Immunocytochemical Analysis of the Mouse Retina

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1 THE JOURNAL OF COMPARATIVE NEUROLOGY 424:1 23 (2000) Immunocytochemical Analysis of the Mouse Retina SILKE HAVERKAMP AND HEINZ WÄSSLE* Max-Planck-Institut für Hirnforschung, D Frankfurt/Main, Germany ABSTRACT Transgenic mice provide a new approach for studying the structure and function of the mammalian retina. In the past, the cellular organization of the mammalian retina was investigated preferentially in primates, cats, and rats but rarely in mice. In the current study, the authors applied 42 different immunocytochemical markers to sections of the mouse retina and studied their cellular and synaptic localization by using confocal microscopy. The markers applied were from three major groups: 1) antibodies against calcium-binding proteins, such as calbindin, parvalbumin, recoverin, or caldendrin; 2) antibodies that recognize specific transmitter systems, such as glycine, -aminobutyric acid, or acetylcholine; and 3) antibodies that recognize transmitter receptors and show their aggregation at specific synapses. Only a few markers labeled only one cell type: Most antibodies recognized specific groups of neurons. These were analyzed in more detail in double-labeling experiments with different combinations of the antibodies. In light of their results, the authors offer a list of immunocytochemical markers that can be used to detect possible changes in the retinal organization of mutant mice. J. Comp. Neurol. 424:1 23, Wiley-Liss, Inc. Indexing terms: horizontal cells; bipolar cells; amacrine cells; ganglion cells; calcium-binding proteins; transmitters; transmitter receptors The mouse retina is becoming an important source for studies of mammalian retinal organization. In the past, mutant mice were used as a model for the study of retinal degeneration (Sidman and Green, 1965; Mullen and La- Vail, 1975; LaVail et al., 1982), and several genes that cause retinal degeneration have been identified in the murine retina (Farber and Danciger, 1997). Many studies on mammalian retinal development have been performed on the mouse retina (Young, 1985; Turner et al., 1990; Xiang et al., 1996; Zhou et al., 1996; Furukawa et al., 1997; Oliver and Gruss, 1997). However, at present (and even more so in the future), transgenic mice provide a new approach for the study of the structure and function of the mammalian retina (Oberdick et al., 1990; Nirenberg and Cepko, 1993; Chiu and Nathans, 1994a,b; Masu et al., 1995; Gustincich et al., 1997; Nirenberg and Meister, 1997; Ueda et al., 1997; Xu et al., 1997; Jacobs et al., 1999; Weng et al., 1999). Therefore, a precise knowledge of the cellular organization of the mouse retina is vital. Mouse retinae have rods and cones (Carter-Dawson and LaVail, 1979), and the proportion of cones has been found to be 1% (Jeon et al., 1998). This suggests that the mouse retina is rod dominated; however, the perspective changes if one considers that the absolute number of cones, which is 3,500 cones per mm 2 across most of the mouse retina (Jeon et al., 1998). The cone density of extrafoveal primate retina, which is 90% of that retina, is 2,000 4,000 cones per mm 2 (Curcio et al., 1990). Similar cone densities have been described in cat and rabbit retinae (outside the central area or streak: Steinberg et al., 1973; Hughes, 1977). Comparable to other mammalian retinae, two cone types have been identified in the mouse that are distributed unevenly, with a high proportion of S cones in the lower retina (Szél et al., 1992, 1993, 1994). In contrast to most mammalian retinae, rodent retinae contain only one type of horizontal cell (Peichl and Gonzáles-Soriano, 1994). Lucifer yellow injections have revealed axon-bearing horizontal cells with a B-type morphology (Suzuki and Pinto, 1986). Horizontal cells of the mouse retina were found to express specific types of neurofilaments (Dräger, 1983; Dräger et al., 1984). More recently it has been shown that neurofilaments label only the axon terminals of horizontal cells. The calciumbinding protein calbindin was found to label horizontal Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: SFB 269-B4. *Correspondence to: Prof. Heinz Wässle, Max-Planck-Institut für Hirnforschung, Deutschordenstrasse 46, D Frankfurt/Main, Germany. waessle@mpih-frankfurt.mpg.de Received 5 November 1999; Revised 18 January 2000; Accepted 18 January WILEY-LISS, INC.

