Ribeye a-mCherry fusion protein: A novel tool for labeling synaptic ribbons of the hair cell

Journal of Neuroscience Methods 197 (2011) 274–278

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Ribeye a-mCherry fusion protein: A novel tool for labeling synaptic ribbons of the hair cell Megan C. West a,b , Brian M. McDermott Jr. a,b,c,d,∗ a

Department of Otolaryngology-Head and Neck Surgery, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA Department of Biology, Case Western Reserve University, Cleveland, OH 44106, USA c Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA d Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA b

a r t i c l e

i n f o

Article history: Received 5 June 2010 Received in revised form 14 November 2010 Accepted 20 November 2010 Keywords: Balance Hair cell Hearing Transgenic Zebrafish

a b s t r a c t Synaptic ribbons are presynaptic cytomatrices that are required for efficient transfer of auditory information from hair cells to the central nervous system. In the hair cell, each electron-dense ribbon tethers numerous synaptic vesicles by fine filaments. The ribbon generally resides juxtaposed to the active zone plasma membrane. A dearth of appropriate tools to visualize the ribbon synapse has limited our knowledge of its development. Here we present the design and implementation of a method to visualize synaptic ribbons in hair cells. This scheme uses a tagged version of the protein Ribeye a, which is specific to ribbons. We generate the DNA construct Tg(pvalb3b:ribeye a-mCherry) to transgenically express the fusion protein Ribeye a-mCherry in zebrafish hair cells. The fusion protein localizes to the basolateral surface of the hair cell with a pattern similar to that of a hair cell labeled with an antiserum that recognizes ribeye proteins. Moreover, using this antiserum to label transgenics that express Ribeye a-mCherry, we demonstrate that the fusion protein and antibody-associated fluorescent signals overlap. In addition, ribbons labeled with the fusion protein are proximal to afferent nerve endings. Finally, the fusion protein labels hair-cell ribbons of zebrafish at different developmental time points. These findings indicate that the fusion protein is an effective tool to label ribbons in live and fixed hair cells, which will make it useful in the study of ribbon synapse development and to characterize zebrafish mutants with defects in synapse formation. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Chemical synapses are asymmetric contact sites between cells that translate presynaptic electrical signals into chemical signals. Communication from sensory receptor cells of the auditory, vestibular, and visual systems to associated afferent neurons occurs through a specialized form of the chemical synapse known as the ribbon synapse (Fuchs et al., 2003; Moser et al., 2006; tom Dieck and Brandstatter, 2006; Schmitz, 2009). Hair cells are sensory receptors, which are indispensable for hearing, balance, and, in some aquatic vertebrates, the detection of water movement. These sensory cells transduce mechanical stimuli into electrical signals and then forward the information, as exocytosed neurotransmitter, across synaptic clefts to afferent nerve endings.

∗ Corresponding author at: Department of Otolaryngology-Head and Neck Surgery, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Tel.: +1 216 844 6036; fax: +1 216 844 5727. E-mail address: [email protected] (B.M. McDermott Jr.). 0165-0270/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2010.11.011

The presynaptic active zone of the chemical synapse is comprised of two major regions: the active zone plasma membrane and the cytomatrix at the active zone (CAZ) (Fejtova and Gundelfinger, 2006). The synaptic ribbon, also termed the presynaptic dense body, is a large CAZ that is shaped like a sphere or a ribbon, depending on the cell type and the species in which it is found (Fuchs et al., 2003; Moser et al., 2006; tom Dieck and Brandstatter, 2006; Schmitz, 2009). Contained within the hair cell, each ribbon is generally oriented adjacent to its active zone plasma membrane (Flock and Jorgensen, 1974). In addition, each ribbon has numerous synaptic vesicles tethered to it by fine filaments (Lenzi et al., 2002). A current model of ribbon function suggests that a major role of this structure is in trafficking synaptic vesicles from the cytoplasm to the active zone plasma membrane (Parsons and Sterling, 2003; Schnee et al., 2005). Another model posits that a ribbon acts to position synaptic vesicles in close proximity to each other to facilitate multivesicular release of neurotransmitter (Parsons and Sterling, 2003). The ribbon synapse contains many of the same proteins that are found at the conventional synapse; however, the RIBEYE protein is found specifically at the ribbon synapse (Schmitz et al., 2000; Piatigorsky, 2001). It is hypothesized that RIBEYE homo-

