Introduction 1972 1972 1976 1979 1991 1991 1992 1992 1993 1993 1993 1994 1994 1995 1995 1999 1981 1981 1993 1993 1994 1995 1997 1997 1997 2000 1999 2002 2005 1989 1992 1999 2001 2001 1988 1990 1992 Materials and methods Subjects Carassius auratus Preparation Dissections took place early in the morning (between 7 a.m. and 9 a.m.) under ambient light. The fish used in this study were therefore all in the light phase of their circadian regime. After cervical transection, the cornea and lens of light-adapted animals were removed, and the remaining eyecups were cut in half through the equator. Half-eyecups were placed vitreous-side-down on a Millipore filter (13 mm diameter, 8 μm pore size; Millipore, Amsterdam, The Netherlands) placed on a filter holder. Suction was applied to remove the vitreous; the sclera and retinal pigment epithelium were peeled away. Light microscopy Retinas were fixed at room temperature for 10 min in 0.1 M phosphate-buffered 4% paraformaldehyde (pH 6.5) and for another 10 min in 0.1 M sodium carbonate-buffered 4% paraformaldehyde (pH 10.4). These short fixation times were chosen to prevent loss of antigenicity. After being rinsed in 0.1 M phosphate buffer (PB, pH 7.4), the tissue was cryoprotected at room temperature for 30 min in PB containing 12.5% sucrose and for 1–2 h in PB containing 25% sucrose. The pieces of retina, still attached to the filter, were embedded in Tissue Tek (Sakura Finetek Europe, Zouterwoude, The Netherlands) in an aluminum boat and frozen in liquid nitrogen. Sections (8–10 μm thick) were cut in a cryostat, mounted on poly-L-lysine-coated slides (Menzel-Gläser, Braunschweig, Germany), dried and stored in a non-frost-free freezer at −20°C until use. 1 Table 1 LM EM aas PKC mGluR Antibody Source Catalog number Lot Type Host Purification Antigen Dilution for LM Dilution for EM a b Sigma-Aldrich, St. Louis, USA P5704 0641H4847 Monoclonal Mouse Protein A Bovine, within aas 296–317 of brain PKC 1:500 1:400 80 mGluR1α Chemicon, Temecula, USA AB1551 196011 Polyclonal Rabbit Affinity Rat, C-terminal, aas 1180–1199 (PNVTYASVILRDYKQSSSTL) 1:100 1:100 140 mGluR2/3 Chemicon AB1553 196011 Polyclonal Rabbit Affinity Rat, C-terminal, aas 860–872 (NGREVVDSTTSSL) 1:100 1:100 100 and 190 mGluR5 Chemicon AB1596 24010139 Polyclonal Rabbit Affinity Rat, C-terminal, aas 1159–1171 (LIIRDYTQSSSSL) 1:100 1:100 116 mGluR7 Upstate, Lake Placid, USA 07–239 25162 Polyclonal Rabbit Protein A Human, C-terminal, aas 899–912 (NSPAAKKKYVSYNN) 1:500 1:500 97 a b 1984 1988 Sections were cover-slipped with Vectashield (Vector Labs, Burlingame, USA) and stored at −20°C. Slides were observed on a Leica DMRD (Leica Microsystems, Wetzlar, Germany) fluorescence microscope equipped with filter sets that were designed for fluorescein isothiocyanate and Cy3. Sections from double-label experiments were observed on an inverted Zeiss Axiovert 100 M microscope equipped with the LSM 510 META laser scanning confocal module (Carl Zeiss Jena, Jena, Germany). Two types of controls were performed. To control for unspecific labeling attributable to secondary antibodies, experiments were performed by omitting the primary antibody. This eliminated all labeling. To control for unspecific labeling attributable to the primary antibodies, preadsorption experiments were performed by mixing, in a 20-fold molar excess, a synthetic peptide having the amino acid sequence against which each mGluR antibody had been raised (see below) with the corresponding primary antibody overnight at 4°C. Incubation proceeded as previously described, with the same concentrations for the primary antisera. Labeling was also completely eliminated in these preadsorption controls. Electron microscopy The same fixation and cryoprotection procedures were used as described for light microscopy. The retinas were peeled off the filter and embedded in Tissue Tek (Sakura Finetek Europe, Zouterwoude, The Netherlands) in an aluminum boat. The boat was frozen in liquid nitrogen. Transverse frozen sections (30–40 μm thick) were obtained on a freezing microtome and collected in PB at room temperature (equivalent to freeze-thawing). 