Dopaminergic transmission in the rat retina: evidence for volume transmission

Journal of Chemical Neuroanatomy 12 (1996) 37 – 50

Dopaminergic transmission in the rat retina: evidence for volume transmission Bo¨rje Bjelkea,*, Menek Goldsteind, Barbro Tinnera, Cecilia Anderssona, Susan R. Sesackb, Harry W.M. Steinbusche, Jow Y. Lewd, Xi Hed, Stan Watsonf, Bjo¨rn Tengrothc, Kjell Fuxea a Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden Dept. Beha6ioural Neuroscience, Uni6ersity of Pittsburgh, Crawford Hall, Pittsburgh, PA 15260, USA c St. Eriks Eye Hospital, 112 82 Stockholm, Sweden d Neurochemistry Research Unit, New York Uni6ersity, Medical Centre, 560 First A6enue, New York, NY 10016, USA e European Graduate School for Neuroscience in Brain & Beha6iour, Uni6ersity of Maastricht, Dept. Psychiatry and Neuropsychology, Maastricht, The Netherlands f Mental Health Research Institute, The Uni6ersity of Michigan, Ann Arbor, MI 48109, USA b

Received 1 May 1996; accepted 18 August 1996

Abstract The study was designed to determine whether dopaminergic neurotransmission in the retina can operate via volume transmission. In double immunolabelling experiments, a mismatch as well as a match was demonstrated in the rat retina between tyrosine hydroxylase (TH) and dopamine (DA) immunoreactive (ir) terminals and cell bodies and dopamine D2 receptor-like ir cell bodies and processes. The match regions were located in the inner nuclear and plexiform layers (D2 ir cell bodies plus processes). The mismatch regions were located in the ganglion cell layer, the outer plexiform layer, and the outer segment of the photoreceptor layer, where very few TH ir terminals can be found in relation to the D2 like ir processes. In similar experiments analyzing D1 receptor like ir processes versus TH ir nerve terminals, mainly a mismatch in their distribution could be demonstrated, with the D1 like ir processes present in the outer plexiform layer and the outer segment where a mismatch in D2 like receptors also exists. The demonstration of a mismatch between the localization of the TH terminal plexus and the dopamine D2 and D1 receptor subtypes in the outer plexiform layer, the outer segment and the ganglion cell layer (only D2 immunoreactivity (IR)) suggests that dopamine, mainly from the inner plexiform layer, may reach the D2 and D1 mismatch receptors via diffusion in the extracellular space. After injecting dopamine into the corpus vitreum, dopamine diffuses through the retina, and strong catecholamine (CA) fluorescence appears in the entire inner plexiform layer and the entire outer plexiform layer, representing the match and mismatch DA receptor areas, respectively. The DA is probably bound to D1 and D2 receptors in both plexiform layers, since the DA receptor antagonist chlorpromazine fully blocks the appearance of the DA fluorescence, while only a partial blockade is found after haloperidol treatment which mainly blocks D2 receptors. These results indicate that the amacrine and/or interplexiform DA cells, with sparse branches in the outer plexiform layer, can operate via volume transmission in the rat retina to influence the outer plexiform layer and the outer segment, as well as other layers of the rat retina such as the ganglion cell layer. Copyright © 1996 Elsevier Science B.V. Keywords: Dopamine receptor; Immunohistochemistry; Mismatch; Catecholamine histochemistry

Abbre6iations: aa, amino acid; ABC, avidin biotin complex; b.wt., body weight; CA, catecholamine; cb, cerebellum; Ch, choroid layer; cs, striatum; D1, dopamine receptor type 1; D2, dopamine receptor type 2; D2(242 – 266), L dopamine receptor type 2 long; D2(273 – 287), L+ S dopamine receptor type 2 long and short; D2(246 – 316), L+S dopamine receptor type 2 long and short; D3, dopamine receptor type 3; D3, dopamine receptor type 4; D5, dopamine receptor type 5; DA, dopamine; DAB, 3%3 diaminobenzidine; FITC, fluorescein isothiocyanate; GC, ganglion cell layer; ICC, immunocytochemistry; INL, inner nuclear layer; IPL, inner plexiform layer; IR, immunoreactivity; ir, immunoreactive; NA, noradrenaline; L, large dopamine cell type; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment of the photoreceptor layer; PAP, peroxidase anti peroxidase; PBS, phosphate buffered saline; R&C, rods and cones; Rec, receptor; S, small dopamine cell type; SPG, sucrose, potassium phosphate, glyoxylic acid; TH, tyrosine hydroxylase. * Corresponding author. Tel.: + 46 8 7287085; fax: + 46 8 337941. 0891-0618/96$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S0891-0618(96)00176-7

