Localization of metabotropic glutamate receptors in the outer plexiform layer of the goldfish retina

Christina Joselevitch Jan Klooster Maarten Kamermans 0 ) Retinal Signal Processing, The Netherlands Institute for Neuroscience (KNAW) , Meibergdreef 47, 1105 BA Amsterdam, The Netherlands We studied the localization of metabotropic glutamate receptors (mGluRs) in the goldfish outer plexiform layer by light-and electron-microscopical immunohistochemistry. The mGluR1 antibody labeled putative ON-type bipolar cell dendrites and horizontal cell processes in both rod spherules and cone triads. Immunolabeling for mGluR2/3 was absent in the rod synaptic complex but was found at horizontal cell dendrites directly opposing the cone synaptic ribbon. The mGluR5 antibody labeled Mller cell processes wrapping rod terminals and horizontal cell somata. The mGluR7 antibody labeled mainly horizontal cell dendrites invaginating rods and cones and some putative bipolar cell dendrites in the cone synaptic complex. The finding of abundant expression of various mGluRs in bipolar and horizontal cell dendrites suggests multiple sites of glutamatergic modulation in the outer retina. - Stimulation of vertebrate photoreceptors by light decreases the release of glutamate (Cervetto and MacNichol 1972; Financial support for this work was provided by Conselho Nacional de Pesquisa (CNPq), Brazil (grant 200915/98-3 to C.J.) Murakami et al. 1972; Kaneko and Shimazaki 1976), which is sensed by glutamate receptors (GluRs) in the dendrites of second-order neurons. Glutamate receptors are roughly divided into ionotropic and metabotropic receptors (Eccles and McGeer 1979). Ionotropic receptors (iGluRs) form an integral ion channel, whereas metabotropic receptors (mGluRs) mediate responses through indirect mechanisms involving signal transduction cascades. So far, eight different mGluRs have been cloned and classified into three groups based on their sequence similarities, second-messenger cascade, and pharmacology: group I comprises mGluR1 (Houamed et al. 1991; Masu et al. 1991) and mGluR5 (Abe et al. 1992) and their splice variants (1ad and 5ab); group II contains mGluR2 and mGluR3 (Tanabe et al. 1992; Tanabe et al. 1993); and group III includes mGluR4 (Tanabe et al. 1993), mGluR6 (Nakajima et al. 1993), mGluR7 (Okamoto et al. 1994; Saugstad et al. 1994), and mGluR8 (Duvoisin et al. 1995), and their splice variants (4ab, 7ab, 6ab, and 8ab). The lack of specific pharmacological tools to distinguish between mGluRs within a group makes it difficult to link a particular receptor type to a certain response property (for reviews, see Thoreson and Ulphani 1995; Schoepp et al. 1999). In the retina, mGluRs play an important role in the transmission and modulation of visual signals. Light responses of ON-type bipolar cells (ON BCs) of a number of species, for instance, are sensitive to substances/drugs that target group III mGluRs (Shiells et al. 1981; Slaughter and Miller 1981; Thoreson and Miller 1993). The receptor activated by these drugs is most likely mGluR6 (Nakajima et al. 1993; Akazawa et al. 1994; Masu et al. 1995; Ueda et al. 1997; Vardi and Morigiwa 1997; Laurie et al. 1997; Vardi et al. 2000). In fish, amphibians, and mammals, group III mGluRs modulate the glutamate release of cones (Koulen et al. 1999; Hirasawa et al. 2002; Hosoi et al. 2005). Furthermore, pharmacological activation of both group I and group III mGluRs modulates horizontal cell (HC) responses (Nawy et al. 1989; Takahashi and Copenhagen 1992; Linn and Gafka 1999), suggesting that multiple mGluRs must be present in the fish outer plexiform layer (OPL). At the moment, however, data available on the identity and distribution of the various mGluRs in the teleost outer retina are scarce (Yazulla et al. 2001; Klooster et al. 2001). We have therefore performed a light and electron microscopy study of the mGluRs present in the goldfish OPL. An antibody against the ON BC marker, protein kinase C (PKC; Negishi et al. 1988; Suzuki and Kaneko 1990; Yazulla and Studholme 1992), has been used in order to identify possible localization in mixed-input ON BCs (ON MBCs). Materials and methods Goldfish (Carassius auratus), 1215 cm standard body length, were obtained from a commercial supplier and maintained at 16C in aerated tanks filled with tap water circulating through a biological filter system. The fish were fed and kept on a 12 h/12 h light-dark cycle. All animal experiments were approved by the ethical committee of the Royal Netherlands Academy of Arts and Sciences, acting in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). 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 vitreousside-down on a Millipore filter (13 mm diameter, 8 m 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 12 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 (810 m thick) were cut in a cryostat, mounted on poly-L-lysine-coated slides (Menzel-Glser, Braunschweig, Germany), dried and stored in a non-frost-free freezer at 20C until use. Retinal sections were washed (2, 10 min) in phosphatebuffered saline (PBS), blocked for 20 min in PBS with 2% normal goat serum (NGS; Jackson ImmunoResearch Lab, West Grove, USA), incubated for 1848 h with the primary antibody (for dilutions, see Table 1) in PBS containing 0.3% Triton X-100 and 5% NGS at 4C. After several washes (15 min, 3) in PBS, sections were incubated in the secondary antibodies diluted in PBS containing 0.3% Triton X-100 for 30 min at 37C (Cy3-conjugated antibody: 1:500, Alexa-conjugated antibody: 1:600). Sections were cover-slipped with Vectashield (Vector Labs, Burlingame, USA) and stored at 20C. 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 con (...truncated)