BRAIN RESEARCH

ELSEVIER Brain Research 695 (1995) 110-116

Research report

Localization of putative dopamine D2-1ike receptors in the chick retina, using in situ hybridization and immunocytochemistry

Baerbel Rohrer *, William K. Stell Neuroscience Research Group, Department of Anatomy and Lions Sight Centre, University of Calgary, 3330 Hospital Drive N. W., Calgary, AB T2N 4N1,

Canada

Accepted 16 May 1995

Abstract

The actions of dopamine are mediated by 5 or more receptor subtypes, any of which may be coupled by G-proteins to adenylate cyclase (Dl-family: stimulatory, D2-family: inhibitory or no action). Postnatal ocular growth in the chick is a vision-dependent mechanism which involves D2-type receptors in either the retina or the retinal pigment epithelium (RPE). Although the dopaminergic amacrine cells are well described in the chick retina, only D2-receptors, but not D 3- and D4-receptors have been clearly localized, and the cells that express them have not been identified. In this study we showed that immunoreactive D2/3-receptor protein is localized to the photoreceptor inner segments, outer and inner plexiform layer and ganglion cell layer, as described previously (Wagner et al., J. Comp. Neurol., 330 (1993) 1-13). D2-receptor mRNA was localized to cell bodies in all nuclear layers of the retina, whereas Da-receptor mRNA was restricted to the inner half of the retina. Immunoreactive D2-type receptors and their mRNA were observed also in the basal region of the RPE. Because of the widespread distribution of both D 2- and D4-receptor mRNA in the chick retina and RPE and the lack of D 3- and D4-receptor-specific antibodies, we were unable to identify which of the D2/3/n-receptor-bearing cells are involved in controlling ocular growth.

Keywords: Dopamine D2-1ike receptor; Chick retina; Immunocytochemistry; In situ hybridization; Form-deprivation myopia

I. Introduction

Dopamine is a major synaptic transmitter and modulator utilized by cells in various locations in the central nervous system. Dopamine interacts with G-protein-coupled recep- tors, through which it modulates the availability of cyclic AMP (cAMP) and other second messenger systems such as phospholipase C [9]. Dopamine-receptors belong to two broad classes [10]: Dl-like, which are positively coupled to adenylate cyclase and lead to an increase in cAMP, and D2-1ike, which inhibit adenylate cyclase and lead to a decrease in cAMP accumulation or act through some other second messenger systems. The application of molecular biological techniques has led to the identification of further dopamine receptor subtypes: D 1 [24] and D 5 [23] in the Dl-family, and D 2 [6], D 3 [19] and D 4 [26] in the D2-family. These five receptor subtypes can all be distinguished by

* Corresponding author. Present address: Howard Hughes Medical Institute, University of California at San Francisco, Rm. U-426, 533 Parnassus & Third Ave., San Francisco, CA 94143-0724, USA. Fax: (1) (415) 566-4969; E-maih rohrer@cgl.ucsf.edu

0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00700-8

their topology, deduced primary structure, size of messen- ger RNA (mRNA), chromosomal location, tissue distribu- tion, and binding kinetics with specific ligands [13].

Dopamine plays a crucial role in visual information processing and other retinal functions (e.g. [18,30]). These diverse functions include horizontal (D1; [11]) and bipolar cell coupling (D1; [17,25]), retinomotor movement (D2; [7]) and melatonin metabolism (D2; [2]). Dopamine is also thought to participate in the regulation of postnatal ocular growth and the pathogenesis of form-deprivation myopia (FDM). Ocular growth is regulated by a vision-dependent retinal feedback loop (e.g. [28]). If this loop is opened by blurring vision, the eye grows excessively and develops severe near-sightedness (myopia), in parallel with a de- crease in dopamine synthesis and release [20,21]. We have demonstrated that intraocular injections of a nonspecific dopaminergic agonist apomorphine, prevents FDM in chicks probably by acting through receptors of the D 2- family located in either retina or retinal pigment epithe- lium (RPE; [14]). It is not clear, however, which retinal neurons and pathways might participate in this phe- nomenon.

B. Rohrer, W.K. Stell / Brain Research 695 (1995) 110-116 111

Dopaminergic amacrine cells have been well charac- terized in the chicken retina [5,22] and D2-receptors have been localized immunocytochemically in the chick pho- toreceptor inner and outer segments, outer plexiform layer (OPL) and inner plexiform layer (IPL; [27]). However, the identity of the cellular targets of receptor specific dopamine agonists (D 2 vs. D 3 VS. D 4) remains obscure.

In this study we have employed immunocytochemistry and in situ hybridization histochemistry to identify cells that express D2-1ike receptors in the chicken retina.