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1. Introduction The morphology of the retina was previously described by Cajal (Cajal, 1904; Polyak, 1941). The distribution of catecholamine amacrine nerve cells of type 1 (large) and type 2 (small) has been shown to vary to a large degree between species (Dowling and Ehinger, 1978a; Tork and Stone, 1979; Brecha et al., 1984; Oyster et al., 1985; Versaux-Botteri et al., 1986; Nguyen-Legros, 1988; Kolb et al., 1990; Wang et al., 1990). The CA terminal network of type 2 neurons also shows a different innervation pattern and a lower tyrosine hydroxylase IR than the type 1 neurons (Mariani, 1991), suggesting different functions for these two types of CA cells in the vertebrate retina, the former of which may mainly represent epinephrine cells and the latter type representing DA cells (Versaux-Botteri et al., 1986). Only sparse TH ir terminals have been observed in the outer plexiform and nuclear layers belonging to interplexiform DA cells (Dowling and Ehinger, 1975; Savy et al., 1989; Nguyen-Legros et al., 1990), except in the fish and the new world monkey retina (Djamgoz and Wagner, 1991; Witkovsky and Schu¨tte, 1991; Witkovsky et al., 1993). The vast majority of TH ir terminals are instead found in the outer part of the internal plexiform layer (Moussafi et al., 1990; Zhu and Straznicky, 1990; Witkovsky and Schu¨tte, 1991). Dopamine is released by light stimulation (Bauer et al., 1980; Kirsch and Wagner, 1989) and it was suggested that it affects the light-dark adaptation (Dearry and Burnside, 1989) by regulating the function of the pigment epithelium, (Besharse et al., 1988), the rods and cones, (Pierce and Besharse, 1988; Witkovsky and Shi, 1990; Zawilska and Nowak, 1994), the horizontal cells (Schorderet and Nowak, 1990), and also the rod AII amacrine cells (Jensen, 1989). These actions except the first one reduce the impact of the rod pathway (Djamgoz and Wagner, 1991; Witkovsky and Schu¨tte, 1991; Witkovsky and Dearry, 1992). Two distinct DA receptor families were described, namely the D1 like receptors and the D2 like receptors (Sibley and Monsma, 1992). The D1 and D5 subtypes belong to the former category (Dearry et al., 1990; Zhou et al., 1990) and the D2, D3 and D4 subtypes belong to the latter category (Bunzow et al., 1988; Sokoloff et al., 1990; Van Tol et al., 1991). The dopamine D1 and D2 subtypes have been localized by receptor autoradiography in the retina of various species including the rat retina (Makman and Dvorkin, 1986; Porceddu et al., 1987; Elena et al., 1989; Denis et al., 1990; Schorderet and Nowak, 1990). The results show that the D1 receptors are mainly located in the inner and outer plexiform layers of the vertebrate, including the rat retina, see also (Behrens and Wagner, 1995). The D2 receptors also seem to be mainly localized in the outer and the inner plexiform layers but also exist in the rod outer

segment of the rat, rabbit, cow, chicken, turtle, frog and fish retina (Vuvan et al., 1993; Wagner et al., 1993). Evidence has been presented that D4 receptors are localized on photoreceptors of mouse retina using in situ hybridization techniques (Cohen et al., 1992). In the rat retina, the DA interplexiform cells with their dendritic networks have been described in detail (Savy et al., 1995). The close relationship between DA cell processes and blood capillary walls has also been established by means of ultrastructural immunocytochemistry (Favard et al., 1990). However, there is a lack of knowledge about the exact topographic relationships between the network of DA neuronal processes and the D1 and D2 receptor containing structures. In the present study, a detailed analysis of the pre- and post-junctional features of the DA amacrine and/or interplexiform neurons of the rat retina has been performed. These studies involve TH immunocytochemistry, CA fluorescence histochemistry and D1 and D2 receptor like immunocytochemistry. The results of these studies provide further insights on the mode of DA communication in the retina. The diffusion and binding of DA in the retina following microinjection into the corpus vitreum also gives indications for the existence of volume transmission (Agnati et al., 1986; Bjelke et al., 1988; Bjelke et al., 1989; Fuxe and Agnati, 1991) as a major mode of communication for DA amacrine and/or interplexiform cells of the rat retina.

2. Material and methods

2.1. Material Sixty male Sprague Dawley rats (BKL/SD) b.wt. 250–350 g (BK Universal, Stockholm, Sweden) were used and kept under standardized conditions, with lights on at 0700 h and off at 1900 h and with free access to food pellets and water. The animals were acclimatised in the animal ward some days before use. All rats were light-adapted before any experimental procedures were started.

2.2. Experimental procedures Microinjections were made into the corpus vitreum close to the retina, of solvent or 0.01–0.1 mmol/ml of 3-hydroxytyramine (dopamine, Sigma, USA) dissolved in 2 ml of 0.1 M phosphate buffered saline (PBS), including 1.1 mM ascorbic acid, using a 10 ml Hamilton syringe. The 26 rats used were exposed to barbiturate anaesthesia (Pentobarbital-sodium 50 mg/kg, i.p., Apoteksbolaget) before the intraocular injection and kept anaesthetised for 5 or 15 min after the DA or solvent injection, at which time the rats were sacrificed.