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multimerizes to form an array that serves as a framework for the ribbon (Magupalli et al., 2008). Zebrafish have two paralogous ribeye genes, ribeye a and ribeye b, which encode proteins with high amino acid sequence similarity to each other and to rat RIBEYE (Schmitz et al., 2000; Wan et al., 2005). Both zebrafish paralogs are expressed in the ear (Wan et al., 2005; Obholzer et al., 2008). The zebrafish is a valuable model for examining the molecular and cellular mechanisms of hearing and balance (Nicolson, 2005). Zebrafish hair cells are morphologically similar to those of amphibians, birds, and mammals. Cross-species similarities also extend to the ribbon synapse (Sidi et al., 2004; Nicolson, 2005). In zebrafish, water movement is detected by hair cells that are housed in neuromast organs. These organs are distributed on the surface of the animal and are constituents of the lateral-line system. Hair cells of the larval zebrafish ear are housed in two maculae and three cristae, which together detect sound stimuli in addition to linear and angular accelerations (Haddon and Lewis, 1996). Because of its large size, the synaptic ribbon serves as an important model for studying the development and function of the CAZ. Electron microscopy of bipolar neurons has revealed that synaptic ribbons are dynamic structures that oscillate in number according to the circadian cycle (Hull et al., 2006). Studies of axolotls have shown that most, but not all, of the ribbons in hair cells localize to the active zone plasma membrane, and some presynaptic dense bodies of the lateral line can move within the cell (Flock and Jorgensen, 1974, 1997). Several approaches have been taken to visualize the ribbon, including the use of Nomarski optics (Flock and Jorgensen, 1974, 1997), immunolabeling (Obholzer et al., 2008), electron microscopy (Lenzi et al., 2002), and the introduction of a fluorescent peptide into cells by patch pipette (Zenisek et al., 2003). The shortcomings of these methods for developmental studies include the use of a fixed tissue instead of a living specimen, the manipulation of a cell by a means that is not compatible with longterm studies, or the use of a model organism that is not genetically tractable. To overcome these limitations, we developed a novel method to visualize the subcellular position of the synaptic ribbon using a genetically tractable system. This method employs the use of transgenic zebrafish that express fluorescently tagged Ribeye a protein to reveal the locations of synaptic ribbons in ear and lateral-line hair cells. This technique is valuable for examining synaptic ribbons within live hair cells in real time and those contained in fixed specimens. 2. Materials and methods 2.1. Fish Two stable transgenic zebrafish lines, Ppv3b-4 (McDermott et al., 2010) and HGn39D (Faucherre et al., 2009), were used in these experiments. They were maintained and bred at 28 ◦ C by standard procedures (Nüsslein-Volhard and Dahm, 2002) and kept with the approval of the Case Western Reserve University Institutional Animal Care and Use Committee. 2.2. Molecular biology All restriction endonucleases used in these experiments were obtained from New England Biolabs. In reverse transcriptionpolymerase chain reaction (RT-PCR) procedures, randomly primed cDNA (Superscript III; Invitrogen) was produced from lagenar RNA of adult zebrafish (McDermott et al., 2007). Polymerase chain reaction (PCR) experiments were performed (Ex Taq DNA Polymerase; Takara Bio or Pfu DNA Polymerase; Stratagene) with the primer pairs listed below:

