction in the visual system of mice.13,14 
Ball et al.18 identified Cavβ2 as the important Cavβ isoform in photoreceptor synapses. They showed that targeted deletion of the cacnb2 gene disrupts synaptic transmission between photoreceptors and bipolar cells as indicated by the absence of the electroretinogram b-wave.18–20 We now characterized the impact of Cavβ2 in respect to the ultrastructure of ribbon-type active zones in photoreceptor ribbon synapses in a mouse strain in which the lethal phenotype of cacnb2 deletion21 was rescued by a cardiomyocyte-specific expression of a Cavβ2 transgene. We show that Cavβ2 is essential for transmission of both, rod and cone photoreceptor ribbon synapses although only the architecture of the rod photoreceptor synapses are severely disrupted in Cavβ2-deficient retinas. 
Materials and Methods
Experimental Animals
All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research; all animal experiments were performed in accordance with German legislation on the protection of animals and were approved by the local ethics committee. For cardiomyocyte specific trangenesis, the β2aN4 cDNA22 was subcloned into the SalI-HindIII Alpha MyHC clone 26 cDNA fragment,23 which was kindly provided by Jeffrey Robbins, PhD (Cincinnati, OH, USA), and that comprise the mouse α myosin heavy-chain (MHC) minimal promoter (Fig. 1A). Four independent transgenic founder lines carrying the β2aN4 cDNA as transgene (β2+/+/β2tg/0) were obtained by standard procedures, genotyped by Southern blots, and mated with β2+/− mice,21 which are viable and fertile, to obtain viable β2−/− mice heterozygous for the β2aN4 transgene expressed. Figure 1B summarizes the β2aN4 transgene and the relevant region of the wild-type and the minus alleles of the β2 gene cacnb2. Genotyping was performed as indicated in Figure 1C using the following oligodesoxynucleotide primers: 1, 5′-GGC CAT CTC CTT CGA GGC-3′; 2, 5′-CAT CAT AAG GAG TGT GC-3′; 3, 5′-TAG CAA CAA GCA TCC TCG CAC-3′; 4, 5′-TGC ATC TTA TCA TGG ATG ATC C-3′; 5, 5′-GTC CAC ATA TCA CAT GTG TGC-3′. All four lines were able to rescue the lethal β2−/− phenotype and one of this lines was used in this study. Mice homozygous for the β2 minus allele (β2−/−) and heterozygous for the β2aN4 transgene (β2tg/0) are referred to as extra-cardiomyocyte β2 knock out mice (ecmβ2 ko). The ecmβ2 ko mice were compared with wild-type controls from the same litter and with 129B6F1 wild-type mice of mixed background (F1 generation of 129/SvJ and C57Bl6/N breedings). 
Figure 1
 
Cardiomyocyte-specific Cavβ2 transgene expression in Cavβ2−/− mice and Cavβ proteins in mouse retina. (A) Scheme of the Cavβ2 transgene used to rescue the lethal cacnb2−/− phenotype. The transgene comprises the cDNA of the Cavβ2 variant predominantly expressed in cardiomyocytes, Cavβ2aN4 (shown in yellow), comprising exon 2C encoding the N4-N-terminus and exons 3 to 14 including exon 7a. The expression of the Cavβ2 transgene is restricted to cardiomyocytes by the use of the mouse α MHC minimal promoter. The intergenic DNA fragment encompassing the α MHC promoter additionally contains the extreme 3′terminus of β MHC and the first three noncoding α-MHC exons. Polyadenylation is provided by the growth hormone (hGH) fragment, which contains approximately 100 bp of 3′UTR 5′ of the polyA signal, AATAAA. (B) The β2 transgene (tg), wild-type (+) allele and minus allele (−) including the position of oligodesoxynucleotides primers 1 to 4 used for genotyping. Exon and intron sequences are not in scale. (C) Summary of expected fragment sizes after genotyping (Table) and identification of the different fragments by agarose gel electrophoresis. The β2+/+ (+/+) and β2−/−/β2aN4tg/0 (−/−tg/0) genotypes refer to wild-type and heterozygous cardiomyocyte-specific transgenesis on β2-deficient genetic background, respectively. (D) Western blot of protein lysates from isolated cardiomyocytes (20 μg per lane) showing β2 wild-type (+/+) and β2 trangene (−/−tg/0) expression. (E) Western blot of protein lysates from isolated cardiomyocytes and cardiac fibroblasts. Expression of the proline 4-hydroxylase β gene (p4hb ∼57 kDa) was used as fibroblast-specific loading control. (F) Western blot of protein lysates from retina of either genotype using the indicated amount of proteins. The β2 protein is only present in wild-type but not in the −/−/tg/0 (ecmβ2 ko) genotype, whereas β3, β4, and β1 are present in equal amounts in retina from both genotypes. Expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (∼36 kDa) was used as loading control.
Figure 1
 
Cardiomyocyte-specific Cavβ2 transgene expression in Cavβ2−/− mice and Cavβ proteins in mouse retina. (A) Scheme of the Cavβ2 transgene used to rescue the lethal cacnb2−/− phenotype. The transgene comprises the cDNA of the Cavβ2 variant predominantly expressed in cardiomyocytes, Cavβ2aN4 (shown in yellow), comprising exon 2C encoding the N4-N-terminus and exons 3 to 14 including exon 7a. The expression of the Cavβ2 transgene is restricted to cardiomyocytes by the use of the mouse α MHC minimal promoter. The intergenic DNA fragment encompassing the α MHC promoter additionally contains the extreme 3′terminus of β MHC and the first three noncoding α-MHC exons. Polyadenylation is provided by the growth hormone (hGH) fragment, which contains approximately 100 bp of 3′UTR 5′ of the polyA signal, AATAAA. (B) The β2 transgene (tg), wild-type (+) allele and minus allele (−) including the position of oligodesoxynucleotides primers 1 to 4 used for genotyping. Exon and intron sequences are not in scale. (C) Summary of expected fragment sizes after genotyping (Table) and identification of the different fragments by agarose gel electrophoresis. The β2+/+ (+/+) and β2−/−/β2aN4tg/0 (−/−tg/0) genotypes refer to wild-type and heterozygous cardiomyocyte-specific transgenesis on β2-deficient genetic background, respectively. (D) Western blot of protein lysates from isolated cardiomyocytes (20 μg per lane) showing β2 wild-type (+/+) and β2 trangene (−/−tg/0) expression. (E) Western blot of protein lysates from isolated cardiomyocytes and cardiac fibroblasts. Expression of the proline 4-hydroxylase β gene (p4hb ∼57 kDa) was used as fibroblast-specific loading control. (F) Western blot of protein lysates from retina of either genotype using the indicated amount of proteins. The β2 protein is only present in wild-type but not in the −/−/tg/0 (ecmβ2 ko) genotype, whereas β3, β4, and β1 are present in equal amounts in retina from both genotypes. Expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (∼36 kDa) was used as loading control.
Table.
 
Primary Antibodies Used in This Study
Table.
 