1 2 2 1986 4 Sampling and production of photomicrographs At least 20 retinal sections obtained from a minimum of five animals were used for a given light microscopy or electron-microscopy experiment (i.e., single-immunolabeling at the light-microscopy level for mGluR1α). About five photomicrographs were taken from the most representative sections in each experiment. Light micrographs of the single-label experiments were acquired as TIFF files directly from the Leica microscope by using a Leica 350 F digital B/W camera (1284×1028 pixels, 72 ppi). Light micrographs of the double-label experiments were acquired as TIFF files from the Zeiss LSM 510 META (1048×1024 pixels, 72 ppi). n Electron micrographs obtained with the FEI electron microscope were directly acquired as TIFF files (1024×1024 pixels, 72 ppi). Electron micrographs obtained from the Philips electron microscope were first printed from the negatives for analysis. The negatives of the prints that were selected for publication were scanned on a sprint scan 4000 scanner (Polaroid Nederland, Breda, The Netherlands) and acquired as TIFF files at 600 ppi. All TIFF files were resampled at 400 ppi and subsequently re-sized and optimized for brightness and contrast by using Photoshop (Adobe Systems, San Jose, USA). Immunochemicals 1 Western blotting g g For sodium dodecyl-sulfate polyacrylamide gel electrophoresis, samples were run through a polyacrylamide stacking gel at 20 mA and through a 13% polyacrylamide gel at 30 mA. Protein standards (Bio-Rad Laboratories, Veenendaal, The Netherlands) were run in adjacent lanes. Gels were electroblotted on a poly-vinylidene-di-fluoride blot membrane (Millipore) overnight at a constant current of 80 mA. After being rinsed with a 0.5 M TRIS buffer containing 1.5 M NaCl and 5% Tween 200, the membrane was blocked in the same buffer containing 2% dry milk for 1 h. Subsequently, the membrane was incubated for 1 h in the primary antibodies (1:100), washed in the TRIS buffer, incubated in an HRP-conjugated goat anti-rabbit IgG (1:5000; Santa Cruz Biotechnology, Santa Cruz, USA), and washed in PBS. The immunoreaction was visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, USA) by using Kodak film. Exposures times were between 1 and 5 min. Results In this study, we aimed at a qualitative description of the mGluR composition in the goldfish OPL. To have a parameter for comparisons with regard to ON BC labeling with the antibodies against the various mGluRs, we first investigated the immunoreactivity pattern yielded by the PKC antibody, a widely used ON BC marker. Therefore, the labeling for PKC will be described first, followed by each of the mGluRs studied. Immunolabeling for PKC 1988 1990 1992 1 Light microscopy 1990 Electron microscopy: rod synapses 1 1 1 1 1 1 1975 1967 1976 1978 1983 1985 2001 2001 Fig. 1 a–c arrowheads arrows asterisks d e arrowheads SR arrows Bars  1967 1976 1975 1 1 1967 1976 1983 1985 Electron microscopy: cone synapses 1 1983 1985 1 Immunolabeling for mGluR1α Western blotting 2 1 2 Fig. 2 ONL OPL INL IPL a arrowheads b pread c ret br ret cer d e Red green d e circles asterisks f yellow Bars  Table 2 Mr aa Source protein Zebrafish matches  Sequence comparison Length source Length zebrafish Mr source Mr zebrafish This study Rat mGluR1(NP_058707) a Source PNVTYASVILRDYKQSSSTL 1199 aa 1158 aa 133 kDa 130 kDa 150 kDa Consensus P+VTYASVILRDYKQSSSTL Zebrafish PSVTYASVILRDYKQSSSTL a Source PNVTYASVILRDYKQSSSTL 271 aa 30 kDa Consensus P+VTYASVILRDYKQSSSTL Zebrafish PSVTYASVILRDYKQSSSTL Rat mGluR2(EDL77281) Hypothetical protein (previousmGluR2 precursor-XP_692887) Source NGREVVDSTTSSL 872 aa 874 aa 96 kDa 97 kDa 100 and230 kDa Consensus NGREVVDSTTSSL Zebrafish NGREVVDSTTSSL Hypothetical protein (XP_001341692) Source NGREVVDSTTSSL 754 aa 85 kDa Consensus NGREVVDSTTSSL Zebrafish NGREVVDSTTSSL Novel protein similar to vertebratemGluR3 (CAM56446) Source NGREVVDSTTSSL 884 aa 98 kDa Consensus NGRE+VDSTTSSL Zebrafish NGREIVDSTTSSL Human mGluR7(AAB51763) Similar to GluR (previously similar tomGluR7-XP_697214) Source NSPAAKKKYVSYNN 915 aa 668 aa 102 kDa 75 kDa 50 kDa Consensus NSPAAKKKYVSYN+ Zebrafish NSPAAKKKYVSYND Rat mGluR5(NP_058708) a Source LIIRDYTQSSSSL 1171 aa 1158 aa 128 kDa 130 kDa 60 and100 kDa Consensus +I+RDY QSSS+L Zebrafish VILRDYKQSSSTL a Source LIIRDYTQSSSSL 271 aa 30 kDa Consensus +I+RDY QSSS+L Zebrafish VILRDYKQSSSTL Similar to mGluR5 (XP_696823) Source LIIRDYTQSSSSL 1173 aa 130 kDa Consensus L++R Y+QSSSSL Zebrafish LVLRHYSQSSSSL a Light microscopy 2 2 2 2 2 2 2 2 Electron microscopy: rod synapses 3 3 3 3 3 3 Fig. 3 a–d SR arrowheads asterisks thick arrow d e f arrowheads thick arrows e arrows thick arrows f Bar  3 3 1967 1976 Electron microscopy: cone synapses 3 3 1967 1976 1983 3 1980 3 Immunolabeling for mGluR2/3 Western blotting 4 1 2 1999 2001 2005 2005 2003 2007 Fig. 4 ONL OPL INL IPL a thick arrows b pread c ret br ret cer d e Red green d e f Bars g h thick arrows thin arrows SR Bars  Light microscopy 4 4 4 4 4 Electron microscopy: rod synapses Electron microscopy: cone synapses 4 1967 1976 1975 Immunolabeling for mGluR5 Western blotting 5 1997 Fig. 5 ONL OPL INL IPL a open arrows thick arrow b pread c ret br ret cer d e Red green d e f Bars g open arrows h open arrows GFAP SR Bars  Light microscopy 5 5 5 5 Electron microscopy 5 1960 1961 1963 1970 5 1977 1994 Immunolabeling for mGluR7 Western blotting 6 1 Fig. 6 ONL OPL INL IPL a arrowheads b pread c ret br ret cer d e Red green d e arrowheads thick arrows f g thick arrows h SR arrowheads thick arrow thin arrows Bars  Light microscopy 6 6 6 6 Electron microscopy: rod synapses 6 Electron microscopy: cone synapses 6 6 6 6 6 Discussion Summarizing scheme 7 Fig. 7 red green blue SR  In the rod spherule, PKC labels invaginating dendrites of putative ON MBCs. The mGluR1α antibody labels similar structures and some lateral elements. Lateral elements of rod triads are also positive for mGluR7. Müller cell processes between the rod spherules are positive for mGluR5. We have found no evidence for mGluR2/3 in the rod synaptic complex. In the cone pedicles, PKC labels fine dendrites at the base of the terminal or invaginating structures away from the ribbons. Elements at similar positions and with similar fine structure are positive for mGluR1α and mGluR7. BC central processes in the cone triads are positive for mGluR1α. Spinules are positive for mGluR1α and mGluR7, and lateral elements are labeled by the mGluR1α, mGluR2/3, and mGluR7 antibodies. Finally, HC central elements are positive for mGluR2/3. Specificity of the antibodies 2 2005 1 http://www.ncbi.nlm.nih.gov/BLAST/ http://www.ch.embnet.org/software/LALIGN_form.html http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html http://www.basic.northwestern.edu/biotools/proteincalc.html To choose good candidates for fish mGluRs among the BLAST hits, we relied on the scores and E-values; the cutoff was E<0.01. We adopted a stringent significance level, because the chance of obtaining random matches with short amino acid sequences was relatively large. All scores used for these comparisons were higher than 1,000. For the C-terminal mGluR5 peptide, the cutoff was lowered to E=0.35, since no candidates could be found with the same stringency as for the remainder of the peptide sequences. 2 2 According to the database search, the C-terminal peptides of mGluR5 and mGluR7 have one fish homolog each. The mGluR5 candidate shows lowest amino acid sequence similarity at the C-terminus, but the lack of overlapping expression patterns between mGluR1 and mGluR5 in the OPL of the goldfish retina excludes cross-reactivity of anti-mGluR5 and mGluR1. 2 mGluRs of Müller cells + 1993 1987 1990 + 1988 + + 2+ 2+ 1997 1994 mGluRs of HCs 1999 1999 We have found that lateral elements in the rod triads are positively labeled for mGluR1α and mGluR7. Since the labeling for mGluR1α is rare, we cannot at this point positively identify these processes as belonging to either rod-driven HCs or MBCs. In the cone pedicle, mGluRs seem to be expressed in a highly organized manner. mGluR1α and mGluR7 tend to be localized to spinules and to dendrites occupying the lateral position in the cone triad, whereas mGluR2/3 has been found at both lateral and central processes directly opposing the synaptic ribbon. 1958 1968 1982 1982 1983 1993 1989 2003 1989 1990 1992 1997 1999 1999 mGluRs of ON MBCs 2001 2001 2001 1997 1999 2000 2003 1981 1987 2004 B 2005 o 1993 i o 2006