2. Material and methods

2.1. Immunocytochemistry

Immunocytochemistry was performed as reported else- where [14-16]. In short, eyes of 10-20-day-old leghorn chicks were enucleated, eyecups were fixed in 4% para- formaldehyde, cryoprotected in 30% sucrose and OCT compound (Tissue Tek), and radial sections were taken on

a cryotome (10 /zm). Sections were incubated overnight at room temperature (RT) in a D2/3-specific receptor anti- body, raised against the deduced amino-terminal oligopep- tide common to the human D 2- and D3-receptors (Chem- icon, Cat. no. AB1502) diluted 1:200 in 0.05 M phosphate-buffered saline (PBS), containing 0.3% Triton X-100 and 0.01% sodium azide (PBS-TX-A). After wash- ing in PBS they were incubated for 2 h at RT in a FITC-conjugated anti-rabbit secondary antibody (Sigma). Additionally, a monoclonal antibody against tyrosine hy- droxylase (obtained from the Developmental Studies Hy- bridoma Bank; University of Iowa; Ref. [8]) was used, with TRIC-conjugated anti-mouse secondary antibody. As controls we omitted the primary antisera. Tissue was cov- erslipped under 50% glycerol (in PBS) and viewed under a fluorescence microscope.

2.2. In situ hybridization

In situ hybridization was performed according to Barthel and Raymond [1] with slight modifications, using digoxy-

Fig. 1. Localization of dopamine D2/3 immunoreactivity in the chick retina. 10 /tm cryosections were stained by indirect immunofluorescence with a human-specific polyclonal D2/3-receptor antibody (B). The basal layer of the RPE showed clusters that were immunopositive for D2/3-receptors, and the inner segments of photoreceptors, ganglion cells and layers in the IPL were distinctly stained. Diffuse labeling can be observed in the IPL. Omission of the primary antibody revealed that the staining in the outer segments of the photoreceptors and the diffuse labeling in the basal layer of the RPE is due to autofluorescence (A). Abbrevations: RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 50 /xm.

112 B. Rohrer, W.K. Stell / Brain Research 695 (1995) 110-116

genin-labeled cRNA probes and detection with alkaline phosphatase-conjugated antibodies. Tissue collection and preparation of sections were performed as above, except in this case it was essential to maintain sterile conditions. We obtained from Dr. H. Van Tol (Clarke Institute of Psychia- try, Toronto, ON, Canada) a 700 bp cDNA fragment of the human D4-receptor sequence, and a full-length rat cDNA (~ 2.4 kb) coding for the long isoform of the D2-receptor from the Office of Technology Management at Oregon Health Sciences University (Portland, OR, USA). In vitro transcription of the antisense and sense RNA probes was performed with a kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol. An alkaline di- gest was performed to reduce the size of the transcripts for better tissue penetration. Tissue was digested with 0.01 mg /ml proteinase K, acetylated with 0.1 M tri- ethanolamide and 0.25% acetic anhydride and dehydrated. 20/zl of hybridization solution (10 mM Tris, pH 7.5, 50% formamide, 300 mM NaCI, 1 mM EDTA, 10% dextran sulfate, 1% blocking reagent; Boehringer Mannheim) con- taining 100 ng of the probe was pipetted onto the sections, covered with a coverslip, sealed with Fluka mountant, and hybridized overnight. Post-hybridization treatment in- cluded 2 × SSC at RT, 50% formamide in 2 × SSC at temperatures indicated in the individual experiments, RNase treatment in 2 × SSC (0.02 mg/ml) at 37°C and rinsing in 2 × SSC (30 min each). The bound probe was detected immunocytochemically with anti-digoxygenin Fab fragment coupled to alkaline phosphatase (Boehringer Mannheim) at 1:500 in maleate buffer (100 mM maleic acid, 150 mM NaC1, pH 7.5) containing 1% blocking agent (Boehringer Mannheim). The subsequent color reaction with NTB/BCIP was performed according to the manu- facturer's protocol (Boehringer Mannheim), and tissue was again coverslipped under glycerol for viewing.

dopaminergic amacrine cells (DA-ACs) revealed that the band of immunoreactive (IR) D2/3-R at 40-42% of IPL depth co-localizes with the dendritic branches of DA-ACs We summarized these results in a sketch in Fig. 2. Some of these receptors might be autoreceptors, which so far have been assumed to be of the D2-type, or receptors on postsynaptic neurites, probably other ACs (in particular vasointestinal polypeptide- (VIP-) containing; [16]), that co-stratify with the TH cells.