Primary Antibodies Used in This Study
Biochemical Studies
Antibodies used for Western blots and immuncytochemistry are listed in the Table. The preparation of lysates and microsomal membrane proteins used for Western blot and immunoprecipitation have been described.22,24 
Immunocytochemistry
Immunocytochemistry was performed on cryostat sections exactly as previously described.5,25,26 Immunolabelled sections were analyzed with a Nikon A1R MP confocal microscope (Nikon GmbH, Düsseldorf, Germany) and the NIS elements software (Nikon GmbH). 
Transmission Electron Microscopy
Transmission electron microscopy was performed as previously described.27 Ultrathin sections were analyzed with a Tecnai 12 Biotwin digital transmission electron microscope (FEI, Hilsboro, OR, USA) operated at 100 kV. 
E-PTA Staining of Active Zones and Postsynaptic Densities
Ethanolic phosphotungstic acid (E-PTA) staining was performed exactly as previously described.28–30 This staining method allows a selective staining of presynaptic active zones and postsynaptic densities (PSDs). In the retina, also synaptic ribbons are stained by that method. Ultrasthin sections were stained for 10 minutes in lead citrate for optimal contrast. 
Electroretinography (ERG)
Electroretinographs were recorded binocularly from ecmβ2 ko mice at the age of 5 weeks postnatally as described previously.31 Mice were anaesthetized using a combination of Ketamine (66.7 mg/kg body weight) and Xylazine (11.7 mg/kg body weight). Their pupils were dilated and single-flash ERG responses were obtained under scotopic (dark-adapted overnight) and photopic (light adapted with a background illumination of 30 cd/m2, starting 10 minutes before recording) conditions. Single white-flash stimuli ranged from −4 to 1.5 log cd*s/m2 under scotopic and from −1.0 to 1.5 log cd*s/m2 under photopic conditions. Ten responses were averaged with interstimulus intervals of 5 seconds (for −4 to −0.5 log cd*s/m2) or 17 seconds (for 0–1.5 log cd*s/m2). 
Fura2-AM Ca2+ Imaging of Acutely Isolated Mouse Photoreceptor Cell Synaptic Terminals
Acutely isolated mouse photoreceptor cells from wild-type and ecmβ2 ko mice (isolated as described in Wahl et al.32) were plated on 2.5-cm glass coverslips and identified as rods based on their typical morphology and size of presynaptic terminals. Cells were incubated for 30 minutes at 37°C in low Ca2+ solution (LCS) (containing 132 mM NaCl, 3 mM KCl, 1 mM MgCl2 × 6H2O, 0.5 mM CaCl2, 10 mM sodium pyruvate, 10 mM glucose, and 10 mM HEPES, pH 7.4; ≈ 300 mOsm/L) to which 0.5% BSA and 5 μM Fura2-AM (Invitrogen, Darmstadt, Germany) have been added. Incubation was performed in the dark. LCS was saturated with 5%CO2/95%O2 before use. Fura2-AM-loaded photoreceptor cells were washed for 10 minutes in 1 mL LCS to remove excess dye prior to experiments. Ca2+ imaging experiments were performed at an inverted Zeiss Axiovert200 microscope (Jena, Germany) setup using a ×63 oil immersion objective (N.A. 1.4) and a camera-based imaging system from TILL Photonics (Oligochrome, Martinsried, Germany), equipped with a fast wavelength switcher. Fura-2 fluorescence emission was monitored at greater than 510 nm after excitation at 340 and 380 nm for 100 ms each at a rate of 0.2 Hz for up to 350 seconds. Synaptic terminals were marked and the ratio of the background-corrected Fura-2 fluorescence at 340 and 380 nm (F340/F380) were plotted versus time. Data acquisition and analysis were accomplished with the Live Acquisition (LA) imaging software vs. 2.4 (TILL Photonics). 
Results
Rescue of the Lethal Cavβ2−/− Phenotype by Cardiomyocyte-Specific Cavβ2 Trangene Expression
Of the four mammalian genes coding for Cavβ-subunits, cardiomyocytes predominantly express the cacnb2 gene encoding the Cavβ2 subunit. Disruption of the cacnb2 gene in the entire embryo18,21 or specifically in cardiomyocytes21 causes embryonic death. In order to characterize Cavβ2 functions in the retina, we rescued the lethal Cavβ2−/− phenotype by heterozygous expression of the major cardiac Cavβ2 isoform N422 as a transgene under the control of the α MHC promoter, which drives transgene expression in mouse cardiomyocytes (Figs. 1A–C). A similar approach has previously been pursued by Ball et al.18 using the Cavβ2a-N3 isoform as a transgene, which has also been denoted as Cavβ2a33 and which is expressed predominantly in the brain but not in the heart.22,34–36 
The mice of the genotype cacnb2−/− and heterozygous for the Cavβ2-N4 transgene (Cavβ2-N4tg/0) are viable. In wild-type hearts the cacnb2 gene is expressed in cardiomyocytes and in cardiac fibroblasts (Figs. 1D, 1E), but in the rescued mouse the transgene is only expressed in the cardiomyocytes but absent in the cardiac fibroblasts (Fig. 1D). Therefore, we denote these mice as extra-cardiomyocyte β2 ko mice (ecmβ2 ko) throughout this study. As shown in Figure 1F the Cavβ2 protein is not detectable in retina from ecmβ2 ko mice, whereas the Cavβ1, Cavβ3, and Cavβ4 proteins are present in very similar amounts as in wild-type. 
Characterization of ecmβ2 KO Mice by Immunofluorescence Light Microscopy
Using semithin resin sections, we noticed that the inner retina appeared largely unchanged at the level of semithin, Richardson-Blue stained sections. The most prominent changes were observed in the outer plexiform layer (OPL) that contains the photoreceptor synapses. The OPL of ecmβ2 ko mice was severely thinned and displayed a very irregular appearance (Fig. 2) as has been described on an independent mouse strain lacking the Cavβ2 protein in retina.18 
Figure 2
 
Retinal sections (0.5-μm thick) from control mice (A, C) and ecmβ2 ko mice (B, D). Major morphologic alterations can be observed in the outer retina of ecmβ2 ko mice. The outer segments (OS)/ inner segments (IS)/ONL is thinner than in control mice. A particular strong defect was observed in the OPL where the photoreceptor synapses are located. The inner retina appears largely unchanged. Scale bars: 150 μm (A–D). PE, pigment epithelium; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 2
 