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2.3. Preparation and characterization of D2 receptor antisera 2.3.1. Antiserum against the short and long form of the D2 receptor (D2 (273 – 287); L + S) For an extensive description of this D2-receptor antiserum (D2(273 – 287); L + S) see (Sesack et al., 1994). Briefly, a synthetic peptide fragment corresponding to amino acids 273–287 of the rat dopamine D2 receptor sequence (Bunzow et al., 1988) (part of the third intracellular loop containing amino acid sequences common to both the long and short form of the D2-receptor) was conjugated via glutaraldehyde to the keyhole limpet hemocyanin and the conjugate was used to immunize rats. The antiserum was characterized with immunoblot analysis and furthermore by immunocytochemistry in cultured cells and brain tissue preparations. 2.3.2. Antiserum against the long form of the D2 receptor (D2 (242 – 266); L). The D2-receptor antiserum (D2(242 – 266); L) was prepared and characterized in the following way. 2.3.2.1. Generation of anti-peptide serum. A peptide with the sequence corresponding to the amino acid residues 242–266 of the D2 receptor was synthesized and purified by high performance liquid chromatography. This peptide was conjugated to keyhole limpet hemocyanin through the thiol group of the cysteine residues by sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The peptide conjugate containing 50–100 mg peptide was emulsified with Freund’s complete adjuvant and injected into female New Zealand rabbits at multiple sites subcutaneously. For subsequent booster injections, the peptide conjugate was emulsified with Freund’s incomplete adjuvant. The antibody titers were screened by enzyme linked immunosorbent assay. This antibody was generated by Drs. J.Y. Lew and M. Goldstein (unpublished data). 2.3.2.2. Purification of the antibody. The antibody was purified by chromatography on affinity gels which were prepared by coupling of the peptide to epoxy-activated Sepharose 6B. Sera containing D2 receptor antibody were incubated with affinity gel in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 5 mg/ml each of leupeptin, pepstatin and aprotinin in 0.5 mM EDTA). After loading the gels on the columns the gels were first treated with 50 mM Tris buffer (pH 7.4 containing 0.5 M NaCl) until the fractions were protein-free. The antibody was eluted from the gels with 4.6 M MgCl2, dialyzed against 4×3.5 l of 50 mM Tris buffer (pH 7.4 containing 0.154 M NaCl) and concentrated to a small volume.

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2.3.2.3. Western blot analysis. Proteins were separated by 10% SDS-PAGE, transfered to nitrocellulose and blocked with 1.5% powdered milk in 50 mM Tris (pH 7.5), containing 154 mM NaCl, 0.1% Tween 20 (TBS– T) for 1 h at room temperature. The blot was incubated with primary antibodies at 1:1000 dilution in 10% normal goat serum for 90 min at room temperature, washed once with 20 mM Tris (pH 7.5) containing 60 mM NaCl, 0.4% Triton X-100, 0.4% SDS, 2 mM EDTA, and 0.4% sodium deoxycholate (RIPEA) and three times with TBS-T. Secondary antibodies, goat anti-rabbit IgG-HRP conjugate (1:1000 dilution), were incubated for 1 h at room temperature and washed as before, and the protein bands were detected by using an enhanced chemiluminescence (ECO) Western blotting detection system. On immunoblots two protein bands with M.W. of approximately 92 kDa and 105 kDa were recognized by the antibody. In extracts derived from the rat striatum but not from the cerebellum a band was found with the M.W. of 92 kDa corresponding to the M.W. of the DA D2 receptor. The band with the M.W. of 105 kDa was not identified. In control experiments the antiserum was pre-absorbed with the D2 peptide used as antigen (50– 500 mg/ml). 2.3.3. Antiserum against the D2 receptor D2(246 – 316); L+S The antibody was raised in rabbit against the rat D2 receptor amino acid sequence 246–316, with a molecular weight of approximately 7000 g/mol, and with a low level of homology to other known G-protein coupled receptors (Burke et al., 1993). This recombinant peptide was made as a fusion protein in a sequence with glutathione S-transferase in a modified pGEX vector. The pGEX vectors were designed to synthesize fusion proteins with a foreign protein fused to the C-terminal of glutathione S-transferase. The fusion proteins were purified by glutathione affinity chromatography. The antibody was affinity purified. In control experiments the antiserum was pre-absorbed with the D2 peptide used as antigen (50–500 mg/ml). 2.4. Preparation and characterization of the D1 receptor antiserum 2.4.1. Antiserum against the D1 receptor D1(356 – 446) The fusion protein was made of a Trp-E C-terminus of the rat DA D1 receptor (Zhou et al., 1990). A segment of the rat D1 receptor (amino acid residues 356–446) was inserted into the fusion vector (EcoRIHindIII fragment). The fusion vector PATH3 containing the TrpE operon was expressed in a bacterial expression system, XL-1 E. coli cells, and induced with indole acrylic acid. The induced fusion protein was purified and isolated on SDS -PAGE gels and used as

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antigen for immunization of rabbits. The sera from rabbits immunized with the DA D1 fusion protein were screened on immunoblots (Western blots) for their ability to recognize DA D1 receptor protein. The immunoblots show that the anti-fusion DA D1(356 – 446) receptor antiserum immunoreacts with several proteins derived from rat striatum and cerebellum. The immunoblot obtained from striatal extracts contains a protein band (72 kDa) corresponding to the DA D1 receptor. This band was not detectable in the cerebellum (Fig. 1) suggesting that it recognizes the D1 receptor. Furthermore, this antiserum with high specificity strongly labels the D1 receptors, e.g. in the strionigral and strioentopeduncular GABA pathway and fails, e.g. to demonstrate the D2 receptors in the nigral DA cell bodies. Thus, this D1 antiserum does not crossreact with D2 receptors. In a few experiments also a well characterized immuno affinity purified rat monoclonal antibody directed to the C-terminal 97 amino acids of the human D1 dopamine receptor fused to a polypeptide fragment of glutathione S-transferase was used (Research Biochemicals International, USA, diluted 1:500) (Smiley et al., 1994; Hersch et al., 1995; Yung et al., 1995). It was obtained from Research Biochemicals International, USA.