2 2 S. HAVERKAMP AND H. WÄSSLE cells more completely (for review, see Peichl and González- Soriano, 1994). This label was abolished in a calbindin knockout mouse (Wässle et al., 1998b). The density of horizontal cells in the mouse retina was found to be 800 per mm 2 in peripheral retina and 1,500 per mm 2 central retina. Horizontal cells comprise 3% of the cells in the inner nuclear layer (INL; Jeon et al., 1998). No detailed study of the types of bipolar cells in the mouse retina has been performed. However, it was expected that, similar to cat, rabbit, primate, and rat retinae, as many as ten cone bipolar cell types and one rod bipolar cell type exist in the mouse retina (Famiglietti, 1981; Cohen and Sterling, 1990a,b; Boycott and Wässle, 1991; Euler and Wässle, 1995). Bipolar cell perikarya are found mainly in the center of the inner plexiform layer (IPL), and they comprise 40% of the cell bodies in the INL (Jeon et al., 1998). Rod bipolar cells of the mouse retina, as in other mammalian retinae, were immunoreactive for the isoform of protein kinase C (PKC ) and the Purkinje cell marker L7 (Berrebi et al., 1991; Masu et al., 1995; Soucy et al., 1998). There are between 20 and 30 different types of amacrine cells in mammalian retinae (Vaney, 1990; Strettoi and Masland, 1996; MacNeil and Masland, 1998). Amacrine cells of the mouse retina form a layer that is two cell bodies thick at the inner part of the INL, and they comprise 41% of all cells in that layer (Jeon et al., 1998). The major subdivision is into glycinergic amacrine cells and -aminobutyric acidergic (GABAergic) amacrine cells, with each comprising approximately half of the amacrine cell population. Glycinergic amacrine cells are mostly small-field amacrine cells (Pourcho and Goebel, 1985; Menger et al., 1998). GABAergic amacrine cells are widefield amacrine cells and colocalize, in addition to GABA, other neuroactive substances, such as peptides. Dopaminergic and cholinergic amacrine cells of the mouse retina and their retinal distributions have been studied (Versaux-Botteri et al., 1984; Wulle and Schnitzer, 1989; Jeon et al., 1998). Studies of the ganglion cells of the mouse retina have been pioneered by U. Dräger and coworkers (Dräger and Olsen, 1980, 1981; Dräger and Hofbauer, 1984; Dräger et al., 1984). They measured the ganglion cell density and found a gradual increase from 2,000 ganglion cells per mm 2 in peripheral retina to 8,000 per mm 2 in central retina (Dräger and Olsen, 1981). They identified displaced amacrine cells in the ganglion cell layer by marking ganglion cells retrogradely (Dräger and Olsen, 1980), and they described the selective staining of large -ganglion cells with antibodies directed against neurofilaments (Dräger and Hofbauer, 1984; Dräger et al., 1984; Peichl et al., 1987). However, a detailed classification of mouse ganglion cells, comparable to that of cat ganglion cells, is still missing (Wässle and Boycott, 1991; Berson et al., 1998). Physiologic studies of mouse ganglion cells have revealed light responses and receptive field organizations similar to those observed in other mammals (Balkema and Pinto, 1982; Stone and Pinto, 1993; Nirenberg and Meister, 1997; Soucy et al., 1998). Comparable to other mammalian retinae, three types of glia cells are present in the mouse retina. Müller cells span the retina vertically and are immunoreactive for vimentin (Dräger, 1983; Schnitzer, 1988). Their cell bodies comprise 16% of the perikarya present in the INL (Jeon et al., 1998). Astrocytes are immunoreactive for glial fibrillary acidic proteins and are found in the optic nerve fiber layer (Schnitzer, 1988). Microglia cells have been labeled with an antibody to the macrophage surface antigen Mac-1 (Dräger, 1983). Their cell bodies form a regular array in the INL (Schnitzer, 1989). Over the years, many immunocytochemical markers that recognize specific cell types have been applied to the retina of the cat, the rabbit, the guinea pig, the primate, and, more recently, the rat. Some of the markers labeled the same cell types across the different species. For instance, rod bipolar cells of all mammals are immunoreactive for PKC (Negishi et al., 1988; Greferath et al., 1990). Other markers, such as calcium-binding proteins, labeled different cell types in different mammals. AII amacrine cells are immunoreactive for parvalbumin (PV) in the rat retina but not in the primate retina (Wässle et al., 1993). Conversely, calretinin labels AII amacrine cells in the primate retina (Wässle et al., 1995) but not in the rat retina. Moreover, some of the markers (i.e., antibodies that recognize transmitter receptors or synapseassociated proteins) are very sensitive to the fixation protocols applied. Therefore, results from other mammalian retinae cannot be transferred automatically to the mouse retina. In the current study, 42 immunocytochemical markers were applied to vertical frozen sections of the mouse retina. The fixation protocols, the secondary antibodies, and the fluorescence microscopy (confocal microscopy) were standardized to make comparisons more straightforward. Many more markers than those shown here are available, and the selection made here shows to some extent a bias toward transmitters and transmitter receptors. Other markers, such as cone opsins, were not used because they have been investigated in great detail previously (Szél et al., 1992, 1993, 1994). MATERIALS AND METHODS Adult mice (C57/BL/6J) ages 8 10 weeks were deeply anesthetized with halothane and decapitated. The procedures were approved by the local animal care committee. The eyes were enucleated, the anterior segments were removed, and the posterior eyecups were immersion fixed. For most of the antibodies listed in Table 1, a fixation of minutes in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), ph 7.4, was chosen. In the case of the recoverin immunostaining, the fixation time was increased to 3 hours. For the n-methyl-d-aspartic acid receptor (NMDAR) antibodies, a fixation in 4% 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide in PB for minutes was necessary. After fixation, the retinae were dissected from the eye cup, cryoprotected in graded sucroses (10%, 20%, and 30%), and sectioned vertically at 12 m on a cryostat. The thickness of the mouse retina and the densities of neurons vary between central and peripheral retina (Jeon et al., 1998). All photomicrographs were taken from sections through the central retina. All antibodies that were applied in the current study are listed in Table 1. Immunocytochemical labeling was carried out by using the indirect fluorescence method. Retinal sections were blocked for 1 hour in a solution containing 10% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.5% Triton-X-100 in PB. The primary antibodies were

3 MOUSE RETINA 3 TABLE 1. List of the Antibodies Applied in the Current Study 1 Antigen Antiserum Source Working dilution Calbindin Rabbit anticalbindin Swant, Bellinzona, Switzerland 1:2,000 1:4,000 Mouse anticalbindin clone CL-300 Sigma Immunochemicals, St. Louis, MO 1:1,000 Pep19 Rabbit anti-pep19 J. I. Morgan, Memphis, TN (Ziai et al., 1986) 1:2,000 PKC Mouse anti-pkc clone MC5 Amersham, Arlington Heights, IL 1:100 PKC Mouse anti-pkc clone MC-3a Seikagaku, Tokyo, Japan 1:100 PKC Mouse anti-pkc clone MC-2a Seikagaku 1:50 GLT-1 Rabbit anti-glt-1 B. J. Kanner, Jerusalem, Israel (Danbolt et al., 1:2, ) Calretinin Rabbit anticalretinin Swant 1:2,000, 1:4,000 Calretinin Mouse anticalretinin Chemicon International Inc., Temecula, CA 1:2,000 Parvalbumin (PV) Rabbit anti-pv Swant 1:1,000 Neurofilament-L (NF-L) Mouse anti-da2 G. Shaw, Gainsville, FL (Shaw et al., 1986) 1:500 Glutamine synthetase (GS) Mouse anti-gs Transduction Laboratories, Lexington, KY 1:500 Glycine Rat antiglycine D. Pow, Brisbane, Australia (Pow et al., 1995) 1:1,000 Glycine transporter (GLY-T1) Rabbit anti-gly-t1 F. Zafra, Madrid, Spain (Zafra et al., 1995) 1:2,000 Go Mouse anti-go mab3073 Chemicon International Inc. 1:500 mab 115A10 (ROB) Mouse anti-mab 115A10 S. Fujita, Machida, Japan (Mori et al., 1985) 1:200 Recoverin Rabbit antirecoverin K.-W. Koch, Jülich, Germany (Lambrecht and Koch, 1:2, ) Caldendrin (Calp) Rabbit anticaldendrin M. Kreutz, Magdeburg, Germany (Seidenbecher et 1:1,000, 1:2,000 al., 1998) GABA Rat anti-gaba D. Pow (Pow et al., 1995) 1:100, 1:200 GABA Rabbit anti-gaba D. Pow (Pow et al., 1995) 1:2,000 1:5,000 GABA Rabbit anti-gaba I. Wulle (Wässle and Chun, 1989) 1:2,000 Tyrosine hydroxylase (TH) Rabbit anti-th Chemicon International Inc. 1:400 Glutamate decarboxylase Mouse anti-gad 65 Roche Diagnostics, Mannheim, Germany 1:100 (GAD 65 ) Choline acetyltransferase Rabbit anti-chat Roche Diagnostics 1:5 (ChAT) Neuronal nitric oxide Mouse anti-bnos Sigma Immunochemicals 1:50, 1:100 synthase (NOS) Substance P (SP) Mouse anti-sp Pharmingen, San Diego, CA 1:100 GlyR 1 Mouse anti-glyr 1 clone mab 2b H. Betz, Frankfurt, Germany (Schröder et al., 1991) 1:100 GlyR all ( 1 4, ) Mouse anti-glyr all clone mab4a H. Betz (Schröder et al., 1991; Kirsch and Betz, 1: ) Gephyrin Mouse antigephyrin clone mab 7a H. Betz (Pfeiffer et al., 1984) 1:100 GABA A 1 (1-16) Rabbit anti- 1 H. Möhler, Zürich, Switzerland (Benke et al., 1991) 1:10,000 GABA A 2 (1-9) Guinea pig anti- 2 H. Möhler (Benke et al., 1991) 1:2,000 GABA A 3 (1-15) Guinea pig anti- 3 H. Möhler (Benke et al., 1991) 1:5,000 GABA A 2 (1-29) Guinea pig anti- 2 H. Möhler (Benke et al., 1996) 1:5,000 GABA C Rabbit anti-gaba C (Enz et al., 1996) 1:100 GluR1 Rabbit anti-glur1 Chemicon International Inc. 1:100 GluR2/3 Rabbit anti-glur2/3 Chemicon International Inc. 1:100 GluR4 Rabbit anti-glur4 Chemicon International Inc. 1:100 GluR6/7 Rabbit anti-glur6/7 Upstate Biotechnology Inc., Lake Placid, NY 1:1,000 NR1 Rabbit anti-nr1 Chemicon International Inc. 1:100 NR1C2 Rabbit anti-nr1c2 Chemicon International Inc. 1:1,000 NR2A Mouse anti-nr2a clone 2F6.3D5 (Hartveit et al., 1994; Laurie et al., 1997) 1:100 NR2B Rabbit anti-nr2b Calbiochem 1:1,000 1 PKC, isoform of protein kinase C; GLT-1, glutamate transporter 1; mab, monoclonal antibody; GABA, -aminobutyric acid. diluted in 3% NGS, 1% BSA, and 0.5% Triton-X-100 in PB applied overnight at room temperature. After washing in PB, secondary antibodies were applied for 1 hour. These included: goat anti-mouse immunoglobulin G (IgG) or anti-rabbit IgG conjugated to either Alexa TM 594 (red fluorescence) or Alexa TM 488 (green fluorescence; Molecular Probes, Eugene, OR) all diluted 1:500 and goat antirat IgG or anti-guinea pig IgG coupled to fluorescein isothiocyanate (FITC; green fluorescence; Dianova, Hamburg, Germany) diluted 1:50. In double-labeling experiments, sections were incubated with a mixture of primary antibodies followed by a mixture of secondary antibodies. Control experiments included the omission of one or both primary antibodies, resulting in labeling by the remaining primary antibody or nonspecific background staining. All fluorescent specimens were viewed by using a Leica TCS SP confocal microscope (Leica, Deerfield, IL) equipped with a kryptonargon laser. Laser lines and emission filters were optimized with the Leica TCS Power Scan software. Brightness and contrast of the images were adjusted using Adobe Photoshop software (version 4.0.1; Adobe Systems, Mountain View, CA). RESULTS For a first screening of the cell classes of the mouse retina, two antisera were found to produce reliable and selective staining: calbindin (CabP-28 kd; Fig. 1a) and Pep19 (Fig. 1b). Calbindin is present in horizontal cells, which have cell bodies, dendrites, and axons that are strongly immunoreactive. The numerous puncta visible in the outer plexiform layer (OPL) in Figure 1a are terminals of horizontal cell axons inserted into rod spherules. Bipolar cells of the mouse retina do not appear to be immunoreactive for calbindin. Amacrine cells at the inner margin of the INL and displaced amacrine cells in the ganglion cell layer (GCL) show variable expression of calbindin. It is shown below that those amacrine cells/displaced amacrine cells that were labeled most prominently represent cholinergic amacrine cells. Ganglion cells also are immunoreactive for calbindin. In the IPL, three prominent layers, or strata, are labeled, and amacrine cells can be seen to contribute dendrites to the respective strata. Pep19 immunofluorescence (Fig. 1b) was not found in horizontal cells; however, many bipolar cell bodies in the outer part of the INL were labeled in a previous study