Retinal sections (0.5-μm thick) from control mice (A, C) and ecmβ2 ko mice (B, D). Major morphologic alterations can be observed in the outer retina of ecmβ2 ko mice. The outer segments (OS)/ inner segments (IS)/ONL is thinner than in control mice. A particular strong defect was observed in the OPL where the photoreceptor synapses are located. The inner retina appears largely unchanged. Scale bars: 150 μm (A–D). PE, pigment epithelium; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
To characterize this phenotype we performed immunolabelling using antibodies against various components of retinal cells. Opsin remained restricted to the outer segments of photoreceptors both in 3-week-old ecmβ2 ko and control mice (Figs. 3A, 3B). Similar results were obtained with 9-month-old animals (data not shown). The vesicular glutamate transporter 1 (vglut1), a protein of the synaptic vesicles, was restricted to the synaptic layers also in the ecmβ2 ko mice indicating that the polarity of photoreceptors is principally maintained (Figs. 3E, 3F). Expression of connexin 36, a gap junction protein important for retinal physiology particularly in the inner retina37 was very similar in ecmβ2 ko and control animals (data not shown). Antibodies against glial fibrillary acidic protein (GFAP), an intermediate protein of activated Müller cells, indicate a slightly stronger GFAP expression in ecmβ2 ko retinas compared with controls (Figs. 3C, 3D). Remarkably, the neurofilament distribution estimated by immunolabelling with antibodies against the small neurofilament subunit was not changed in the outer retina of ecmβ2 ko mice (Figs. 3G, 3H). Neurofilaments are mainly expressed in horizontal cell processes in the outer retina.38 Their normal distribution in the ecmβ2 ko retina indicates absence of sprouting of secondary neurons into the outer nuclear layer (ONL), a frequent feature in mouse models of neurodegeneration (e.g., in mice deficient in genes encoding cysteine-string protein [CSP],38 Cav1.4,7 bassoon,39 or CAST40). 
Figure 3
 
Cryostat section of retinas from ecmβ2 ko and control mice immunolabelled with the indicated antibodies against opsin (A, B), GFAP (C, D), vesicular gultamate transporter 1 (vglut1; [E, F]), and small neurofilament subunit (NR4; [G, H]). In contrast to the Cav1.4 ko mouse, there is no significant sprouting of the immunolabelled horizontal cells into the ONL. The NR4-immunolabelled outer retinas of control (G) and ecmβ2 ko (H) appeared very similar. Scale bars: 10 μm (A, B); 20 μm (C–F); 5 μm (G, H). OLM, outer limiting membrane.
Figure 3
 
Cryostat section of retinas from ecmβ2 ko and control mice immunolabelled with the indicated antibodies against opsin (A, B), GFAP (C, D), vesicular gultamate transporter 1 (vglut1; [E, F]), and small neurofilament subunit (NR4; [G, H]). In contrast to the Cav1.4 ko mouse, there is no significant sprouting of the immunolabelled horizontal cells into the ONL. The NR4-immunolabelled outer retinas of control (G) and ecmβ2 ko (H) appeared very similar. Scale bars: 10 μm (A, B); 20 μm (C–F); 5 μm (G, H). OLM, outer limiting membrane.
However, RIBEYE, a main component of the synaptic ribbon, showed a strongly altered distribution in ecmβ2 ko retina. Whereas in the OPL of wild-type retinas, the typical horseshoe-shaped immunolabelling pattern of synaptic ribbons was present (Fig. 4A), abnormally few and rather dot-like-shaped RIBEYE-labelled synaptic ribbons were present in the OPL of ecmβ2 ko retinas (Fig. 4B), indicative of immature, unanchored ribbons.4 At the postsynapse, mGluR6 was abnormally localized (Figs. 4C, 4D). Whereas in the wild-type tissue, mGluR6 immunoreactive punctate were directly opposing the RIBEYE-immunolabelled active zones (Fig. 4C), in the ecmβ2 ko the number of mGluR6 containing puncta was strongly reduced (Fig. 4D) and only few RIBEYE and mGluR6-labelled structures could be identified. The reduced RIBEYE and mGluR6 immunolabelling in Figures 4B and 4D may correspond to reduced amounts of RIBEYE and mGluR6 proteins. Densitometric analyses of the immunostain of Western blots (Fig. 4E) reveals that the amount of RIBEYE protein in protein lysates obtained from retina was reduced by approximately 41% to 58.6 ± 5.8 (n = 6) compared with lysates from controls, whereas the amount of mGluR6 was not significantly different (n = 3). 
Figure 4
 
Cryostat section of retinas from ecmβ2 ko and control mice immunolabelled with antibodies against RIBEYE (A, B) and double-immunolabelled with the antibodies (polyclonal rabbit) against mGluR6 and against RIBEYE(B)-domain/CtBP2 (mouse monoclonal) (C, D) to label synaptic ribbons and Western blot using retina protein lysates ([E] protein per lane as indicated) from wild-type and ecmβ2 ko mice with the indicated antibodies. (C–D) Synaptic ribbons are largely absent from photoreceptor synapses in the OPL while ribbons were abundantly found in the inner retina, in the IPL. Rabbit antibodies were detected by goat anti-rabbit antibodies conjugated to Alexa568 (red channel); mouse antibodies were detected with chicken anti-mouse antibodies conjugated to Alexa488 (green channel). Scale bars: 20 μm (A, B); 5 μm (C, D).
Figure 4
 
Cryostat section of retinas from ecmβ2 ko and control mice immunolabelled with antibodies against RIBEYE (A, B) and double-immunolabelled with the antibodies (polyclonal rabbit) against mGluR6 and against RIBEYE(B)-domain/CtBP2 (mouse monoclonal) (C, D) to label synaptic ribbons and Western blot using retina protein lysates ([E] protein per lane as indicated) from wild-type and ecmβ2 ko mice with the indicated antibodies. (C–D) Synaptic ribbons are largely absent from photoreceptor synapses in the OPL while ribbons were abundantly found in the inner retina, in the IPL. Rabbit antibodies were detected by goat anti-rabbit antibodies conjugated to Alexa568 (red channel); mouse antibodies were detected with chicken anti-mouse antibodies conjugated to Alexa488 (green channel). Scale bars: 20 μm (A, B); 5 μm (C, D).
Abnormal Active Zones and Synaptic Architecture of Rod Photoreceptor Synapses
The reduction of RIBEYE protein in Western Blots and the light microscopy indicate a major disruption of the rod photoreceptor synapse. To further corroborate this finding we analyzed the ultrastructure of the photoreceptor synaptic terminals with transmission electron microscopy in 3 weeks (Fig. 5) and 9-month-old animals (Fig. 6). The photoreceptor presynaptic terminals of 3-week-old ecmβ2 ko retinas were located at their correct position in the OPL and synaptic vesicle density was normal, but the ultrastructural morphology of the presynaptic terminals was severely altered. No synaptic ribbon was present in the majority of the presynaptic terminals. In addition, the typical morphology of the photoreceptor synapse with the invaginated postsynaptic dendritic complex disappeared. The terminals were no longer invaginated by the postsynaptic dendritic plexus but flat. Similar results were obtained with retinas from 9-month- old animals (Fig. 6). The PSD-95 protein underlying the presynaptic photoreceptor plasma membrane41 was no longer restricted to the presynaptic terminal (Fig. 7A) but was also found in other photoreceptor compartments (e.g., at the outer limiting membrane; Fig. 7B). Dystroglycan is normally present close to the presynaptic ribbon in finger-like extensions of the photoreceptor terminal that protrude into the invaginated postsynaptic dendritic plexus but was almost absent in the synaptic complexes of photoreceptors from ecmβ2 ko animals (Figs. 7C, 7D). 
Figure 5
 