2.5. Immunohistochemistry The rats were transcardially perfused, under deep barbiturate anaesthesia (Pentobarbital-sodium 50 mg/ kg). For all antisera, except for the anti-dopamine antiserum and the anti-D2(273 – 287); L +S receptor antiserum, the following solutions were used; 50 ml 0.9% saline containing 500 IE heparin was used for preperfusion, immediately followed by 500 ml 4% formalin including 14% v/v saturated picric acid. Perfusion pressure was set to 0.4 Bar, using a specially designed air driven perfusion devise. The eye balls were rapidly dissected out, the cornea, the lens and the corpus vitreum were removed, while the retina attached to the sclera were immersed in the fixative for 60 min and thereafter transferred into 10% sucrose in 0.1 M PBS and kept refrigerated until sectioning. Some retinas were submitted to 60-mm thick sectioning in a cryostat and incubated glassmounted on chrome alun coated slides, while other retinas were processed free floating. For dopamine immunohistochemistry, a 50 ml Krebs solution with 10 mM ascorbic acid (pH 6.2) was used for preperfusion, immediately followed by 5% glutaraldehyde in phosphate buffer including 10 mM ascorbic acid (pH 7.4). After 30 min of immersion in the fixative the eye balls were vibratome sectioned in a 0.05 M Trizma buffer containing 10 mM ascorbic acid. For vibratome sectioning the opened eye balls were embedded in 35% gelatin and sectioned in 60-mm thick slices and processed free floating.

For D2 like immunohistochemistry with the D2(273 – 287); L+ S antiserum the following fixation procedure was used. A preperfusion, consisting of 10 ml saline with heparin (1000 U/ml), was followed by 50 ml of 3.75% acrolein (Polyscience), in 2% formaldehyde in 0.1 M phosphate buffer (pH 7.4) and then by an additional 200 ml of 2% formaldehyde, in which the retinas also were immersed for 30 min. The retinas were vibratome sectioned, 60-mm thick, using the same embedding technique as described for the dopamine immunohistochemistry. The following antibodies were used for immunohistochemical studies, mouse monoclonal anti-tyrosine hydroxylase (1:1000; Incstar, USA), rabbit anti Dopamine (1:800; from Dr. H.W.M. Steinbusch) (Steinbusch et al., 1991), a rat antiserum against the dopamine D2(273 – 287); L+ S receptor (1:1000; from Dr. S.R. Sesack) (Sesack et al., 1994), a rabbit antiserum against the dopamine D2(242 – 266); L receptor (1:200; from Dr. M. Goldstein), a rabbit antiserum against the rat dopamine D2 receptor (1:200, Dr. Watson) (Burke et al., 1993), a rabbit antiserum against the dopamine D1(356 – 446) receptor (1:200) from Dr. M. Goldstein, a rat anti-human monoclonal antibody against the D1 receptor (1:500) from Research Biochemicals International, USA. The following secondary antisera were used: FITC or Texas Red conjugated antisera (Jackson) for immuno fluorescence, biotin conjugated antisera (Vector) for ABC immunocytochemistry (ICC) and goat anti rabbit IgG (Nordic) for dopamine ICC. The avidin/biotin (ABC) technique (ABC kit, Vector) was used, except for the dopamine stainings, where the peroxidase anti peroxidase (PAP) technique was employed, in both cases with 3%3 diaminobenzidine (DAB) as a chromogen. The stained free floating sections were mounted on chrome alum coated slides, dehydrated and coverslipped with Mountex (Go¨teborgs termometerfabrik, Sweden). For immunofluorescence, the sections were coverslipped in a glycerin medium using p-phenylendiamine as an antifading agent (50 mg p-phenylendiamine, No. P-6001 Sigma, and 5 mg ascorbic acid dissolved in 5 ml 0.1 M PBS (pH 9.0), and mixed with 45 ml cooled glycerin).

2.6. CA fluorescence histochemistry To visualize the injected dopamine the original FalckHillarp method (Falck et al., 1962; Andersson et al., 1985) was used. The entire eyeballs was quickly removed and frozen in liquid propane, kept in liquid nitrogen until freezedrying and reacted with paraformaldehyde vapor according to the standard procedure. The material was paraffin embedded, microtome sectioned and coverslipped with xylen-entellan as a mounting medium (Andersson et al., 1985). The SPG method (Torre and Surgeon, 1976) was used alternatively, since it is less labor requiring, and

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Fig. 1. Western blot of the D1-DA receptor antiserum showing the recognition of two proteins (72 and 84 kDa). The presence of the 72 kDa band in striatum (cs) but not in cerebellum (cb) suggests that this band represents the D1 receptor. Fig. 2. (A,B,C,D) Overview (panel A) of the rat retina showing the two types of TH immunoreactive cells; to the right, the large cell type (L), located in the inner plexiform layer (IPL), and to the left, the small cell type (S), located in the inner nuclear layer (INL). The respective cells are shown in higher magnification in panel B and C. The cell somata are located in the inner part of the INL with the somata oriented towards the outer part of retina. The dense TH immunoreactive terminal plexus (panel A and D) are mainly located in the outer part of the IPL with single branches reaching out to the INL and the outer plexiform layer (OPL) indicated by arrowheads (panel B and D). Scattered TH ir terminals with large varicosities also extend to the border of the ganglion cell layer (GC) as indicated by arrowheads (panel D). Bars indicate 50 mm and 25 mm.