4 Figure 1

5 MOUSE RETINA (Berrebi et al., 1991). Some amacrine cells were weakly labeled. Other cells that represent cholinergic amacrine cells (see below) were strongly immunoreactive. Label in the IPL once again was stratified. The two narrow strata represent the bands in which cholinergic amacrine cells have their dendrites. The broad varicose band close to the ganglion cell layer represents the axon terminals of rod bipolar cells. In the GCL, displaced (cholinergic) amacrine cells and ganglion cells show variable degrees of labeling intensity. A more selective staining of certain populations of cells in the mouse retina was obtained by using the markers presented in Figure 2. Similar to other mammalian retinae, PKC immunoreactivity was present in rod bipolar cells (Fig. 2a). In addition, some amacrine cells (see Fig. 3d) and photoreceptors (Fig. 3d, arrow) were labeled with mouse anti-pkc (clone MC5; purchased from Amersham, Arlington Heights, IL). A more detailed analysis of the photoreceptor labeling showed that S-cones were labeled selectively (Wikler et al., 1998). The labeling of S-cones was not present with mouse anti-pkc (clone MC-3a; purchased from Seikagaku, Tokyo, Japan). The antibody against glutamate transporter 1 (GLT-1; EAAT2) labeled cones (strongly) and rods (weakly) in the outer nuclear layer (Fig. 2b). Rod spherules and cone pedicles in the OPL were labeled prominently. The cell bodies and descending axons of many bipolar cells were labeled in the INL. A horizontal band of putative bipolar cell axon terminals was found in the approximate center of the IPL. No label was found in the amacrine cell layer or the GCL. This was in contrast to immunostaining for calretinin (Fig. 2c). Many different types of amacrine cells, displaced amacrine cells, and possibly all ganglion cells were labeled. Three characteristic strata, similar to the calbindin strata (Fig. 1a), were labeled in the IPL. The inner and outer strata, once again, represent the two cholinergic strata. PV also labeled amacrine cells and ganglion cells (Fig. 2d); however, no distinct stratification was present in the IPL. Large, putative -ganglion cells and their primary dendrites were labeled more prominently than the other ganglion and amacrine cells. The light neurofilament protein DA-2 was found in the axon terminals of horizontal cells in the OPL (Fig. 2e), in a network of processes in the IPL, and in the optic nerve fiber layer. Some ganglion cell bodies and their primary dendrites also were labeled. Glutamine synthetase (GS) labeled exclusively Müller cells (Fig. 2f) to their full extent, with their cell bodies in the center of the INL and their radial processes terminating at the outer and inner limiting membranes. Müller cells also were labeled with antibodies against vimentin (not shown). Fig. 1. Confocal fluorescence photomicrographs of vertical frozen sections through mouse retinae that were labeled immunocytochemically. The retinal layers are indicated (ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer). a: Section immunostained for calbindin. Horizontal cells, amacrine cells, displaced amacrine cells, and ganglion cells are labeled. The arrow indicates a brightly fluorescent amacrine cell that projects a process into the band labeled in the center of the IPL. b: Section immunostained for Pep19. Bipolar cells, amacrine cells, displaced amacrine cells, and ganglion cells are labeled. The bipolar cells with somata in the outer INL and axonal varicosities in the inner IPL are rod bipolar cells. Scale bars 25 m. Glycine-immunoreactive amacrine and bipolar cells It has been shown in other mammalian retinae that glycine immunoreactivity is prominent in glycinergic amacrine cells. They release glycine as their transmitter and show high-affinity uptake of glycine. However, weaker glycine immunoreactivity also is present in ONcone bipolar cells, which receive glycine through gap junctions from AII amacrine cells (for review, see Vaney et al., 1998). Figure 3a shows glycine immunoreactivity in the mouse retina. Prominent fluorescence is present in amacrine cells, which form two rows of perikarya adjacent to the IPL. Weaker fluorescence also is present in bipolar cells in the center of the INL. The section shown in Figure 3a also was labeled for the glycine transporter GLY-T1 (Fig. 3b). Comparison of the labeled perikarya in Figure 3a,b shows that only the strongly glycine-immunoreactive amacrine cells expressed GLY- T1; the weakly labeled bipolar cells in Figure 3a apparently did not express the glycine transporter. The cell body of a glycinergic interplexiform cell is marked by an arrow in Figure 3a,b. To characterize further the glycine-immunoreactive bipolar cells, double-labeling experiments were performed with glycine and PKC (Fig. 3c,d), with glycine and GLT-1 (Fig. 3e,f), and with glycine and G-protein Go (Fig. 3g,h). PKC labeled rod bipolar cells and a few amacrine cells (Fig. 3d). Comparison of the PKC labeling with the glycine labeling of rod bipolar cells in Figure 3c shows no apparent colocalization, and it is obvious that the PKC labeled amacrine cells (Fig. 3d, arrows) were not immunoreactive for glycine in Figure 3c. The comparison of bipolar cells labeled by glycine (Fig. 3e) with those labeled by GLT-1 (Fig. 3f) was not as simple: Some of the GLT-1- immunoreactive bipolar cells (Fig. 3f, arrowheads) expressed glycine, and others did not (Fig. 3f, arrows). If it is assumed that glycine-immunoreactive bipolar cells are ON-cone bipolar cells, then it would be predicted that GLT-1 labels a mixed population of putative ON-cone and OFF-cone bipolar cells. The G-protein Go has been shown in cat and primate retinae to be expressed in ON-cone bipolar cells and in rod bipolar cells (Vardi et al., 1993; Vardi, 1998). Go is involved in the signaling cascade downstream of mglur6, the metabotropic glutamate receptor (mglur) of ON-bipolar cells (Nomura et al., 1994). We found also (Fig. 3h) an expression of Go in putative ON-cone bipolar cells in the mouse retina. The section also was double labeled for glycine (Fig. 3g), and comparison of the stained bipolar cells in Figure 3g,h shows that bipolar cells in the center of the INL (putative cone bipolar cells) appear to be double labeled. Rod bipolar cells in the outer part of the INL express Go (Fig. 3h) but not glycine. Unfortunately, there is some crosstalk of the secondary antibody in Figure 3g,h: The FITC antirat antibody recognizes both the antiglycine antibody (made in rat) and the anti-go (made in mouse). However, because the Go produces strictly membrane staining, whereas glycine fills the whole perikaryon, the two markers remain distinguishable nonetheless. Taken together, these experiments suggest that the glycinergic bipolar cells are ON-cone bipolar cells. 5