Transmission electron microscopy of photoreceptor synapses from 3-week-old animals (ecmβ2 ko and control mice, as indicated). (A, D) show photoreceptor synapses from control mice; (B, C, E, F) show photoreceptor synapses from ecmβ2 ko mice. Scale bars: 1 μm (A–C); 500 nm (D), 300 nm (E); 250 nm (F). sr, synaptic ribbon; ss, synaptic sphere; nu, photoreceptor nucleus; r, rod terminal; ho, bi, dendritic tips of bipolar cells and horizontal cells; v, vessel.
Figure 5
 
Transmission electron microscopy of photoreceptor synapses from 3-week-old animals (ecmβ2 ko and control mice, as indicated). (A, D) show photoreceptor synapses from control mice; (B, C, E, F) show photoreceptor synapses from ecmβ2 ko mice. Scale bars: 1 μm (A–C); 500 nm (D), 300 nm (E); 250 nm (F). sr, synaptic ribbon; ss, synaptic sphere; nu, photoreceptor nucleus; r, rod terminal; ho, bi, dendritic tips of bipolar cells and horizontal cells; v, vessel.
Figure 6
 
Transmission electron microscopy of photoreceptor synapses from 9-month-old animals (ecmβ2 ko and control mice, as indicated). (A, B) Show photoreceptor synapses from control mice; (C–F) show photoreceptor synapses from ecmβ2 ko mice. Scale bars: 500 nm (A, B), 1 μm (C, D), 500 nm (E, F).
Figure 6
 
Transmission electron microscopy of photoreceptor synapses from 9-month-old animals (ecmβ2 ko and control mice, as indicated). (A, B) Show photoreceptor synapses from control mice; (C–F) show photoreceptor synapses from ecmβ2 ko mice. Scale bars: 500 nm (A, B), 1 μm (C, D), 500 nm (E, F).
Figure 7
 
Cryostat section of retinas from control (A, C, E1–E3) and ecmβ2 ko mice (B, D, F1–F3) immunolabelled with antibodies against PSD-95 (A, B), a presynaptic scaffold protein, antibodies against β-dystroglycan (C, D), and double immunolabelled with antibodies against RIBEYE (E1/F1) and β-dystroglycan (E2/F2) to label synaptic ribbons and dystrophin/dystroglycan-positive fingerlike-extensions of the presynaptic terminal into the postsynaptic dendritic plexus. Scale bars: 10 μm (A, B), 5 μm (C, D), 2 μm (E1–E3, F1–F3).
Figure 7
 
Cryostat section of retinas from control (A, C, E1–E3) and ecmβ2 ko mice (B, D, F1–F3) immunolabelled with antibodies against PSD-95 (A, B), a presynaptic scaffold protein, antibodies against β-dystroglycan (C, D), and double immunolabelled with antibodies against RIBEYE (E1/F1) and β-dystroglycan (E2/F2) to label synaptic ribbons and dystrophin/dystroglycan-positive fingerlike-extensions of the presynaptic terminal into the postsynaptic dendritic plexus. Scale bars: 10 μm (A, B), 5 μm (C, D), 2 μm (E1–E3, F1–F3).
E-PTA staining reveals structural details of active zones and postsynaptic densities28,29,42,43 and was applied to further evaluate the structural changes at the active zones of rod photoreceptor synapses in retina samples from 3-week-old control and ecmβ2 ko mice. The synaptic ribbons, arciform densities and postsynaptic densities were clearly visible in photoreceptor synapses in control retinas, while, as expected, other components of the synapse (e.g., synaptic vesicles) were barely visible by E-PTA staining (Fig. 8).28,29,42,43 In contrast, the E-PTA stained structures gave a different pattern in ecmβ2 ko retinas. The absence of ribbons from rod synapses was confirmed but ribbons in cone synapses were still present. Most remarkably, a presynaptic active zone with dense projections was still detectable in ecmβ2 ko retina (Fig. 8), which resembled active zones from conventional synapses.28,29,44–46 The ultrastructural morphology of synaptic ribbons from cone synapses and synapses of the inner plexiform layer of the retina appeared unaffected (Figs. 8, 9) and no major ultrastructural changes were observed for cone photoreceptor and bipolar cell ribbon synapses (Figs. 8, 9), although peanut agglutinin (PNA) staining of ecmβ2 ko cone synapses was altered (Fig. 10). In summary, these results indicate that subtle morphologic alterations are also present at cone synapses. 
Figure 8
 
E-PTA–stained photoreceptor synapses of ecmβ2 ko and control mice. In photoreceptor synapses of control mice (A, B), synaptic ribbons and the arciform densities, the photoreceptor homologs of the presynaptic projections of active zones, are clearly visible. In photoreceptor synapses of ecmβ2 ko (C), synaptic ribbons are absent. Also, proper arciform densities are absent. Instead, dense projections as typically seen in conventional synapses are present (see also Fig. 9). Square/box in (C) is enlarged and rotated by 90° in (D) and (E), respectively. Cone photoreceptor synapses appeared unaffected by Cavβ2 deletion (F). Scale bars: 500 nm (A), 1 μm (B–D), 400 nm (E), 2.5 μm (F).
Figure 8
 
E-PTA–stained photoreceptor synapses of ecmβ2 ko and control mice. In photoreceptor synapses of control mice (A, B), synaptic ribbons and the arciform densities, the photoreceptor homologs of the presynaptic projections of active zones, are clearly visible. In photoreceptor synapses of ecmβ2 ko (C), synaptic ribbons are absent. Also, proper arciform densities are absent. Instead, dense projections as typically seen in conventional synapses are present (see also Fig. 9). Square/box in (C) is enlarged and rotated by 90° in (D) and (E), respectively. Cone photoreceptor synapses appeared unaffected by Cavβ2 deletion (F). Scale bars: 500 nm (A), 1 μm (B–D), 400 nm (E), 2.5 μm (F).
Figure 9
 
E-PTAstained synapses from the inner retina. (A, B) show ribbon synapses (A) and conventional synapses (B) in the IPL of control retinas. In (C, D) ribbon synapses in the IPL of ecmβ2 ko mice are demonstrated. In contrast to the OPL, ribbon synapses of the IPL appear largely unchanged. Scale bars: 100 nm (A–D).
Figure 9
 
E-PTAstained synapses from the inner retina. (A, B) show ribbon synapses (A) and conventional synapses (B) in the IPL of control retinas. In (C, D) ribbon synapses in the IPL of ecmβ2 ko mice are demonstrated. In contrast to the OPL, ribbon synapses of the IPL appear largely unchanged. Scale bars: 100 nm (A–D).
Figure 10
 
Cryostat section of ecmβ2 control (A, B) and ko mice (C, D) stained with fluorescent PNA to label cone synapses in the OPL. While cone synapses are readily visible in the OPL of control retinas (arrows in [A, B]), the cone-typical PNA signal is strongly reduced in the OPL of ecmβ2 ko mice (arrows in [C, D]). This finding indicates that cone synapses are also altered in ecmβ2 ko mice although they look normal at the ultrastructural level. Scale bars: 10 μm (A, D); 5 μm (B, C).
Figure 10
 