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the results can be obtained in 1 day compared to the standard Falck-Hillarp method, involving at least a 3-day procedure. When using the SPG method, the entire eyeball was quickly removed, frozen with dry ice, cryostat sectioned (14-mm thick), glassmounted and air dried immediately. The slides were then dipped into the SPG (sucrose, potassium phosphate, glyoxylic acid) solution and immediately dried, covered with light mineral oil and reacted for 90 min at 90°C in an oven. The slides were then coverslipped and evaluated in a fluorescence microscope.

3. Results In agreement with previous work (Dowling and Ehinger, 1975; Dowling and Ehinger, 1978b; Brecha et al., 1984; Oyster et al., 1985; Versaux-Botteri et al., 1986; Nguyen-Legros, 1988; Savy et al., 1989; Kolb et al., 1990; Steinbusch et al., 1991) the CA positive cell somata consisted of two types, a small and a large type (Fig. 2B,C) and the cell somata were located in the inner part of the inner nuclear layer with branches oriented mainly towards the inner layers in the retina (Figs. 2 and 3). Also in line with previous work (see

Fig. 3. (A,B) Catecholamine (CA) fluorescence, shown as a light blue fluorescence obtained by the SPG method (panel A). A CA positive nerve cell of the large type (L) located in the inner nuclear layer (INL) with its terminal plexus in the outer part of the inner plexiform layer (IPL). It is possible to see a punctate cytoplasmic CA fluorescence. Scattered terminal puncta are also present in the outer nuclear layer, indicated by arrowheads. In panel B, a dopamine ir nerve cell of the large type (L) is shown together with the terminal plexus. The same distribution within the retinal layers is seen as in panel A. Bars indicate 50 mm and 25 mm, respectively.

above) the vast majority of the TH ir terminals was located in the outer part (sublamina 1) of the inner plexiform layer (Fig. 2) with only a few scattered fibers present in the ganglion cell layer, in the inner nuclear layer and in the outer plexiform layer. All the terminals were rich in varicosities (Figs. 2 and 3).

3.1. Characterization of the DA receptor subtypes and their relationship to TH ir ner6e terminal structures 3.1.1. D2 -like immunoreacti6ity The D2(273 – 287); L+ S antiserum against the rat D2 receptor showed a cytoplasmic labelling of a discrete nerve cell population localized in the inner part of the inner nuclear layer with the same morphological fea tures as the large DA nerve cells (Fig. 4). The D2 receptor-like ir terminal network also showed a similar distribution to that of the TH ir terminals with a dominant presence in the outer part (sublamina 1) of the inner plexiform layer. The acrolein used is a very strong fixative limiting the penetration of the D2(273 – 287); L+ S antiserum. Therefore, only a surface staining is shown in Fig. 4, explaining the presence of only a very sparse D2 receptor-like ir terminal network. The outer segment shows a strong D2-like IR. The D2(242 – 266); L and the D2(246 – 316);L+S receptor antisera showed similar patterns of IR as seen with D2(273 – 287); L+ S in the inner plexiform and nuclear layers. Thus, they showed a weak to moderate cytoplasmic labelling of TH ir nerve cell bodies as seen in double immunolabelled sections (Fig. 5). In addition, with these two antisera radial D2 like ir branches formed terminal-like processes in the outer plexiform layer (Fig. 6) and in the ganglion cell layer, where also ir cell bodies were found. As shown in single and double labelled sections very few TH ir terminals were present in the outer retina, thus, demonstrating a topological mismatch between the DA terminal field and the D2 like ir processes (Fig. 2 and data not shown). After pre-absorption with the corresponding D2 peptide (50– 500 mg/ml) the labelling was markedly reduced (D2(242 – 266); L) or fully disappeared (D2(246 – 316); L +S) in all parts of the retina (Fig. 6) 3.1.2. D1 -like immunocytochemistry Using the D1(356 – 446) receptor antiserum, it was possible to demonstrate D1 receptor IR in the outer segment of the rods and cones, in a distinct layer of processes in the outer plexiform layer, as well as in bands of puncta in the internal plexiform layer (Fig. 7). A few D1 receptor ir cellbodies were also identified in the outer part of the inner nuclear layer. The appearance of D1 receptor-like IR was fully prevented when the D1(356 – 446) antiserum had been absorbed with a peptide from the C terminal portion of the D1 receptor (500 mg/ml) (Fig. 8). A double immunolabelling procedure with TH

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Fig. 4. (A,B) D2-like IR in a thin surface staining of a rat retina section, using a D2 antiserum (amino acid sequence 273–287). A large nerve cell soma (arrow), with a cytoplasmic D2 IR is located in the inner nuclear layer (INL) and D2 ir varicose terminals (arrowheads) in the outer part of the inner plexiform layer are seen (panel A). In panel B, a few scattered D2 ir varicose terminals (arrowheads) are present also in the outer nuclear and plexiform layer. D2-like IR is also seen in the outer segment (OS). Bar indicates 25 mm.