6 Fig. 2. Confocal fluorescence photomicrographs of vertical frozen sections through mouse retinae that were immunocytochemically labeled (for retinal layers, see Fig. 1). a: Section immunostained for the isoform of protein kinase C (PKC ). Blue cones (arrow) and rod bipolar cells are labeled. b: Section labeled for the glutamate transporter GLT-1. In the outer retina rods, cones, and their synaptic terminals appear to be labeled. In the inner retina putative cone bipolar cells are labeled. c: Calretinin immunofluorescence is present in amacrine, displaced amacrine, and ganglion cells. d: Parvalbumin (PV) labeling is weak in some amacrine cells and is prominent in large ( -like) ganglion cells. e: Neurofilament (NF-L) immunofluorescence can be seen in processes within both plexiform layers, in some ganglion cells, and in optic nerve fibers. f: Glutamine synthetase (GS) labeling is restricted to Müller cells. Scale bars 25 m.

7 MOUSE RETINA Immunocytochemical characterization of bipolar cells Many bipolar cells were found to be immunoreactive for Pep19 (Fig. 1b), and we wanted to subdivide this population further. A section that was double labeled for Pep19 and PKC is shown in Figure 4a,b. The cell bodies of the PKC -immunoreactive rod bipolar cells were found at the outer margin of the INL (Fig. 4b). All of these cell bodies also were immunoreactive for Pep19 (Fig. 4a). Pep19 also labeled the descending axons of rod bipolar cells and their axon terminals in the inner IPL. However, as shown by the bipolar cells denoted by arrows in Figure 4a, Pep 19 labeled other bipolar cells in addition to rod bipolar cells. Therefore, further double-labeling experiments were performed, and Figure 4c,d shows a section that was double labeled for Pep 19 (Fig. 4c) and glycine (Fig. 4d). Those bipolar cells with cell bodies in the outer INL (mostly rod bipolar cells; Fig. 4c) were not immunoreactive for glycine (Fig. 4d), which was expected due to the lack of colocalization between glycine and PKC (Fig. 3c,d). Very few cell bodies of cone bipolar cells in the center of the INL appeared to be double labeled. One putative bipolar cell, however, that appeared to be double labeled is indicated by an arrowhead in Figure 4d. Because glycine immunoreactivity is most likely in ON-cone bipolar cells, this would suggest that Pep19 labels rod bipolar cells and OFF-cone bipolar cells. This also is supported by the pattern of labeling found in the IPL of Figure 1b: Rod bipolar cell axon terminals in the inner IPL and the outer IPL, where OFF-cone bipolar cells axons terminate, were labeled strongly. However, it also is obvious from Figure 1b that a small band in the outermost IPL was labeled only weakly, possibly indicating that those OFF-cone bipolar cells with axons that terminate in this band may not have been labeled. To separate the bipolar cell types further, mouse retinae also were immunostained with the monoclonal antibody (mab) 115A10. This antibody was raised against a crude homogenate of rabbit olfactory bulb (ROB; Onoda and Fujita, 1987) and has been shown in different mammalian species to label rod bipolar cells and putative ON-cone bipolar cells (Greferath et al., 1990; Martin and Grünert, 1992). A section of mouse retina that was double labeled for Pep19 and mab 115A10 is shown in Figure 4e,f. mab 115A10, as expected, labeled many cell bodies of putative rod bipolar cells in the outer part of the INL (Fig. 4f) that also were immunoreactive for Pep19 (Fig. 4e). Many putative cone bipolar cell bodies in the center of the INL also were labeled by mab 115A10; however, these were not double labeled for Pep19. We also applied antibodies against recoverin to sections of the mouse retina (Fig. 5a). In the rat, two bipolar cell types and the photoreceptors express high amounts of recoverin (Milam et al., 1993; Euler and Wässle, 1995). Photoreceptors of the mouse retina were labeled brightly, and three types of bipolar cells were labeled more faintly. One bipolar cell type had a bushy axon terminal in the outer one-third of the IPL and most likely represented an OFF-cone bipolar cell. A second type had also a bushy axon terminal more in the center of the IPL, probably also an OFF-cone bipolar cell. Double-labeling experiments with glycine showed that neither type was immunoreactive for glycine (not shown). The third type had a stratified, wide axon terminal in the inner IPL close to the GCL and a small cell body in the outer INL. This type is reminiscent of the blue cone bipolar cells of the primate retina (Boycott and Wässle, 1991) and is glycine-positive (not shown). A new calcium-binding protein, caldendrin, has been described recently (Seidenbecher et al., 1998). Figure 5b shows the expression of caldendrin in a section through the mouse retina. Bipolar, amacrine, and ganglion cells are labeled. The bipolar cells that were stained have a rather large cell body in the center of the INL, and their axons extend into a bushy, varicose terminal system in the outer one-third of the IPL, suggesting that they are OFFcone bipolar cells. Their axon terminals occupy a similar position in the IPL to that of one of the recoverin-labeled OFF-cone bipolar cell types shown in Figure 5a. Because both antisera were from the same species, we could not test directly whether they were identical types. However, a comparison of Figure 5c with Figure 5d shows that the caldendrin-positive OFF-cone bipolar cells were not immunoreactive for glycine. In the primate retina antibodies against PKC were found to label the majority of bipolar cells (Grünert et al., 1994; Kolb and Zhang, 1997). Figure 7f shows that PKC immunofluorescence in the mouse retina outlined the perikarya of most neurons in the INL and the GCL. Müller cell bodies in the center of the INL were not labeled. Prominent label also was found, with the exception of three darker horizontal bands, over most of the IPL. In summary, rod bipolar cells were labeled by the antibody against PKC. They also were immunoreactive for Pep19 and Go, and they were recognized by mab 115A10. ON-cone bipolar cells expressed glycine immunoreactivity and were labeled by Go and mab 115A10. OFF-cone bipolar cells could express caldendrin and/or Pep19. Mixed populations of ON-cone and OFF-cone bipolar cells were recognized by the antisera against GLT-1 and recoverin. Immunocytochemical characterization of GABAergic amacrine The light microscopic appearance of GABA immunofluorescence in the mouse retina was critically dependent on the specific antibody applied. Figure 6a,c,e shows the different patterns of labeling that were obtained. All three antisera against GABA label amacrine cells, which are found in two rows of cell bodies at the inner margin of the INL. Few displaced amacrine cells also are labeled in the GCL. The labeling of the cholinergic amacrine cells, which have been shown in other mammals also to express GABA, is particularly variable (Brecha et al., 1988; Kosaka et al., 1988; Vaney and Young, 1988). It is prominent in Figure 6c and more reduced in Figure 6a,e. Most of the displaced GABAergic amacrine cells represent ON-cholinergic cells (Voigt, 1986). The sections shown in Figure 6 were double labeled for GABA and other markers to further subdivide the amacrine cells. An amacrine cell that was immunoreactive for tyrosine hydroxylase (TH) is shown in Figure 6b. The major dendritic tree of these cells is restricted to the outermost part of the IPL, and many inner plexiform processes can be seen leaving the TH-immunoreactive plexus and extending toward the OPL (Wulle and Schnitzer, 1989). The cell also is weakly immunoreactive for GABA (Fig. 6a, arrow). Figure 6c,d shows a section that was double labeled for GABA and glycine. We observed no colocalization of GABA and glycine, which is in 7