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5 -X-RibeyeA: 5 -ATC CCG GGA CCA TGT TGA TCT CCA GTA AGC AGT TG-3 . 3 -P-RibeyeA: 5 -ATT AAT TAA GGT ATA CAT TTT GTC TTG CAG GCC G-3 . RPG 8: 5 -GGA CTG GGC ATG GGT GAC ATT G-3 . RPG 4: 5 -CTT TAC CTG CAG TTC CTC AGT CAA T-3 . 5 -p-mCh: 5 -GGT TAA TTA AAG GCA TGG TGA GCA AGG GCG AGG AG-3 . 3 -a-mCh: 5 -TTG GCG CGC CTT ACT TGT ACA GCT CGT CCA TGC-3 . 2.3. Construction of the Ribeye a-mCherry fusion protein expression vector The first step in making the Ribeye a-mCherry expression plasmid, Tg(pvalb3b:ribeye a-mCherry), was to insert the mCherry cDNA (Shaner et al., 2005) into pMT/SV/PV (Chou et al., in press). pMT/SV/PV harbors a segment of DNA that contains the parvalbumin 3b promoter, which had been excised from the Ppv3b-4 construct (McDermott et al., 2010). This segment, in pMT/SV/PV, is flanked on each end by a region of the Tol2 transposable element (Balciunas et al., 2006). PCR was used to add a PacI restriction site to one terminus of the mCherry cDNA, and an AscI site to the other, using the primers 5 -p-mCh and 3 -a-mCh; this product was inserted into pCR-Blunt II-TOPO (Invitrogen). The resulting vector was digested with AscI and PacI to remove the modified mCherry cDNA; this cDNA was ligated (T4 ligase; Promega) into AscI- and PacI-digested pMT/SV/PV to form pMT/SV/PV/mCh. In the next step of constructing Tg(pvalb3b:ribeye a-mCherry), the coding region of ribeye a cDNA (GenBank number: AY878349) was assembled from two partial segments. The cDNA fragments X8 RP1 or 4P 3.3 were amplified from lagenar cDNA by PCR using the primer pairs 5 -X-RibeyeA and RPG 8 or 3 -P-RibeyeA and RPG 4, respectively. The X8 RP1 and 4P 3.3 amplicons were ligated into pCR-Blunt II-TOPO (Invitrogen) and pCRII (Invitrogen), respectively. Both of the resulting plasmids were digested with HindIII, and the X8 RP1 fragment was ligated into the pCRII vector that contained 4P 3.3. The X8 RP1-4P 3.3 fragment, which contained the ribeye a cDNA, was removed from pCRII using XmaI and PacI. This fragment was ligated into XmaI- and PacI-digested pMT/SV/PV/mCh. The resulting construct encodes the fusion protein Ribeye a-mCherry. 2.4. Production of transgenic zebrafish that express Ribeye a-mCherry in somatic cells Tg(pvalb3b:ribeye a-mCherry) DNA at 250 ng/␮l and Tol2 transposase RNA at 25 ng/␮l (Balciunas et al., 2006) in 0.1 M KCl were injected into embryos at the one-cell stage. Both Ppv3b-4 and HGn39D lines were injected. To visually monitor injections, phenol red was added to each injection solution to make the final concentration of the tracer 0.05%. 2.5. Immunolabeling and imaging Zebrafish injected with Tg(pvalb3b:ribeye a-mCherry) DNA, which exhibited mosaic transgene expression in somatic cells, were collected at 5 and 28 days postfertilization (dpf) and fixed using 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 ◦ C overnight. The larvae were rinsed with PBS, permeabilized in 3% Triton X-100 in PBS overnight at room temperature, bathed in blocking solution (5% goat serum in PBS) for 4–6 h at room temperature, and labeled overnight at 4 ◦ C with Ribeye b antiserum (Obholzer et al., 2008) diluted 1:100 in blocking solution. Larvae were washed at room temperature with blocking solution five times over a period of 6 h. The secondary antibody (Cascade Blue goat anti-rabbit IgG (H+L); Invitrogen) was diluted 1:200 in blocking solution and incu-