IR demonstrated a marked functional mismatch in the distribution of the TH ir terminals (mainly present in the inner plexiform layer) versus that of the D1 receptor ir processes mainly present in the outer plexiform layer and in the outer segment of the photoreceptors. Similar results on localization and distribution of D1 like immunoreactivity were obtained in the rat retina with the commercially available rat-anti-D1 monoclonal antibody but with less strong labelling.

Fig. 5. (A,B) Double immunofluorescence labelling of the same retina section with a rabbit D2(242 – 266); L-antiserum (aa. 242 – 266) (FITC), (panel A) and a mouse monoclonal TH antibody (Texas red) (panel B). A cell body showing a weak D2(242 – 266); L like IR (arrow) (panel A) and is surrounded by strong TH IR (arrow), located in large terminal processes in the inner plexiform layer (IPL) (panel B). Bar indicates 20 mm.

3.2. DA microinjections into the corpus 6itreum 3.2.1. CA fluorescence in retina DA was injected in concentrations of 0.01 – 0.1 mmol/ ml in a volume of 2 ml into the corpus vitreum adjacent

to the retina. A concentration-related appearance of a band of CA fluorescence was observed, 5 and 15 min later, in the outer plexiform layer, which became very strong after the highest concentration of injected DA.

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Fig. 6. (A,B,C) D2-like IR (panel A) found in the rat retina, using a D2 antiserum (aa. 246 – 316) and immunofluorescence procedures (FITC). D2 like ir cell somata are observed (indicated by arrows) in the inner part of the inner nuclear layer (INL) with the somata oriented towards the outer part of the retina. A strong band of D2-like ir processes is present in the outer plexiform layer (OPL) (indicated with arrowheads). A dense plexus of strongly D2 like ir processes is also observed in the ganglion cell layer (GC). After preabsorption (panel B) with the D2 peptide (500 mg/ml), used as antigen, there is no IR to be demonstrated. Panel C shows the D2 like IR in lower magnification. Bars indicate 25 mm.

Substantial increases of CA fluorescence also took place within the inner plexiform layer, and the CA fluorescence now covered the entire width of the layer. Thus, also in this area a concentration-related appearance of CA fluorescence was found in the entire layer after the DA microinjection, making it difficult to visualize the extensive CA nerve terminal plexus in the outer part of this layer. However, with lower concentrations, the extraneuronal CA fluorescence was visualized together with the CA nerve terminal networks in the outer part (see Fig. 9).

3.2.2. Effects of systemic Chlorpromazine and haloperidol pretreatement on the apperance of DA fluorescence A complete blockade of the appearance of the DA fluorescence in the inner and outer plexiform layers was observed after corpus vitreum injections of DA accompanied by systemic chlorpromazine treatment (6.5 mg/ kg i.p., 30 min earlier) (see Fig. 10) blocking both D1 and D2 like receptors. After the preferential D2 receptor antagonist haloperidol (5 mg/kg i.p., 30 min earlier), only a partial blockade of the appearance of DA

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Fig. 7. (A,B) Double immunofluorescence labelling of the same retina section with a rabbit D1(356 – 446)-Rec antiserum (FITC) from Dr. Goldstein, (panel A) and a mouse monoclonal TH antibody (Texas red) (panel B). The strongly D1-like immunoreactive processes of the outer plexiform layer (OPL) (panel A) with associated D1 positive cell bodies in the inner nuclear layer (INL) (arrows) show a functional mismatch with the TH ir terminal plexus (panel B). In contrast, the strongly TH ir terminal plexus (arrows) in the inner plexiform layer (IPL) with TH ir cell bodies in the INL (panel B) codistributes in part with bands of weak D1-like IR as demonstrated with the present antiserum D1(356 – 446) (panel A). Layer of rods and cones (R&C). Bar indicates 50 mm.

fluorescence was found in the inner and outer plexiform layers (data not shown).

4. Discussion TH and DA immunocytochemistry as well as CA fluorescence histochemistry was utilized to further document the presynaptic features of the DA amacrine and/or interplexiform cells with their terminal varicose plexus, predominantly found within the outer part of the inner plexiform layer. Only a few branches extend into the inner nuclear layer and the outer plexiform layer, forming single varicose processes (Djamgoz and Wagner, 1991; Witkovsky and Dearry, 1992). The cellular localization of D2 and D1 like receptors within the rat retina and in their relationship to the presynaptic