8 8 S. HAVERKAMP AND H. WÄSSLE Figure 3

9 MOUSE RETINA agreement with findings in other mammalian retinae (Fletcher and Kalloniatis, 1996; Marc et al., 1998). The colocalization of GABA and glutamic acid decarboxylase 65 (GAD 65 ) is shown in Figure 6e,f. In the majority of amacrine cell bodies, GABA and GAD65 appear to be colocalized. The cell body close to the OPL (Fig. 6e, indicated by arrow) represents a putative misplaced amacrine cell and not a horizontal cell: It does not express GAD65. The two cholinergic strata that express GABA immunoreactivity strongly in the IPL in Figure 6e appear to be devoid of GAD65. Hence, there is not a 1:1 match between GABA and GAD65, suggesting that some amacrine cells express the other isoform of GAD, namely, GAD67 (Vardi and Auerbach, 1995), or that GAD65 is not sent to the processes in some cases. Stratification of amacrine cell dendrites in the IPL The calcium-binding proteins calbindin (Fig. 1a) and calretinin (Fig. 2c) as well as Pep19 (Fig. 1b) showed a characteristic stratified pattern of expression in the IPL. To analyze the different strata, double-labeling experiments were performed taking the two cholinergic bands as references (Fig. 7a f). The section shown in Figure 7a,b was labeled for choline acetyltransferase (ChAT) and for Pep19. The cell bodies of normal and displaced cholinergic amacrine cells and their two strata in the IPL (Fig. 7a) also are immunoreactive for Pep19 (Fig. 7b). The cell bodies of cholinergic amacrine cells pop out in the Pep19 labeling, because they are the most intensively labeled perikarya. In turn, we double labeled a section for Pep19 (Fig. 7c) and calbindin (Fig. 7d): Once again, the cholinergic amacrine cells, which are labeled prominently for Pep19 (Fig. 7c), also are immunoreactive for calbindin. The cholinergic bands in the IPL are congruent with two of the three bands that are immunoreactive for calbindin. Sections also were double labeled (not shown) for calbindin and calretinin (Fig. 2c). The trilaminar pattern of the IPL turned out to be congruent for calbindin and calretinin (not shown). When antibodies against PKC were applied to the mouse retina (Fig. 7f), many bipolar, amacrine, and ganglion cells were labeled. Therefore, PKC is a rather nonspecific marker of neurons of the inner retina. Fig. 3. Confocal fluorescence photomicrographs of vertical frozen sections through mouse retinae that were double-labeled for glycine (GLY; left) and for other markers (right). Pairs of double-labeled sections here and in Figures 4 10 are indicated by a double-letter code: The letter in parentheses refers to the other member of the pair. a: Glycine immunofluorescence is prominent in amacrine cells at the INL/IPL border. It is weaker in bipolar cells in the center of the INL. b: The same section shown in a immunolabeled for the glycine transporter GLY-T1. Label is restricted to amacrine cells. Comparison of a and b shows that all labeled amacrine cells are double labeled. Arrows indicate an interplexiform cell that sends processes into the OPL. c: Glycine immunofluorescence. d: The same section shown in c immunolabeled for PKC. Rod bipolar cells and some amacrine cells (arrows) are labeled. Comparison of c and d shows no apparent colocalization. e: Glycine immunofluorescence. f: The same section shown in e immunolabeled for glutamate transporter 1 (GLT-1). Comparison of e and f shows that some bipolar cells are double labeled (arrowheads) and others are not (arrows). g: Glycine immunofluorescence. h: The same section shown in g immunolabeled for the G-protein Go. Comparison of g and h shows bipolar cells that are double labeled (arrowheads) and others that are not (arrows). Scale bars 25 m. The section shown in Figure 7e,f was double labeled for calbindin and PKC. The IPL is stained in a complementary fashion by the two markers: The three strongly calbindin-immunoreactive bands (Fig. 7e) show a reduced expression of PKC (Fig. 7f). We also applied markers that recognize specific amacrine cell populations to sections of the mouse retina, such as antibodies against enkephalin or somatostatin, and found that their distribution across the IPL was closely similar to what has been shown previously in the rat and mouse retina. Therefore, they are not illustrated here. Generally speaking, the IPL of the mouse retina appears to be thicker than that of the rat retina, and the different sublayers appear to be more distinct. This is illustrated for a retinal section that was double labeled for neuronal nitric oxide synthase (NOS) and for Pep19 (Fig. 8a,b). NOS-immunoreactive processes form a loose network throughout the IPL and appear to be concentrated in three bands (Fig. 8a,b, arrows) that only partially overlap with the strata revealed by Pep19. A section that was double labeled for NOS and calretinin (Fig. 8c,d) shows that the strongly NOS-immunoreactive amacrine cell (Fig. 8d, arrow) provides a thick primary dendrite aiming for the middle stratum of calretinin immunofluorescence. Figure 8e,f shows a section double labeled for substance P (SP) and calretinin. SP immunoreactivity was found in a small population of mostly normally placed amacrine cells that showed a more punctate staining pattern. Their dendrites were stratified in three bands throughout the IPL, similar to the NOS-immunoreactive processes shown in Figure 8a. Some of the SP-immunoreactive cells were colocalized with calretinin-immunoreactive amacrine cells (Fig. 8e,f, arrowheads), and others were not (Fig. 8e,f, arrows). The SP-immunoreactive bands within the IPL did not show much overlap with the calretinin-immunoreactive strata. Localization of transmitter receptors in the mouse retina GABA and glycine receptors. Glutamate is the major excitatory transmitter of the retina, and GABA and glycine are the major inhibitory transmitters. Molecular cloning has shown that the receptors to which these transmitters bind consist of multiple subunits and types. The different receptors are expressed and clustered at specific synapses and define the signal transfer at these synapses (for reviews, see Brandstätter et al., 1998; Wässle et al., 1998a). Several spontaneous mutants of transmitter receptors are known that show deficits in the retina (spastic mouse: Pinto et al., 1994; oscillator mouse: Wässle et al., 1998a; lurcher mouse: Zuo et al., 1997). Several mutants have been created by gene-directed mutagenesis of transmitter receptors or synapse-associated proteins (Essrich et al., 1998; Feng et al., 1998). This growing field demands a catalogue of the distribution of transmitter receptors in the normal mouse retina. The glycine receptor (GlyR) is a pentameric complex comprised of five subunits. Three ligand-binding subunits and two subunits form a Cl -selective channel. To date, at least four different subunits ( 1, 2, 3, and 4) and one subunit have been identified (Betz, 1991). Similar to other mammalian retinae, the GlyR 1 subunit has a characteristic distribution (Fig. 9a). Large synaptic hot spots form a broad band in the outer half of the IPL, whereas the inner half of the IPL contains only small hot spots. The large hot spots in the outer IPL most likely 9