Cryostat section of ecmβ2 control (A, B) and ko mice (C, D) stained with fluorescent PNA to label cone synapses in the OPL. While cone synapses are readily visible in the OPL of control retinas (arrows in [A, B]), the cone-typical PNA signal is strongly reduced in the OPL of ecmβ2 ko mice (arrows in [C, D]). This finding indicates that cone synapses are also altered in ecmβ2 ko mice although they look normal at the ultrastructural level. Scale bars: 10 μm (A, D); 5 μm (B, C).
Synaptic Transmission at the Photoreceptor Synapse
It is unlikely that synaptic transmission can take place between rod photoreceptors and postsynaptic neurons considering the morphologic changes in retina from ecmβ2 ko mice. We therefore recorded depolarization-induced Ca2+ influx in acutely isolated photoreceptor cell synaptic terminals and performed full-field ERG (Fig. 11). We found that basic Ca2+ levels (Fura2 F340/F380 ratio) were not different in cells isolated from wild-type and ecmβ2 ko mice, but a major reduction of depolarization-induced Ca2+ influx (Figs. 11A, 11B) indicating disruption of functional voltage-gated Ca2+ channels. 
Figure 11
 
Depolarization-induced Ca2+ influx in acutely isolated photoreceptor cell synaptic terminals (A, B) and full-field ERG (CF). Ratiometric Ca2+ imaging from Fura-2-loaded synaptic terminals of photoreceptor cells isolated from ecmβ2 ko (red) and wild-type mice (black). (A) Changes of Fura-2 F340/F380 ratio normalized to the ratio right before application of 50 mM KCl. Cells were bathed in low Ca2+ solution and 2 mM Ca2+ and 50 mM KCl were added as indicated. (B) F340/F380 (i.e., intracellular Ca2+) at low external Ca2+, 2 mM Ca2+, and upon 50 mM KCl-dependent depolarization in the presence of 2 mM Ca2+. Data represent means ± SEM of the indicated number of experiments. *Indicates a significant difference (P < 0.05) between ecmβ2 ko and wild-type. (C–F) Functional assessment of ecmβ2 ko and corresponding control mice based on electroretinography. Representative ERG intensity series for scotopic (dark-adapted, [C] and photopic [light-adapted, (E)]) responses in wild-type (black) and ecmβ2 ko mice (red). Quantitative evaluation of the scotopic (D) and photopic (F) b-wave amplitude data, shown as box- and whisker-plot, for the entire group (controls, n = 4; ecmβ2 ko mice, n = 3).
Figure 11
 
Depolarization-induced Ca2+ influx in acutely isolated photoreceptor cell synaptic terminals (A, B) and full-field ERG (CF). Ratiometric Ca2+ imaging from Fura-2-loaded synaptic terminals of photoreceptor cells isolated from ecmβ2 ko (red) and wild-type mice (black). (A) Changes of Fura-2 F340/F380 ratio normalized to the ratio right before application of 50 mM KCl. Cells were bathed in low Ca2+ solution and 2 mM Ca2+ and 50 mM KCl were added as indicated. (B) F340/F380 (i.e., intracellular Ca2+) at low external Ca2+, 2 mM Ca2+, and upon 50 mM KCl-dependent depolarization in the presence of 2 mM Ca2+. Data represent means ± SEM of the indicated number of experiments. *Indicates a significant difference (P < 0.05) between ecmβ2 ko and wild-type. (C–F) Functional assessment of ecmβ2 ko and corresponding control mice based on electroretinography. Representative ERG intensity series for scotopic (dark-adapted, [C] and photopic [light-adapted, (E)]) responses in wild-type (black) and ecmβ2 ko mice (red). Quantitative evaluation of the scotopic (D) and photopic (F) b-wave amplitude data, shown as box- and whisker-plot, for the entire group (controls, n = 4; ecmβ2 ko mice, n = 3).
Full-field ERG measurements allow the assessment of both scotopic (i.e., dark adapted, rod-dominated) and photopic (i.e., light adapted, usually cone-driven) responses.31 We observed that the ERG amplitudes of ecmβ2 ko mice are greatly reduced under both scotopic and photopic conditions (Figs. 11C–F). In the scotopic standard flash recording, the b-wave was severely reduced, and a small remaining component became visible at −2 log (cd*s/m2) stimulus intensity (Figs. 11C, 11D). Under photopic, cone-dominated conditions no ERG amplitudes could be observed (Figs. 11E, 11F). As the positive b-wave is generated mainly by ON-bipolar cells, these results indicate that signal transmission from photoreceptors to ON-bipolar cells is strongly diminished. This is supported by the findings that the Ca2+ influx is abolished (Figs. 11A, 11B) and eventually, glutamate release being disturbed. 
Discussion
Here, we show that in rod photoreceptors, synaptic ribbons completely disappeared from the rod presynaptic terminals in Cavβ2-deficient retinas, while synaptic vesicles and their density were normal. The morphologic changes were already apparent at 3-week-old animals indicating that the loss of the synaptic ribbon complex is not caused by degeneration. Even in samples from 9-month-old mice very few, if any, signs of synaptic degeneration resembling human CSNB2 were apparent. These results point to a Cavβ2 function for anchoring and/or stabilization of synaptic ribbons in rod synapses. This assumption is further supported by our finding that ribbons never developed appropriately even early during development. In the absence of Cavβ2, only conventional, active zones were formed by rod photoreceptors but not the typical ribbon-type active zones although E-PTA staining demonstrated that active zones were not completely absent. Presynaptic dense projections that looked similar to presynaptic grids in conventional synapses were still present in Cavβ2-deficient retinas, but the ribbon-type active zone was never built in rod photoreceptors. Taken together these morphologic findings demonstrate that the Cavβ2 is essential for the development of ribbon-type active zones in rod photoreceptor synapses. Furthermore, the level of RIBEYE protein was reduced in Cavβ2-deficient retinas. Thus, both structure and expression levels of RIBEYE strongly depend upon Cavβ2. There was no complete reduction in the expression of RIBEYE protein because synaptic ribbons in the inner retina were unaffected by Cavβ2 deletion. Interestingly, while clustering of postsynaptic mGluR6 was strongly altered in the OPL of Cavβ2-deficient mice, overall protein levels were unchanged in Western blot analyses. These data indicate that the mGluR6 receptor is still expressed but no longer enriched at the postsynaptic complex of the photoreceptor synapse. The loss of the ribbon-type active zone goes in parallel with a disruption of functional voltage-gated Ca2+ channels because depolarization-induced Ca2+ entry is abolished in the presynaptic terminals of Cavβ2-deficient photoreceptors. 
The Cavβ2 protein contributes to the targeting of the pore forming Cav1.4 protein to the plasma membrane and modulates the kinetics of Cav currents. Accordingly, most of the described phenotypes could be explained by the lack of the Cav1.4 protein at the active zone. Like the deletion of Cavβ2 the deletion of the Cav1.4 subunit leads to a developmental malformation of the synaptic ribbon complex.7,47 But unlike in the Cav1.4-deficient retina, no sprouting of secondary neurons is detectable in the ecmβ2 ko retina. Sprouting of retinal bipolar and horizontal cells is a common feature in Cav1.4 ko mice7 and the lack of sprouting in ecmβ2 ko could be caused by some residual Cav1 protein present at the photoreceptor active zone as it is the case in the inner ear (for Cav1.3) when Cavβ2 is absent.48 Like in Cav1.4-deficient retina,49 rearrangement of the PSD-95 and dystrophin/dystroglycan-based synaptic cytoskeleton was observed in ecmβ2 ko mice. These data demonstrate that intact, fully-assembled Cav channels are essential not only for initiating synaptic vesicle exocytosis by Ca2+-influx but also contribute as an important structural component to the morphological architecture of the presynaptic terminal. 
Accordingly, the loss of the invaginated shape of the presynaptic terminal and its conversion into a flat form could be a consequence of a disturbed connection between the Cav-organized presynaptic cytoskeleton and the extracellular synaptic matrix. Such a molecular link has been demonstrated for the neuromuscular junction.50–54 Because most of those synaptic components are also present in the photoreceptor synapse,50,52,54 the synaptic architecture of the photoreceptor synapse might be influenced by a similar protein network. Remarkably, cone photoreceptors and retinal bipolar cells appeared to be spared from major structural changes. These neurons probably could employ different Cavβ isoforms.19 But this issue has not yet been thoroughly investigated. 
Deletion of just one β subunit, Cavβ2, eliminates depolarization-dependent Ca2+ influx in photoreceptor cells even though Cavβ1, Cavβ3, and Cavβ4 are still present in the retina19 (Fig. 1F). This finding indicates that it is mainly the Cavβ2, which in photoreceptors is associated with Cav1.4 and that the other βs do not compensate for the loss of Cavβ2 in these cells. A preferred association of a β subunit and a given Cavα1 subunit has also been observed in cardiac myocytes (Cavβ2 and Cav1.221,22,24), skeletal muscle (Cavβ1 and Cav1.155), and inner hair cells (Cavβ2 and Cav1.348), but, in most cases, Cavβs seem not to prefer a given Cavα1.56 Additional investigations are required to solve this issue and to answer the questions whether the lack of Ca current is the cause of structural abnormality observed in the ecmβ2 ko mice or whether the heteromultimeric Ca2+ channel complex may be an indispensable component of the pre- and postsynaptic structure. 
Although the ultrastructure of cone synapses and bipolar cell ribbon synapses appeared completely normal cone function was also severely affected in retina of ecmβ2 ko mice according to photopic ERG recordings (Fig. 11). Only the PNA staining of ecmβ2 ko cone synapses were different compared with controls indicating that Cavβ2 is also important for cone synapse function but less essential as structural component for cone synapse architecture. Eventually, other Cavβ-subunits may substitute Cavβ2 for this structural role in cone photoreceptor synapses but cannot compensate defects in voltage-gated Ca2+ entry. Alternatively, various Cavβ-subunits may be integrated in a differential manner in the presynaptic cytoskeleton of rod and cone photoreceptor synapses. Future studies have to discriminate between these possibilities. 
Acknowledgments
The authors thank Stefanie Buchholz, Sandra Plant, Sylvia Brundaler, and Gabi Kiefer for excellent technical assistance. They also thank Tanja Volz and Tom Janke for mouse breeding. 
This work was supported by the German Research Foundation, DFG (HFSP grant [Strasbourg, France] to FS; CRC894 to FS, AB, PW and VF; RTG 1326 to FS, AB, PW, and VF; Bonn, Germany). 
Disclosure: R. Katiyar, None; P. Weissgerber, None; E. Roth, None; J. Dörr, None; V. Sothilingam, None; M. Garcia Garrido, None; S.C. Beck, None; M.W. Seeliger, None; A. Beck, None; F. Schmitz, None; V. Flockerzi, None 
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Figure 1
 