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features of the DA amacrine and/or interplexiform neurons were investigated. In line with previous D2 receptor binding data, see (Schorderet and Nowak, 1990), based on quantitative receptor autoradiography, D2-like ir nerve cell bodies and processes, at least some of which show TH IR, were found in the inner part of the inner nuclear layer with the three D2 antibodies used in the present study. Thus, there appears to exist a D2 autoreceptor within the amacrine and/or interplexiform DA neurons, probably involved in the control of DA synthesis and release. It is possible that the D2 long form is responsible for part of the IR, since the D2 IR was demonstrated also with the D2(242 – 266); L antibody against the insert (Schorderet and Nowak, 1990). With two of the D2 receptor antibodies used (raised against the D2(242 – 266); L receptor and especially the rat D2 receptor, aminoacid sequence 246–316) a D2 labelling was observed also in processes of the inner and outer plexiform layer and the ganglion cell layer, where also D2 ir cell bodies were demonstrated. These findings are in agreement with those reported by Wagner et al. (Wagner et al., 1993). Furthermore, D2 receptor binding has been reported to take place in the outer plexiform layer of the rat retina (Elena et al., 1989; Denis et al., 1990; Schorderet and Nowak, 1990). The fact that the D2 insert antibody also gave positive results in the outer plexiform layer opens up the possibility that the D2 long form exists also in processes of nerve cells also in this layer. The inability of the antibody D2(273 – 287); L+ S to visualize many of the D2 like ir structures, found with the other two antibodies may be related to the difference in fixation procedure, leading to a masking of the corresponding epitope because of the existence of a special conformational state of the D2 receptor in many nerve cells after this fixation, which no longer could bind to this antiserum. It could also be due to differences in the titers between these antibodies. D2 like IR could be demonstrated in the outer segment of the photoreceptor layer with one of the D2 antibodies used (D2(273 – 287); L+ S), in agreement with previous work (Wagner et al., 1993). These results would be compatible with the finding that [3H]-spiperone binding sites exist in the photoreceptor layer (Makman and Dvorkin, 1986; Porceddu et al., 1987; Denis et al., 1990; Schorderet and Nowak, 1990) which may mainly represent D2-like receptors such as D4 receptors (Cohen et al., 1992). Also, the D2-like IR could in some instances with the antiserum D2(242 – 266); L not be fully blocked by preabsorption with the corresponding peptide (Wagner et al., 1993) suggesting some degree of unspecific staining. DA mediated actions on photoreceptors in the rat retina have been demonstrated, involving for example a reduction of the light sensitive pool of cAMP (Cohen and Blazynski, 1990).

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B. Bjelke et al. / Journal of Chemical Neuroanatomy 12 (1996) 37–50

Fig. 8. (A,B) D1-like IR obtained in the rat retina using a D1(356 – 446) antiserum and immunofluorescence procedures (FITC). Panel A shows the D1(356 – 446) like IR mainly as strongly ir processes forming a narrow band in the outer plexiform layer (OPL). D1 like ir cell bodies (indicated by arrows in the outer part) are also seen in inner nuclear layer (INL) as well as in the ganglion cell layer (GC). Weak bands of D1 IR are found in the inner plexiform layer IPL. After pre-absorption with the C terminal part of the D1 receptor (500 mg/ml), there is a clear reduction of D1(356 – 446) IR in all layers (panel B). Outer segment (OS). Bar indicates 50 mm.

Specific D1(356 – 446) like IR was also found in the inner and especially outer plexiform layers localized as horizontal bands built up of fine processes. These regions are known to contain D1 receptor binding sites (Elena et al., 1989). Also distinct D1 like ir nerve cell bodies were located in the inner nuclear layer, probably representing horizontal nerve cell bodies. Similar results were also obtained with the commercially available monoclonal rat-anti-D1 receptor antibody. Thus, D1 receptors may directly control the horizontal nerve cell function, producing an uncoupling of their gap junctions (Witkovsky and Shi, 1990; Djamgoz and Wagner, 1991). It seems possible that the high affinity D2 and D1 receptors in the outer plexiform layer, functioning via inhibition and stimulation of adenylate cyclase respectively, (Qu et al., 1989) can play a role in these interactions (Djamgoz and Wagner, 1991), which may be of relevance for the role of DA in light adaptation (Nowak and Zurawska, 1989). According to the present analysis with demonstration of D1 like IR in the outer segments D1 receptors may also have a role in directly controlling photoreceptor function. The major findings of the present analysis of the preand post-synaptic features of the amacrine and/or interplexiform DA cells of the rat retina were obtained in

the double immunolabelling experiments and in the experiments with diffusion of DA into the retina from the corpus vitreum after microinjection of DA. Thus, in the double immunolabelling experiments showing TH and D2, and TH and D1(356 – 446) like immunoreactivities, distinct functional transmitter/receptor mismatches could be documented. Thus, very few TH and DA ir terminals and CA fluorescent terminals extended into the inner part of the inner plexiform layer and ganglion cell layer and outer plexiform layer. In the latter layer, a distinct band of D2 and D1 like ir processes was observed including D1 receptor ir nerve cell bodies in the outer part of the inner nuclear layer. These findings were supported by the results obtained in the DA diffusion experiments, involving microinjections of DA into the corpus vitreum. Thus, in these experiments a strong diffuse DA fluorescence appeared not only in the inner plexiform layer, but also in the outer plexiform layer, precisely where D2 and D1 receptor-like immunoreactivities had been demonstrated. A total prevention of the development of the band of DA fluorescence in the inner and outer plexiform layers was observed after corpus vitreum injections of DA accompanied by prior systemic chlorpromazine treatment blocking both D1 and D2 receptors (O8 gren et al.,