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Fig. 1. Labeling synaptic ribbons in hair cells with Ribeye a-mCherry fusion protein. (A) A schematic of the construct Tg(pvalb3b:ribeye a-mCherry) that was developed to express Ribeye a-mCherry in hair cells for the labeling of synaptic ribbons is shown. Flanking the promoter and cDNA are segments of the miniTol2 transposon (purple), which permit a high rate of transgene integration into the genomes of somatic cells (Balciunas et al., 2006). A graphic of a hair cell that expresses Ribeye a-mCherry to label ribbons (red), which reside close to regions of plasma membrane that are contacted by afferent nerve endings (green), is displayed. (B) A hair cell from an anterior macula of a zebrafish that was fixed at 5 dpf is shown. This hair cell expresses Ribeye a-mCherry fusion protein (red) and GFP (green), each under control of the parvalbumin 3b promoter (McDermott et al., 2010). GFP allows demarcation of the cell’s boundaries. Puncta that contain the Ribeye a-mCherry fusion protein are in close proximity to the basolateral surface. (C) In a live larva, ribbons of a neuromast are labeled with Ribeye a-mCherry. An HGn39D doubly transgenic zebrafish at 5 dpf, expressing GFP in afferent neurons of the posterior lateral line and Ribeye a-mCherry fusion protein in associated hair cells, reveals that most of the labeled ribbons (red) are proximal to the nerve endings (green). (D) Positions of Ribeye a-mCherry-labeled ribbons (red) within a live hair cell (green) of the posterior lateral line in a Ppv3b-4 transgenic zebrafish at 5 dpf are displayed. All labeled ribbons are juxtaposed to the basolateral surface. Arrowhead shows the positions of two ribbons, in a single hair cell, that reside in close proximity to each other. (E and F) Images reveal overlapping fluorescent signals from Ribeye a-mCherry and those produced by immunolabeling with Ribeye b antiserum at the basolateral surfaces of lateral-line hair cells in zebrafish at 28 (E) and 5 dpf (F). Doubly transgenic zebrafish expressing GFP (green) and Ribeye a-mCherry (red) in neuromast hair cells were labeled with antiserum raised against Ribeye b (cyan). When the three colors overlap, they appear as off-white. In F, the cell on the left does not express the transgene, but its ribbon is immunolabeled. (G) Ectopic expression of Ribeye a-mCherry in a doubly transgenic peridermal cell is shown. This cell is from a larva that was labeled at 5 dpf with Ribeye b antiserum (cyan), and it contains GFP (green) and Ribeye a-mCherry (red). Ribeye a-mCherry forms fluorescent puncta that are labeled with Ribeye b antiserum. Insets show enlarged images of immunolabeled structures that contain the fusion protein. All scale bars are 2 ␮m. Asterisks indicate the positions of the apical surfaces of the hair cells.