Cardiomyocyte-specific Cavβ2 transgene expression in Cavβ2−/− mice and Cavβ proteins in mouse retina. (A) Scheme of the Cavβ2 transgene used to rescue the lethal cacnb2−/− phenotype. The transgene comprises the cDNA of the Cavβ2 variant predominantly expressed in cardiomyocytes, Cavβ2aN4 (shown in yellow), comprising exon 2C encoding the N4-N-terminus and exons 3 to 14 including exon 7a. The expression of the Cavβ2 transgene is restricted to cardiomyocytes by the use of the mouse α MHC minimal promoter. The intergenic DNA fragment encompassing the α MHC promoter additionally contains the extreme 3′terminus of β MHC and the first three noncoding α-MHC exons. Polyadenylation is provided by the growth hormone (hGH) fragment, which contains approximately 100 bp of 3′UTR 5′ of the polyA signal, AATAAA. (B) The β2 transgene (tg), wild-type (+) allele and minus allele (−) including the position of oligodesoxynucleotides primers 1 to 4 used for genotyping. Exon and intron sequences are not in scale. (C) Summary of expected fragment sizes after genotyping (Table) and identification of the different fragments by agarose gel electrophoresis. The β2+/+ (+/+) and β2−/−/β2aN4tg/0 (−/−tg/0) genotypes refer to wild-type and heterozygous cardiomyocyte-specific transgenesis on β2-deficient genetic background, respectively. (D) Western blot of protein lysates from isolated cardiomyocytes (20 μg per lane) showing β2 wild-type (+/+) and β2 trangene (−/−tg/0) expression. (E) Western blot of protein lysates from isolated cardiomyocytes and cardiac fibroblasts. Expression of the proline 4-hydroxylase β gene (p4hb ∼57 kDa) was used as fibroblast-specific loading control. (F) Western blot of protein lysates from retina of either genotype using the indicated amount of proteins. The β2 protein is only present in wild-type but not in the −/−/tg/0 (ecmβ2 ko) genotype, whereas β3, β4, and β1 are present in equal amounts in retina from both genotypes. Expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (∼36 kDa) was used as loading control.
Figure 1
 