3.2. Localization o lD 2- and D4-receptor specific mRNA

In situ hybridization with the two dopamine receptor probes gave similar, but not identical staining patterns. Fig. 3A and B compare the results of experiments performed with equal amounts of De-receptor antisense and sense (control) probe under identical conditions of hybridization (50% formamide, 50°C) and post-hybridization (50°C). No staining was obtained with the sense probe, but specific staining could be identified in virtually all cells, in all nuclear layers, with the antisense probe. Cells in the GCL, INL and outer nuclear layer (ONL), plus photoreceptor inner segments, the basal side of the RPE (arrow), and the choroid were labeled with the antisense probe, indicating that those cells were actively transcribing D2-receptor mRNA. The distribution of D4-receptor-specific labeling was more restricted. Similarly, Fig. 3C and D compare the results of hybridization (50% formamide, 52°C) with D 4-

r e c e p t o r antisense and sense probes, respectively. Again there was no staining with the sense probe but we could identify immunopositive cells in the GCL and INL with a

3. Results

3.1. Distribution of D2 / 3-receptor protein

Labeling with the D2/3-receptor antiserum showed spe- cific staining in the inner segments of photoreceptors, the inner nuclear layer (INL) and ganglion cell layer (GCL), as well as the inner plexiform layer (IPL; Fig. 1B). In the basal layer of the retinal pigment epithelium we observed intensely stained areas. In the IPL, distinct synaptic bands parallel to the vitreal surface were stained at 17-25%, 40-42% and 50-52% of depth (measured form distal to proximal). Fig. 1A shows the background autofluorescence after omission of the primary antibody. The autofluo- rescence in the basal layer of the RPE is diffuse as compared to the clustered staining of the D2/3-receptors immunofluorescence, and appears slightly brighter than it really is, because of printing.

Double staining with tyrosine hydroxylase to identify

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Fig. 2. Sketch of synaptic layer in the [PL. This drawing represents data from double-staining immunocytochemistry. The D2/a-receptor antibody was used to identify dopamine-receptors in the IPL (of. Fig. ]), and a monoclona] antibody against TH revealed the branching pattern of the dopaminergic amacrine cells. The lamination of the dopaminergi¢ recep- tors was expressed with respect to the width of the IPL, which was taken as ]00%. The D;/3-receptors at 40% depth overlaps with dendritic branches of the ~ - A G s , suggesting the |ocalization of autoreceptors. For abbreviations see Fig. 1.

B. Rohrer, W.K. Stell/Brain Research 695 (1995) 110-116 113

gradient declining towards the outer retina. In the chick retina, most of the amacrine cells are located in the proximal one-third of the INL and in the GCL, whereas the

cell bodies of bipolar and Mueller cells are restricted to the distal two-thirds of the INL. Therefore the Da-receptor-im- munoreactive cells in the INL are probably mainly amacrine

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Fig. 3. In situ hybridization to localize message for dopamine D 2- (A, B) and D4-receptors (C, D) in the chick eye. 5 ng of DIG-labeled probe per #1 of solution was used for hybridization (50°C (D 2) or 52°C (D4), and 50% formamide), and alkaline phosphatase conjugated antibodies were used for visualization. D2-receptor mRNA was distributed throughout the retina (A), whereas the D4-receptor seems to be more confined to the inner retina (C). Both probes weakly detected message in the basal layer of the RPE (arrows). No specific staining could be observed using the sense probes (B, D). For abbreviations see Fig. 1. Scale bar = 50 p.m.

114 B. Rohrer, W.K. Stell / Brain Research 695 (1995) 110-116

cells. In the outer coats of the eye, the basal cytoplasm of the RPE (arrow), cells in the choroid and scleral chondro- cytes express D 4 mRNA.

4. Discussion

Using a combination of immunocytochemistry and in situ hybridization, we have determined the distribution of dopamine D2-type receptors and their mRNA (or their sites of synthesis) in the chick retina.

4.1. Comparison with previous immunocytochemical re- suits

We were able to confirm the immunocytochemical re- sults on chick retina reported previously [27], and observed even more striking examples of discrete laminar labeling in the IPL. As the localization reported here was identical to the one demonstrated by Wagner et al. [27] in a preabsorption-controlled study, we did not further charac- terize the antibody staining. Because the antiserum used by Wagner et al. [27] should not recognize the D3-receptor, we assume that our own localization indicates specifically D2-receptors, but we cannot exclude the possibility that D3-receptors are expressed and localized in the very same subset of neurons as D2-receptors. We also observed in- tense labeling of discrete patches of D2/3-receptors in the basal, unpigmented part of the RPE, but not on the apical, heavily pigmented side. However, due to the pigmentation, description and interpretation of RPE staining is generally difficult and thus all physiological conclusions have to be tentative.