10 10 S. HAVERKAMP AND H. WÄSSLE Fig. 4. Confocal fluorescence photomicrographs of vertical frozen sections through mouse retinae that were double labeled for Pep19 (left) and for other bipolar cell markers (right). a: Section labeled for Pep19. b: The same section shown in a labeled for PKC. Rod bipolar cells are immunoreactive for both Pep19 and PKC (arrowheads). Other bipolar cells (arrow) express only Pep19. c: Section labeled for Pep19. d: The same section shown in c labeled for glycine. Most of the bipolar cells are not double labeled. One double-labeled bipolar cell is indicated by an arrowhead. e: Section labeled for Pep19. f: The same section shown in e immunostained with monoclonal antibody (mab) 115A10 directed against rabbit olfactory bulb (ROB). Only rod bipolar cells appear to be double labeled (arrowhead): The cone bipolar cells that are immunoreactive for Pep19 are different from those labeled by mab 115A10 (arrows). Scale bars 25 m. represent the glycinergic synapses between AII amacrine cells and OFF-cone bipolar cells (Sassoé-Pognetto et al., 1994). An antibody that recognizes all GlyR subunits known so far reveals many more glycinergic synapses throughout the IPL (Fig. 9b). A synapse-associated protein that plays an important role in aggregating both glycine and GABA A receptors at their postsynaptic densities is gephyrin (Kirsch and Kröger, 1996). Gephyrin immunoreactivity in the mouse retina is confined to the IPL and is clustered at numerous postsynaptic sites (Fig. 9c). Molecular cloning studies have demonstrated that the GABA A receptor, like the GlyR, is composed of five struc-

11 MOUSE RETINA 11 Fig. 5. Confocal fluorescence photomicrographs of vertical frozen sections through mouse retinae. a: Recoverin immunofluorescence is prominent in the ONL in the rods and cones. Cell bodies of three different types of putative cone bipolar cells are labeled more weakly in the INL. Their axons terminate at different levels within the IPL. b: Caldendrin immunoreactivity is restricted to the inner retina. Among other cells, putative OFF-cone bipolar cells are labeled prominently. c,d: Section that was double labeled for caldendrin and glycine. The strongly caldendrin-immunoreactive bipolar cells are not double labeled (arrows). Scale bars 25 m.

12 12 S. HAVERKAMP AND H. WÄSSLE Fig. 6. Confocal fluorescence photomicrographs of vertical frozen sections through mouse retinae that were double labeled for -aminobutyric acid (GABA; left) and for other markers (right). a: section labeled for GABA (antibody; rat anti-gaba; D. Pow). Amacrine cells and displaced amacrine cells are labeled. b: The same section shown in a labeled for tyrosine hydroxylase (TH). A dopaminergic amacrine cell is strongly immunoreactive. It also shows weak GABA immunofluorescence (arrow in a). c: Section labeled for GABA (antibody; rabbit anti-gaba; D. Pow). Prominent immunofluorescence is present in the cholinergic amacrine cells. d: The same section shown in c labeled for glycine (GLY). No colocalization of glycine and GABA was observed. e: Section labeled for GABA (antibody; rabbit anti-gaba; I. Wulle). f: The same section shown in e labeled for glutamic acid decarboxylase 65 (GAD 65 ). There are many colocalizations; however, the GABAergic cell in the outer INL (e, arrow) is not double labeled. Scale bars 25 m. turally related subunits that form a Cl -conducting pore. To date, six subunits, four subunits, three subunits, and one subunit have been cloned (for reviews, see MacDonald and Olsen, 1994; Sieghart, 1995). GABA C receptors consist of subunits ( 1, 2, and 3) that also form (homooligomeric or heterooligomeric) Cl -conducting pores (for review, see Enz and Cutting, 1998). Recently, it has been suggested that GABA C receptor subunits can coassemble with the 2 subunit of GABA A receptors (Ekema and Lu, 1999). We have shown previously in the rat retina that the three subunits of the GABA A receptor, 1, 2, and 3,

13 MOUSE RETINA 13 Fig. 7. Confocal fluorescence photomicrographs of vertical frozen sections through mouse retinae that were double labeled to analyze the pattern of stratification in the IPL. a: Cholinergic amacrine cells are labeled with antibodies against choline acetyltransferase (ChAT). b: The same section shown in a labeled for Pep19. The cholinergic cells and the two strata of dendrites in the IPL are double labeled. c: Section labeled for Pep19. d: The same section shown in c labeled for calbindin. The cholinergic amacrine cells, which are strongly immunoreactive for Pep19, are double labeled. Two of the three calbindin-immunoreactive strata in the IPL represent the cholinergic bands. e: Section labeled for calbindin. f: The same section shown in e labeled for PKC. Three bands of weaker labeling for PKC in the IPL correspond to the bands of strong calbindin immunofluorescence in e. Scale bars 25 m. exhibit distinct patterns of stratification in the IPL and are clustered in different synaptic hot spots (Greferath et al., 1995; Koulen et al., 1996). The same segregation apparently also is present in the mouse retina (Fig. 9d f). The GABA A 2 subunit has a distinct band of higher density at the cholinergic ON-stratum, where the 3 subunit shows reduced expression. The section that shows GABA A 2 immunofluorescence (Fig. 9d) also was double labeled for gephyrin (Fig. 9c). A point-by-point comparison of the two photomicrographs shows that the great majority of 2-immunoreactive puncta also expressed gephyrin. However, there are more gephyrin-immunoreactive puncta, because gephyrin also is involved in the clustering of GlyRs. Recently, a gephyrin