Cardiomyocyte-specific Cavβ2 transgene expression in Cavβ2−/− mice and Cavβ proteins in mouse retina. (A) Scheme of the Cavβ2 transgene used to rescue the lethal cacnb2−/− phenotype. The transgene comprises the cDNA of the Cavβ2 variant predominantly expressed in cardiomyocytes, Cavβ2aN4 (shown in yellow), comprising exon 2C encoding the N4-N-terminus and exons 3 to 14 including exon 7a. The expression of the Cavβ2 transgene is restricted to cardiomyocytes by the use of the mouse α MHC minimal promoter. The intergenic DNA fragment encompassing the α MHC promoter additionally contains the extreme 3′terminus of β MHC and the first three noncoding α-MHC exons. Polyadenylation is provided by the growth hormone (hGH) fragment, which contains approximately 100 bp of 3′UTR 5′ of the polyA signal, AATAAA. (B) The β2 transgene (tg), wild-type (+) allele and minus allele (−) including the position of oligodesoxynucleotides primers 1 to 4 used for genotyping. Exon and intron sequences are not in scale. (C) Summary of expected fragment sizes after genotyping (Table) and identification of the different fragments by agarose gel electrophoresis. The β2+/+ (+/+) and β2−/−/β2aN4tg/0 (−/−tg/0) genotypes refer to wild-type and heterozygous cardiomyocyte-specific transgenesis on β2-deficient genetic background, respectively. (D) Western blot of protein lysates from isolated cardiomyocytes (20 μg per lane) showing β2 wild-type (+/+) and β2 trangene (−/−tg/0) expression. (E) Western blot of protein lysates from isolated cardiomyocytes and cardiac fibroblasts. Expression of the proline 4-hydroxylase β gene (p4hb ∼57 kDa) was used as fibroblast-specific loading control. (F) Western blot of protein lysates from retina of either genotype using the indicated amount of proteins. The β2 protein is only present in wild-type but not in the −/−/tg/0 (ecmβ2 ko) genotype, whereas β3, β4, and β1 are present in equal amounts in retina from both genotypes. Expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (∼36 kDa) was used as loading control.
Figure 2
 
Retinal sections (0.5-μm thick) from control mice (A, C) and ecmβ2 ko mice (B, D). Major morphologic alterations can be observed in the outer retina of ecmβ2 ko mice. The outer segments (OS)/ inner segments (IS)/ONL is thinner than in control mice. A particular strong defect was observed in the OPL where the photoreceptor synapses are located. The inner retina appears largely unchanged. Scale bars: 150 μm (A–D). PE, pigment epithelium; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 2
 
Retinal sections (0.5-μm thick) from control mice (A, C) and ecmβ2 ko mice (B, D). Major morphologic alterations can be observed in the outer retina of ecmβ2 ko mice. The outer segments (OS)/ inner segments (IS)/ONL is thinner than in control mice. A particular strong defect was observed in the OPL where the photoreceptor synapses are located. The inner retina appears largely unchanged. Scale bars: 150 μm (A–D). PE, pigment epithelium; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 3
 
Cryostat section of retinas from ecmβ2 ko and control mice immunolabelled with the indicated antibodies against opsin (A, B), GFAP (C, D), vesicular gultamate transporter 1 (vglut1; [E, F]), and small neurofilament subunit (NR4; [G, H]). In contrast to the Cav1.4 ko mouse, there is no significant sprouting of the immunolabelled horizontal cells into the ONL. The NR4-immunolabelled outer retinas of control (G) and ecmβ2 ko (H) appeared very similar. Scale bars: 10 μm (A, B); 20 μm (C–F); 5 μm (G, H). OLM, outer limiting membrane.
Figure 3
 
Cryostat section of retinas from ecmβ2 ko and control mice immunolabelled with the indicated antibodies against opsin (A, B), GFAP (C, D), vesicular gultamate transporter 1 (vglut1; [E, F]), and small neurofilament subunit (NR4; [G, H]). In contrast to the Cav1.4 ko mouse, there is no significant sprouting of the immunolabelled horizontal cells into the ONL. The NR4-immunolabelled outer retinas of control (G) and ecmβ2 ko (H) appeared very similar. Scale bars: 10 μm (A, B); 20 μm (C–F); 5 μm (G, H). OLM, outer limiting membrane.
Figure 4
 
Cryostat section of retinas from ecmβ2 ko and control mice immunolabelled with antibodies against RIBEYE (A, B) and double-immunolabelled with the antibodies (polyclonal rabbit) against mGluR6 and against RIBEYE(B)-domain/CtBP2 (mouse monoclonal) (C, D) to label synaptic ribbons and Western blot using retina protein lysates ([E] protein per lane as indicated) from wild-type and ecmβ2 ko mice with the indicated antibodies. (C–D) Synaptic ribbons are largely absent from photoreceptor synapses in the OPL while ribbons were abundantly found in the inner retina, in the IPL. Rabbit antibodies were detected by goat anti-rabbit antibodies conjugated to Alexa568 (red channel); mouse antibodies were detected with chicken anti-mouse antibodies conjugated to Alexa488 (green channel). Scale bars: 20 μm (A, B); 5 μm (C, D).
Figure 4
 
Cryostat section of retinas from ecmβ2 ko and control mice immunolabelled with antibodies against RIBEYE (A, B) and double-immunolabelled with the antibodies (polyclonal rabbit) against mGluR6 and against RIBEYE(B)-domain/CtBP2 (mouse monoclonal) (C, D) to label synaptic ribbons and Western blot using retina protein lysates ([E] protein per lane as indicated) from wild-type and ecmβ2 ko mice with the indicated antibodies. (C–D) Synaptic ribbons are largely absent from photoreceptor synapses in the OPL while ribbons were abundantly found in the inner retina, in the IPL. Rabbit antibodies were detected by goat anti-rabbit antibodies conjugated to Alexa568 (red channel); mouse antibodies were detected with chicken anti-mouse antibodies conjugated to Alexa488 (green channel). Scale bars: 20 μm (A, B); 5 μm (C, D).
Figure 5
 
Transmission electron microscopy of photoreceptor synapses from 3-week-old animals (ecmβ2 ko and control mice, as indicated). (A, D) show photoreceptor synapses from control mice; (B, C, E, F) show photoreceptor synapses from ecmβ2 ko mice. Scale bars: 1 μm (A–C); 500 nm (D), 300 nm (E); 250 nm (F). sr, synaptic ribbon; ss, synaptic sphere; nu, photoreceptor nucleus; r, rod terminal; ho, bi, dendritic tips of bipolar cells and horizontal cells; v, vessel.
Figure 5
 
Transmission electron microscopy of photoreceptor synapses from 3-week-old animals (ecmβ2 ko and control mice, as indicated). (A, D) show photoreceptor synapses from control mice; (B, C, E, F) show photoreceptor synapses from ecmβ2 ko mice. Scale bars: 1 μm (A–C); 500 nm (D), 300 nm (E); 250 nm (F). sr, synaptic ribbon; ss, synaptic sphere; nu, photoreceptor nucleus; r, rod terminal; ho, bi, dendritic tips of bipolar cells and horizontal cells; v, vessel.
Figure 6
 
Transmission electron microscopy of photoreceptor synapses from 9-month-old animals (ecmβ2 ko and control mice, as indicated). (A, B) Show photoreceptor synapses from control mice; (C–F) show photoreceptor synapses from ecmβ2 ko mice. Scale bars: 500 nm (A, B), 1 μm (C, D), 500 nm (E, F).
Figure 6
 
Transmission electron microscopy of photoreceptor synapses from 9-month-old animals (ecmβ2 ko and control mice, as indicated). (A, B) Show photoreceptor synapses from control mice; (C–F) show photoreceptor synapses from ecmβ2 ko mice. Scale bars: 500 nm (A, B), 1 μm (C, D), 500 nm (E, F).
Figure 7
 