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Fig. 9. (A,B,C) Catecholamine (CA) fluorescence in the rat retina visualized by the SPG method after injection of 2 ml dopamine (0.01 mmol/ml) into the corpus vitreum 5 min before decapitation (panel A). The endogenous dopamine varicose terminal plexus (arrowheads) is present in the inner plexiform layer (IPL) some distance from the center of the dopamine injection. In the outer plexiform layer (OPL) an appearance of dopamine fluorescence is seen (*). In the choroid layer (Ch) a CA fluorescent plexus is seen representing noradrenergic (NA) terminals (arrows). When injecting 2 ml of dopamine (0.1 mmol/ml) into the corpus vitreum the endogenous CA fluorescent terminals can no longer be seen due to the appearance of a diffuse CA fluorescence in the inner plexiform layer (IPL), probably bound to DA receptors (panel B). A strong diffuse CA fluorescence appears in the outer plexiform layer (OPL). In panel C, the DA fluorescence in the outer plexiform layer (OPL), probably bound to DA receptors, is shown in higher magnification close to the center of the dopamine injection (0.1 mmol/ml). Bars indicate 25 mm and 50 mm.

1994). Only a partial disappearence was observed with a high dose of a preferential D2 antagonist, haloperidol. Therefore, it seems a likely hypothesis that in the rat retina, DA can diffuse and inter alia become bound to putative D2 and D1 receptors within the DA D2 and DA D1 receptor mismatch regions in the retina. These include also the ganglion cell layer and the inner plexiform layer, where both synaptic and nonsynaptic D1 and D2 receptors may exist, while mainly nonsynaptic D1 and D2 receptors exist in the outer plexiform layer (Djamgoz and Wagner, 1991; Wagner et al., 1993). Finally, D1 and D2 like IR were demonstrated in the outer segment of the photoreceptors, which also may represent DA receptors for volume transmission (Agnati et al., 1986; Bjelke et al., 1988; Bjelke et al., 1989; Fuxe and Agnati, 1991). Thus, volume transmission for DA appears to exist in many regions of the rat retina (Witkovsky and Schu¨tte, 1991). The demonstration of extracellular DA concentrations in the Xenopus Laevis retina should

also be mentioned (Witkovsky et al., 1993). In this paper, relevant extracellular DA concentrations were demonstrated in the outer retina, containing D1 and D2 receptors but lacking DA terminals. In this way, volume transmission by DA neurons appears to be used to control diskshedding and retinomotor movements such as cone contraction, and pigment dispersal in the Xenopus retina (Wagner et al., 1993; Witkovsky et al., 1993). It should also be considered that the region with the D2 and D1 mismatch receptors in the outer plexiform layer represents a trap for the diffusing DA (since a distinct band of DA fluorescence is formed in the outer plexiform layer), so that much lower DA concentrations will reach the photoreceptors. It should therefore be considered that the DA receptors in the outer segment, to become functional, must possess a very high affinity for DA. A paracrine function for DA released from amacrine DA cells of type II has also been postulated in the primate retina (Mariani, 1991).

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B. Bjelke et al. / Journal of Chemical Neuroanatomy 12 (1996) 37–50

Fig. 10. (A,B,C) Catecholamine (CA) fluorescence in the rat retina visualized by the SPG method in the normal rat (panel A) and after injection of 2 ml of dopamine (0.05 mmol/ml) into the corpus vitreum 5 min earlier (panel B). In panel B, the endogenous dopamine varicose terminal plexus present in the inner plexiform layer (IPL) can no longer be seen due to the appearance of a diffuse CA fluorescence in IPL (arrowheads), probably due to dopamine bound to DA receptors. A strong CA fluorescence also appears in the outer plexiform layer OPL (B). In the choroid layer, CA fluorescent plexus are seen representing noradrenergic terminals (oblique arrow). When injecting chlorpromazine (panel C) (20 mmol/kg) systemically 30 min before the dopamine injection, almost no CA fluorescence was seen in the inner and outer plexiform layers (OPL). Bar indicates 50 mm.

In addition, a strong diffuse extraneuronal DA fluorescence appeared within the entire inner plexiform layer upon diffusion of DA from the corpus vitreum. With increased concentrations, this DA fluorescence became so intense that it became difficult to visualize the DA containing terminal branches of the DA cells in the outer part of the inner plexiform layer. It seems likely that a displacement phenomenon of internal DA stores, can contribute to the development of this strong diffuse CA fluorescence in this region. The major mechanism may, however, be the binding of DA to D2 autoreceptors on the DA amacrine and/or interplexiform cell bodies and terminal processes and to associated postsynaptic D2 and D1 receptors (Dowling and Ehinger, 1978a; Elena et al., 1989). Taken together, the present study shows D1 and D2 like IR within the adjacent inner plexiform layer, the ganglion cell layer, the outer plexiform layer and possibly even the outer segment of the photoreceptors. The findings therefore give support to the hypothesis that volume transmission may be an important mode of communication in the amacrine and/or interplexiform DA cells of the rat retina (Witkovsky and Schu¨tte,

1991), involving a possible diffusion of DA from the terminal branches of the amacrine and/or interplexiform neurons in the inner retina into the extracellular space to reach via diffusion also high affinity D2 and D1 mismatch receptors (Elena et al., 1989).

Acknowledgements This work has been supported by a grant from S:t Eriks Eye Hospital, a grant (04X-715) from the Swedish Medical Research Council, grant USPHS MH50314, a grant from Marianne and Marcus Wallenberg foundation and a grant from the American Parkinson’s Disease Association. We want to thank Robert England and Kiomars Delfani for their substantial contributions.

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