14 14 S. HAVERKAMP AND H. WÄSSLE Fig. 8. Confocal fluorescence photomicrographs of vertical frozen sections through mouse retinae that were double labeled. a: Nitric oxide synthase (NOS) is found in a sparse population of amacrine cells. Their processes form three broad bands in the IPL (arrows). b: The same section shown in a labeled for Pep19. The NOSimmunoreactive cell also expresses Pep19 (arrow). c: The NOSimmunoreactive amacrine cell projects a prominent process to the center of the IPL. d: The same section shown in c labeled for calretinin. The arrow indicates the NOS-immunoreactive cell from c that appears to be double labeled. e: Section labeled for substance P (SP). f: The same section shown in e labeled for calretinin. Some of the substance P-immunolabeled amacrine cells are labeled by calretinin (arrowheads), and others are not (arrow). Scale bars 25 m. knockout mouse became available (Feng et al., 1998), and it will be interesting to study the clustering of GABA A 2 and GlyR subunits in the retina of such mice. We showed previously that GABA C receptors also are clustered in synaptic hot spots in both the OPL and the IPL. We found that GABA C receptors are not aggregated in the same synapses with GABA A receptor subunits. Recently, it has been described in an artificial expression system that GABA C receptor subunits form heterooligomeric channels when they are coexpressed with the 2 subunits of GABA A receptors (Ekema and Lu, 1999). To determine whether this also happens in vivo, we double

15 MOUSE RETINA labeled retinal sections for GABA C receptors and the GABA A 2 subunits. Both cluster in synaptic hot spots; however, a point-by-point comparison of Figure 9g,h shows that they are clustered at different synapses. Therefore, we conclude that, in vivo, no heterooligomeric channels that contain GABA C subunits and GABA A 2 subunits occur in synaptic hot spots. This result also was confirmed in the rat retina (not shown). In conclusion, glycine receptors are clustered at postsynaptic sites in the IPL of the mouse retina. Synapses at which the 1 subunits are expressed include the AII 3 OFF-cone bipolar cell synapse. The different subunits of GABA A receptors also are clustered at postsynaptic sites, and the 1, 2, and 3 subunits do not appear to be colocalized. GABA C receptors also are clustered at postsynaptic sites, but they are not coexpressed with GABA A receptor 2 subunits at the same synapses. Ionotropic glutamate receptors. Ionotropic glutamate receptors (GluRs) possess integral ion channels that are permeable to cations. Pharmacologically, they can be classified by the action of agonists, such as -amino-3- hydroxy-5-methyl-4-isoxazole-propionate (AMPA), kainate, and NMDA. Molecular cloning has revealed that the AMPA subunits are GluR1, GluR2, GluR3, and GluR4, and the kainate subunits are GluR5, GluR6, GluR7, KA-1, and KA-2. Three subunit families have been described for the NMDA receptors: NR1 (with eight known splice variants), NR2 (with the NR2A, NR2B, NR2C, and NR2D subunits), and NR3A (Hollmann and Heinemann, 1994). The distributions of eight different GluR subunits in the mouse retina are shown in Figure 10. Labeling has a punctate appearance, indicating that GluRs, like GABA receptors and GlyRs, are clustered at postsynaptic sites. It must be emphasized that the clusters are very sensitive to prolonged fixation; therefore, as described above (see Materials and Methods), only short fixation times were applied. The AMPA receptor subunit, GluR1 (Fig. 10a), is present in both the OPL and the IPL. In the OPL, an aggregation of GluR1 at the positions of cone pedicles can be observed (Fig. 10a, arrowheads). A few GluR1- immunoreactive puncta also can be found postsynaptic to rod spherules (Brandstätter et al., 1999; Hack et al., 1999). The GluR1-immunoreactive puncta in the IPL have a stratified distribution. Some amacrine cells (Fig. 10a, arrow) and ganglion cells are labeled extrasynaptically. The GluR2/3 and GluR4 subunits also are found in discrete clusters both in the OPL and the IPL. In the OPL, many puncta appear to be associated with rod spherules; in the IPL, different patterns of stratification are found: GluR2/3 appears more stratified (Fig. 10b), whereas, for GluR4, a broad band of reduced density of puncta appears in the center of the IPL (Fig. 10c). The kainate receptor subunit, GluR6/7, is found in both the OPL and the IPL (Fig. 10d), very similar to what has been observed in our laboratory in the rat retina (Brandstätter et al., 1997). Together, the two antisera applied here against the NR1 subunit of the NMDA receptor recognize all eight splice variants. Both antisera produced punctate label in the IPL with some indication of stratification (Fig. 10e,f). Label in the OPL with the NR1 antiserum was difficult to interpret (Fig. 10e). However, NR1C2 immunoreactivity showed a regular distribution of aggregates corresponding to the pattern of cone pedicles (Fig. 10f, arrowheads). In the rat retina, we recently showed by using electron microscopy that this corresponds to a presynaptic localization of NR1C2 in cone pedicles (Fletcher et al., 2000). A section that was double labeled for the NR2A and NR2B subunits is shown in Figure 10g,h. In the IPL, both subunits appear to be clustered at postsynaptic sites; however, at most of the synapses, they are not found together. This becomes apparent in a point-by-point comparison of Figure 10g with Figure 10h. In conclusion, all three major types of ionotropic GluRs AMPA, kainate, and NMDA receptors are expressed in the mouse retina. In the IPL, they are clustered at postsynaptic sites. Different subunits exhibit different patterns of stratification across the IPL, suggesting that they may be involved with different synaptic circuits and cell types. The analysis of GluRs in the OPL is more complex, and electron microscopy is needed to describe the expression of GluRs on cone pedicles and rod spherules in more detail. DISCUSSION It was the primary aim of this study to present markers that recognize specific cell types of the mouse retina. Such specific markers appear to be useful and necessary not only in studies of normal mouse retina but even more so in studies of mutant mice retinae. Ideally, one would like to have a marker that selectively stains only one of the approximately cell types present in the mouse retina. However, only few markers, such as choline acetyltransferase (ChAT), which is specific for cholinergic amacrine cells, were found to label exclusively a particular cell type. Many of the antibodies described here recognize more than one cell type. It also would be desirable that such antibodies across species label the same type of neuron consistently (Hendry and Calkins, 1998). Unfortunately, this is not the case, and some particularly bad examples of such cross-species variability are the calciumbinding proteins calbindin, PV, calretinin, and recoverin. Finally, it is important that the expression of the markers appears early during the embryonic development of the retina so that the lineage of certain cell types can be followed. The catalog of antibodies applied in the current study is by no means complete, and it is biased toward markers of the inner retina. Many photoreceptor-specific markers are available that have been neglected here (Wikler and Rakic, 1990; Wikler et al., 1996, 1997). The same is true for antibodies that recognize different neuroactive substances in GABAergic amacrine cells, such as somatostatin, neuropeptide Y, enkephalin, and others (Brecha, 1983). We did not present data obtained with antibodies against synapse-associated proteins, such as synapsins, synaptophysin, synaptic vesicle protein 2, or postsynaptic density proteins. Finally, we neglected markers that may recognize specific ganglion cell classes, such as Thy-1 (Barnstable et al., 1983; Barnstable and Dräger, 1984), neurofilament proteins (Peichl, 1991), CaMII kinase (Ochiishi et al., 1994), or POU domains (Xiang et al., 1996). Many of the antibodies applied in the current study are not available commercially; nonetheless; we hope that they will be provided by the laboratories and persons listed in the table of antibodies. During this study, we also realized that some antibodies, when purchased from different companies, have variable specificities and cross reactivities. The antibody against PKC that was purchased 15