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bated overnight with larvae at 4 ◦ C. Next, the larvae were rinsed twice in blocking solution, for a period of 1 h for each wash, and then stored in mounting medium (VectaShield; Vector Laboratory). For live animal imaging, larvae at 5 dpf were anesthetized with 3aminobenzoic acid ethyl ester methanesulfonate (Sigma) and then mounted in low-melting-point agarose (Sigma) (Chan et al., 2009). Larvae were imaged using an inverted microscope (DM IRE2; Leica), with a 20× or a 40× objective lens, and a confocal laser scanner (TCS SP2; Leica). 3. Results and discussion We have designed and implemented a method that enables the visualization of synaptic ribbons of hair cells in live or in fixed zebrafish larvae for developmental studies. As a starting point for this method, we generated the DNA construct Tg(pvalb3b:ribeye a-mCherry) to direct the expression of the fusion protein Ribeye amCherry in hair cells (Fig. 1A). This vector contains the parvalbumin 3b promoter to permit expression in hair cells (McDermott et al., 2010). RT-PCR was used to amplify the coding region of the ribeye a cDNA using RNA isolated from adult zebrafish maculae. The ribeye a cDNA, lacking its stop codon, was ligated to mCherry cDNA to encode Ribeye a fused, at its C-terminus, to the fluorescent protein tag. The Ribeye a isoform was selected for these studies because it is expressed in hair cells of the inner ear (Wan et al., 2005); in addition, ribeye proteins are known to localize to ribbons with high specificity (Schmitz et al., 2000; Obholzer et al., 2008). We therefore anticipated that the Ribeye a-mCherry fusion protein would be an effective ribbon-labeling tool. The Tg(pvalb3b:ribeye a-mCherry) plasmid was used to generate transgenic zebrafish that mosaically expressed this transgene. The construct was injected into embryos of the Ppv3b-4 transgenic line, which express green fluorescent protein (GFP) in hair cells (McDermott et al., 2010); this transgenic background was selected because it allows for the delimitation of hair-cell boundaries during imaging. Hair cells of 5- and 28-dpf larvae that expressed Ribeye amCherry were imaged using confocal laser-scanning microscopy. These doubly transgenic fish exhibited a startle response and typical swimming behaviors, indicating that there was no gross disruption of either hearing or vestibular function as a result of fusion protein expression (data not shown). In maculae (Fig. 1B), cristae (data not shown), or lateral-line organs (Fig. 1D), the hair cells that expressed this fusion protein showed robustly labeled puncta in close proximity to the basolateral membranes. A similar pattern has been observed in hair cells labeled with antiserum raised against Ribeye b (Obholzer et al., 2008). When doubly transgenic zebrafish expressing GFP and Ribeye a-mCherry in neuromast hair cells were labeled with Ribeye b antiserum, the mCherry signal overlapped with the antibody-associated fluorescence (Fig. 1E and F). In hair cells that expressed Ribeye a-mCherry, all ribbons recognized by the Ribeye b antiserum were also labeled with the fusion protein (number of ribbons, N = 77). This suggests that the fusion protein is an effective tool for identifying the subcellular locations of the ribbons. A small percentage of fluorescent structures (4.4% of fluorescent structures, N = 180) were found as massive aggregates (Figure S1); these formations may be attributed to excessive Ribeye a-mCherry fusion protein expression. This occasionally occurs in animals where transgene distribution is mosaic among cells, and the amount of transgenic protein can vary from cell to cell. When the massive aggregates were exposed to Ribeye b antiserum, they were also effectively immunolabeled (data not shown). Hair cells that expressed mCherry protein using the parvalbumin 3b promoter (McDermott et al., 2010) displayed fluorescence throughout the whole cell, not sphere-like labeling close to the basolateral surface, indicating that the mCherry protein on its own does not label ribbons (data not shown).

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To determine whether the Ribeye a-mCherry-labeled ribbons were in close proximity to afferent nerve endings, we generated doubly transgenic zebrafish that mosaically expressed the fusion protein transgene in hair cells and consistently expressed GFP in afferent neurons of the posterior lateral line. For this study, we used the HGn39D transgenic line (Faucherre et al., 2009). Afferent neurons carry information from hair cells towards the central nervous system, and their nerve endings contact regions of the plasma membranes of hair cells that are associated with synaptic ribbons. Live imaging using confocal laser-scanning microscopy of doubly transgenic animals revealed that the majority of ribbons were proximal to afferent nerve endings (Fig. 1C). This finding further confirms that the Ribeye a-mCherry fusion protein labels ribbons, indicating that it will be an effective tool for characterizing synapse development. Rat RIBEYE aggregates to form discrete sphere-like structures when it is expressed in COS-7 cells or in cells of the R28 retinal precursor cell line (Magupalli et al., 2008). Because the version of the parvalbumin 3b promoter that we used drives expression in skin cells in addition to hair cells (McDermott et al., 2010), we looked for sphere-like formations in the periderm of zebrafish that expressed the fusion protein using the Ppv3b-4 transgenic background. Skin cells that expressed Ribeye a-mCherry displayed fluorescent sphere-like structures (Fig. 1G) that resembled those observed in cultured cells expressing tagged RIBEYE (Magupalli et al., 2008). This demonstrates that the zebrafish and the rat orthologous proteins form morphologically similar assemblages. Indirect immunofluorescence demonstrated that the Ribeye b antiserum recognized an epitope in peridermal cells that expressed Ribeye a-mCherry (Fig. 1G), and that the antibody-associated signal overlapped with the emissions of the fusion protein. No fluorescent puncta were observed in peridermal cells of Ppv3b-4 transgenics, which did not express Ribeye a-mCherry, upon labeling with Ribeye b antiserum (data not shown). These results indicate that this antiserum recognizes both Ribeye a and Ribeye b. This finding is not wholly unexpected because of the high amino acid sequence similarity of these proteins. In summary, we have developed a novel method to visualize synaptic ribbons of the zebrafish ear and lateral line. After cloning the coding region of the ribeye a cDNA and ligating it to mCherry cDNA, we expressed the chimeric protein using a hair-cell promoter. Live or fixed hair cells that contained this fusion protein had fluorescent ribbons in close proximity to the basolateral surface of each cell. Moreover, when hair cells expressing Ribeye amCherry were immunolabeled with Ribeye b antiserum, an overlap between fusion protein- and antibody-associated fluorescent signals was revealed. In addition, Ribeye a-mCherry-labeled ribbons localized proximal to afferent nerve endings. These findings indicate that Ribeye a-mCherry is an effective tool for labeling ribbons in zebrafish hair cells and will serve as a useful tool for real-time and fixed-tissue analyses. Use of this method will promote a more comprehensive understanding of the subcellular mechanisms of ribbon synapse development, the characterization of particular zebrafish mutants, and forward and reverse mutagenesis screens.