Cryostat section of retinas from control (A, C, E1–E3) and ecmβ2 ko mice (B, D, F1–F3) immunolabelled with antibodies against PSD-95 (A, B), a presynaptic scaffold protein, antibodies against β-dystroglycan (C, D), and double immunolabelled with antibodies against RIBEYE (E1/F1) and β-dystroglycan (E2/F2) to label synaptic ribbons and dystrophin/dystroglycan-positive fingerlike-extensions of the presynaptic terminal into the postsynaptic dendritic plexus. Scale bars: 10 μm (A, B), 5 μm (C, D), 2 μm (E1–E3, F1–F3).
Figure 7
 
Cryostat section of retinas from control (A, C, E1–E3) and ecmβ2 ko mice (B, D, F1–F3) immunolabelled with antibodies against PSD-95 (A, B), a presynaptic scaffold protein, antibodies against β-dystroglycan (C, D), and double immunolabelled with antibodies against RIBEYE (E1/F1) and β-dystroglycan (E2/F2) to label synaptic ribbons and dystrophin/dystroglycan-positive fingerlike-extensions of the presynaptic terminal into the postsynaptic dendritic plexus. Scale bars: 10 μm (A, B), 5 μm (C, D), 2 μm (E1–E3, F1–F3).
Figure 8
 
E-PTA–stained photoreceptor synapses of ecmβ2 ko and control mice. In photoreceptor synapses of control mice (A, B), synaptic ribbons and the arciform densities, the photoreceptor homologs of the presynaptic projections of active zones, are clearly visible. In photoreceptor synapses of ecmβ2 ko (C), synaptic ribbons are absent. Also, proper arciform densities are absent. Instead, dense projections as typically seen in conventional synapses are present (see also Fig. 9). Square/box in (C) is enlarged and rotated by 90° in (D) and (E), respectively. Cone photoreceptor synapses appeared unaffected by Cavβ2 deletion (F). Scale bars: 500 nm (A), 1 μm (B–D), 400 nm (E), 2.5 μm (F).
Figure 8
 
E-PTA–stained photoreceptor synapses of ecmβ2 ko and control mice. In photoreceptor synapses of control mice (A, B), synaptic ribbons and the arciform densities, the photoreceptor homologs of the presynaptic projections of active zones, are clearly visible. In photoreceptor synapses of ecmβ2 ko (C), synaptic ribbons are absent. Also, proper arciform densities are absent. Instead, dense projections as typically seen in conventional synapses are present (see also Fig. 9). Square/box in (C) is enlarged and rotated by 90° in (D) and (E), respectively. Cone photoreceptor synapses appeared unaffected by Cavβ2 deletion (F). Scale bars: 500 nm (A), 1 μm (B–D), 400 nm (E), 2.5 μm (F).
Figure 9
 
E-PTAstained synapses from the inner retina. (A, B) show ribbon synapses (A) and conventional synapses (B) in the IPL of control retinas. In (C, D) ribbon synapses in the IPL of ecmβ2 ko mice are demonstrated. In contrast to the OPL, ribbon synapses of the IPL appear largely unchanged. Scale bars: 100 nm (A–D).
Figure 9
 
E-PTAstained synapses from the inner retina. (A, B) show ribbon synapses (A) and conventional synapses (B) in the IPL of control retinas. In (C, D) ribbon synapses in the IPL of ecmβ2 ko mice are demonstrated. In contrast to the OPL, ribbon synapses of the IPL appear largely unchanged. Scale bars: 100 nm (A–D).
Figure 10
 
Cryostat section of ecmβ2 control (A, B) and ko mice (C, D) stained with fluorescent PNA to label cone synapses in the OPL. While cone synapses are readily visible in the OPL of control retinas (arrows in [A, B]), the cone-typical PNA signal is strongly reduced in the OPL of ecmβ2 ko mice (arrows in [C, D]). This finding indicates that cone synapses are also altered in ecmβ2 ko mice although they look normal at the ultrastructural level. Scale bars: 10 μm (A, D); 5 μm (B, C).
Figure 10
 
Cryostat section of ecmβ2 control (A, B) and ko mice (C, D) stained with fluorescent PNA to label cone synapses in the OPL. While cone synapses are readily visible in the OPL of control retinas (arrows in [A, B]), the cone-typical PNA signal is strongly reduced in the OPL of ecmβ2 ko mice (arrows in [C, D]). This finding indicates that cone synapses are also altered in ecmβ2 ko mice although they look normal at the ultrastructural level. Scale bars: 10 μm (A, D); 5 μm (B, C).
Figure 11
 
Depolarization-induced Ca2+ influx in acutely isolated photoreceptor cell synaptic terminals (A, B) and full-field ERG (CF). Ratiometric Ca2+ imaging from Fura-2-loaded synaptic terminals of photoreceptor cells isolated from ecmβ2 ko (red) and wild-type mice (black). (A) Changes of Fura-2 F340/F380 ratio normalized to the ratio right before application of 50 mM KCl. Cells were bathed in low Ca2+ solution and 2 mM Ca2+ and 50 mM KCl were added as indicated. (B) F340/F380 (i.e., intracellular Ca2+) at low external Ca2+, 2 mM Ca2+, and upon 50 mM KCl-dependent depolarization in the presence of 2 mM Ca2+. Data represent means ± SEM of the indicated number of experiments. *Indicates a significant difference (P < 0.05) between ecmβ2 ko and wild-type. (C–F) Functional assessment of ecmβ2 ko and corresponding control mice based on electroretinography. Representative ERG intensity series for scotopic (dark-adapted, [C] and photopic [light-adapted, (E)]) responses in wild-type (black) and ecmβ2 ko mice (red). Quantitative evaluation of the scotopic (D) and photopic (F) b-wave amplitude data, shown as box- and whisker-plot, for the entire group (controls, n = 4; ecmβ2 ko mice, n = 3).
Figure 11
 
Depolarization-induced Ca2+ influx in acutely isolated photoreceptor cell synaptic terminals (A, B) and full-field ERG (CF). Ratiometric Ca2+ imaging from Fura-2-loaded synaptic terminals of photoreceptor cells isolated from ecmβ2 ko (red) and wild-type mice (black). (A) Changes of Fura-2 F340/F380 ratio normalized to the ratio right before application of 50 mM KCl. Cells were bathed in low Ca2+ solution and 2 mM Ca2+ and 50 mM KCl were added as indicated. (B) F340/F380 (i.e., intracellular Ca2+) at low external Ca2+, 2 mM Ca2+, and upon 50 mM KCl-dependent depolarization in the presence of 2 mM Ca2+. Data represent means ± SEM of the indicated number of experiments. *Indicates a significant difference (P < 0.05) between ecmβ2 ko and wild-type. (C–F) Functional assessment of ecmβ2 ko and corresponding control mice based on electroretinography. Representative ERG intensity series for scotopic (dark-adapted, [C] and photopic [light-adapted, (E)]) responses in wild-type (black) and ecmβ2 ko mice (red). Quantitative evaluation of the scotopic (D) and photopic (F) b-wave amplitude data, shown as box- and whisker-plot, for the entire group (controls, n = 4; ecmβ2 ko mice, n = 3).
Table.
 
Primary Antibodies Used in This Study
Table.
 
Primary Antibodies Used in This Study
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