16 16 S. HAVERKAMP AND H. WÄSSLE Figure 9

17 MOUSE RETINA from Amersham recognizes rod bipolar cells and S-cones, whereas the antiserum against PKC that was purchased from Seikagaku does not cross react with S-cones. We also noticed that different antibodies against GABA cross react with GABAergic amacrine cells in specific ways, thus producing different staining patterns. We also want to emphasize that the fixation protocol has a decisive influence on the quality of the labeling. In the case of transmitter receptors, for instance, punctate (that is, synaptic) staining became apparent only with short fixation times. Generally, and contrary to expectations, short fixation resulted in more intense immunofluorescence. Throughout this study, we applied secondary antibodies coupled to the Alexa/indocarbocyanine family of dyes (Molecular Probes). Their bright, nonfading fluorescence is far superior to that of conventional FITC- or rhodamine-coupled secondary antibodies. In the discussion below, we review the staining of the major cell types of the mouse retina and compare this with other mammalian retinae. Horizontal cells Most mammals have two types of horizontal cells: an axon-bearing type that contacts cones and rods and an axonless type that contacts cones (Peichl et al., 1998). Antisera against calcium-binding proteins have been shown in various mammals to label horizontal cells (Rabié et al., 1985; Röhrenbeck et al., 1987, 1989; Pasteels et al., 1990; Peichl and Gonzáles-Soriano, 1994; Massey and Mills, 1996). In the primate retina, H1 and H2 horizontal cells were immunoreactive for PV. Calbindin labeled H2 horizontal cells and two types of cone bipolar cells (Röhrenbeck et al., 1989; Grünert et al., 1994). Both A-type and B-type horizontal cells of the cat retina were immunoreactive for PV; only A-type horizontal cells were labeled by calbindin (Röhrenbeck et al., 1987). In the rabbit retina, both A-type and B-type horizontal cells and a cone bipolar cell were found to be immunoreactive for calbindin (Röhrenbeck et al., 1987; Massey and Mills, 1996). Rat horizontal cells were weakly immunoreactive for PV (depending on the specific antiserum applied) and were strongly immunoreactive for calbindin. In the mouse retina (Fig. 10), calbindin appears to be a reliable marker of horizontal cells, and they usually are the most intensively Fig. 9. Confocal fluorescence photomicrographs of vertical frozen sections through mouse retinae that were immunolabeled for various transmitter receptors and gephyrin. a: Section labeled for the 1 subunit of the glycine receptor (GlyR 1; clone mab 2b) showing punctate staining in the IPL. b: Section labeled with antibody mab 4a, which is believed to recognize all subunits of the glycine receptor. c,d: Section that was double labeled for gephyrin and the 2 subunit of the GABA A receptor. Many immunofluorescent puncta express both gephyrin and GABA A 2. e: Section labeled for the 1 subunit of the GABA A receptor. Extrasynaptic labeling of bipolar and amacrine cells can be seen. In the OPL, a dashed band of cone pedicles is labeled prominently. The punctate label in the IPL has a characteristic pattern of stratification. f: Section labeled for the 3 subunit of the GABA A receptor, which is absent from the OPL. The two strata of reduced puncta density in the IPL represent the location of the cholinergic bands. g,h: Section that was double labeled for the 2 subunit of the GABA A receptor and for the subunits of the GABA C receptor. Superposition of the two confocal photomicrographs (not shown) demonstrated that the puncta shown in g and h were not colocalized. Scale bars 25 m. fluorescent neurons in calbindin-labeled sections (for review, see Peichl and Gonzáles-Soriano, 1994). Our laboratory recently studied the retina of a calbindin null mutant mouse, and, as expected, calbindin immunoreactivity was absent from the retina (Wässle et al., 1998b). Bipolar cells There are approximately ten types of cone bipolar cells and one type of rod bipolar cell in all mammalian retinae studied (cat: Kolb et al., 1981; McGuire et al., 1984; Cohen and Sterling, 1990a,b; rabbit: Famiglietti, 1981; Mills and Massey, 1992; Merighi et al., 1996; primate: Boycott and Wässle, 1991; rat: Euler and Wässle, 1995). The major subdivision of cone bipolar cells is into ON-cone and OFFcone bipolar cells. Axon terminals of OFF-cone bipolar cells stratify in the outer part of the IPL, and those of ON-bipolar cells terminate in the inner IPL (Euler et al., 1996). Immunocytochemical markers have been applied to study specific populations of bipolar cells in primate, rabbit, and rat retinae. Rod bipolar cells in all mammals appear to be immunoreactive for PKC (Negishi et al., 1988; Greferath et al., 1990; Vaney et al., 1991; Grünert et al., 1994). Markers also have been described for cone bipolar cells; however, the labeled subtypes appear to be different depending on the species. Recoverin, for instance, has been found to label selectively flat, midget bipolar cells in the primate retina (Milam et al., 1993; Grünert et al., 1994), whereas two types of cone bipolar cells, ON-cone and OFF-cone, are stained in ox, rat, and rabbit retinae (Milam et al., 1993; Euler and Wässle, 1995; Massey and Mills, 1996). Calbindin immunoreactivity was described for H2 horizontal cells, OFF-cone bipolar cells (DB3), and ON-cone bipolar cells in primates (Grünert et al., 1994). In the rabbit retina, A-type and B-type horizontal cells and an ON-cone bipolar cell were calbindinimmunoreactive (Massey and Mills, 1996). Other markers appear to be specific for certain species, such as CCK, which labels blue cone bipolar cells and ON-midget bipolar cells in primates (Kouyama and Marshak, 1992; Wässle et al., 1994), or CD15, which labels a specific population of bipolar cells in the rabbit retina (Brown and Masland, 1999). These results show that some markers for bipolar cells are specific for certain species or label different cell types in different species. Rod bipolar cells. Rod bipolar cells of the mouse retina, similar to other mammals, are immunoreactive for PKC (Figs. 2b, 3d, 4b) and for L7, a Purkinje cell marker (Berrebi et al., 1991). The expression of L7 by rod bipolar cells also was used for a transgenic approach: with the lacz reporter gene coupled to the L7 promoter, rod bipolar cells could be labeled selectively in the retina of these mutant mice (Oberdick et al., 1990). Other markers for rod bipolar cells described in the current study, such as Pep19, PKC, mab 115A10 (ROB), and Go, also recognized some additional cone bipolar cell types. ON-cone bipolar cells. Based on the results from the rat retina, we assume that cone bipolar cells with axons that terminate in the inner IPL are ON-cone bipolar cells (Euler et al., 1996; Hartveit, 1997). They were found to be immunoreactive for Go (Fig. 3h), which agrees with results from cat and monkey retinae (Vardi et al., 1993; Vardi, 1998). They also were immunoreactive for mab 115A10 (Fig. 4), which also has been shown in primate and rat retinae to label ON-cone bipolar cells (Grünert et al., 1994; Euler and Wässle, 1995). Ueda and colleagues 17

18 18 S. HAVERKAMP AND H. WÄSSLE Figure 10