Acknowledgments The HGn39D zebrafish line was a gift of Dr. K. Kawakami at The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan. The pRSET-B mCherry plasmid was a gift from Dr. R. Tsien at the University of California-San Diego, La Jolla, California. The Ribeye b antiserum was a gift from Dr. T. Nicolson at the Oregon Health and Science University, Portland, Oregon. The miniTol2 vector was a gift from Dr. S. Ekker at the University of Minnesota, Minneapolis, Minnesota. We would like to thank Ms. S. Chou for

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producing the Tol2 transposase RNA, Mrs. C. Fernando for assistance with zebrafish husbandry, and Dr. C. Benedict-Alderfer, Dr. K. Alagramam, and members of our laboratory for reviewing this manuscript. This work was supported by a grant from the Center for Clinical Research and Technology at University Hospitals Case Medical Center, the Case Research Institute Vision Fund, and the Basil O’Connor Starter Scholar Research Award Grant No. 5-FY07-663 from the March of Dimes Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneumeth.2010.11.011. References Balciunas D, Wangensteen KJ, Wilber A, Bell J, Geurts A, Sivasubbu S, et al. Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genetics 2006;2:e169. Chan PK, Lin CC, Cheng SH. Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos. BMC Biotechnol 2009;9:11. Chou S, Hwang P, Gomez G, Fernando CA, West MC, Pollock LM, Lin-Jones J, Burnside B, McDermott Jr BM. Fascin 2b is a component of stereocilia that lengthens actinbased protrusions. PLoS ONE; in press. Faucherre A, Pujol-Marti J, Kawakami K, Lopez-Schier H. Afferent neurons of the zebrafish lateral line are strict selectors of hair-cell orientation. PLoS One 2009;4:e4477. Fejtova A, Gundelfinger ED. Molecular organization and assembly of the presynaptic active zone of neurotransmitter release. Results Probl Cell Differ 2006;43:49– 68. Flock A, Jorgensen JM. Synaptic body movements in the sensory cells of lateral line organs in the urodele amphibian Ambystoma mexicanum. Hear Res 1997;104:177–82. Flock A, Jorgensen JM. The ultrastructure of lateral line sense organs in the juvenile salamander Ambystoma mexicanum. Cell Tissue Res 1974;152:283–92. Fuchs PA, Glowatzki E, Moser T. The afferent synapse of cochlear hair cells. Curr Opin Neurobiol 2003;13:452–8. Haddon C, Lewis J. Early ear development in the embryo of the zebrafish, Danio rerio. J Comp Neurol 1996;365:113–28. Hull C, Studholme K, Yazulla S, von Gersdorff H. Diurnal changes in exocytosis and the number of synaptic ribbons at active zones of an ON-type bipolar cell terminal. J Neurophysiol 2006;96:2025–33.

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