By double-labeling for D2/3-receptors and TH, we demonstrated that at least one of the D2/3-IR bands co- stratifies with the dendritic branches of DA-ACs at 40-42% depth of the IPL. We have reported elsewhere [26] that dendrites of VIP-IR amacrine cells also co-stratify in this layer. These amacrines are plausible targets for D2-rece p- tor mediated synaptic actions of dopamine. The relative absence of co-stratification of the other IR D2/3-receptor synaptic bands with branches of DA-ACs might suggest that the main dopamine receptor type in the inner retina is of the Dl-type. On the other hand it supports the concept that dopamine may act in the retina diffusely as a local hormone, as well as focally as a classical neurotransmitter [3].

4.2. Analysis of in situ hybridization results

In situ hybridization was performed with probes against mammalian dopamine receptors. Since there is a signifi- cant amount of sequence identity between homologous dopamine receptors in different species [13], these probes were assumed to show the same specificity in the chick. The Northern blots prepared under high stringency condi-

t ions (D2:0.1 × SSC, 0.1% SDS, 62°C and D 4 : 0 . 1 × SSC, 0.1% SDS, 68°C) strongly recognized the appropriate mRNA band as well as some weak, unrelated bands. Therefore, in situ hybridization was performed at high stringency (50% formamide and 50°C or 52°C for D 2- and D4-receptor probe, respectively) to ensure specificity. Hy- bridization at 2°C or higher eliminated binding altogether.

All the cells that were immunopositive for the D2/3-re- ceptor protein also expressed D2-receptor mRNA. This lends further support to the specificity of the probe. How- ever, we were not able to attribute any of the staining to D3-receptors by simple exclusion. In the rat brain, the absence of overlap in the distribution of D 2- and D3-rece p- tor mRNAs, suggests that they are expressed by different cells [19]. We would like to suggest, therefore, that D3-re- ceptors are absent from the chick retina, but we cannot exclude the possibility that some of the D2/3-positive cells co-express both receptor subtypes. On the other hand, in situ hybridization showed clearly that D4-receptors are confined to the inner retina, perhaps even ACs only. Additionally, we were able to show that D 2- and D4-rece p- tor mRNA was weakly expressed and localized to the basal part of the RPE, supporting the immunocytochem- istry results with the D2/3-receptor antibody. Under our experimental conditions no labeling for D4-receptors was identified in the photoreceptor layer, although D4-receptors have recently been identified in the control of melatonin synthesis by chick photoreceptors [31]. This could be because of a low expression of this receptor subtype and/or the thin shape of the chick photoreceptors, which complicates anatomical studies.

4.3. Relevance to FDM

These experiments were performed as part of an effort to determine possible target cells for the D2-1ike action of dopamine in FDM (see above). In a previous paper, we suggested that dopamine might act through receptors in the RPE [14]. However, due to the heavy pigmentation of the RPE in leghorn chicken, we were only able to detect dopamine receptors on the basal side of the RPE using either immunocytochemistry or in situ hybridization. We were not able to determine whether dopamine receptors were also present on the apical side using either of the two methods. We can therefore still not confirm or rule out the possibility of the involvement of the RPE in the FDM- blocking action of D2-agonists. There are at least two other possible mechanisms. Dopamine was found to alter the center-surround balance in bipolar cells [12], enhancing the antagonistic influence of the surround and thereby the visual acuity of the bipolar cells. Alternatively, exogenous apomorphine could prevent FDM by mimicking the role of high spatial frequency channels or mechanisms in the distal retina. According to the results reported here, the most likely candidate for mediating a distal retinal action would be the D 2- or perhaps D3-receptors, but dopaminer-

B. Rohrer, W.K. Stell / Brain Research 695 (1995) 110-116 115

gic agonists might also act upon D2- , D 3- or D4-receptors on non-dopaminergic ACs, such as the enkephalin contain- ing amacrine cells [4,29].

4.4. Conclusion

In order to gain insight as to which type of D2-1ike receptor and possibly which retinal cells are involved in the dopaminergic control of ocular growth, we analyzed the distribution pattern of D2/3-receptor protein as well as D 2- and Dn-receptor mRNAs in the chick retina. However, the widespread expression of those receptors as revealed by in situ hybridization and the lack of specific antibodies for D 3- and D4-receptors made it impossible to identify restricted (one or a few) cells or circuits that might medi- ate the growth-regulating actions of D2-1ike agonists. It appears that a great many (perhaps all) neurons in the chick retina are responsive to dopamine in some way. A clear understanding of the functional roles of dopamine, for example in FDM, will require identification and char- acterization of specific transduction mechanisms in subsets of cells which are targets for dopaminergic regulation.

Acknowledgements

We thank Dr. H. Van Tol for the human D4-receptor clone, and the Alberta Heritage Foundation and the Marigold Foundation (Calgary) for financial support.

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