Radioimmunoligand characterization and immunohistochemical localization of dopamine D2 receptors on rods in the rat retina

Brain Research, 614 (1993) 57-64 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

57

BRES 18901

Radioimmunoligand characterization and immunohistochemical localization of dopamine D 2 receptors on rods in the rat retina T u y e n V u v a n a M i c h e l G e f f a r d b, P h i l i p p e D e n i s c, A x e l l e S i m o n a and Jeanine Nguyen-Legros a a INSERM Unitg de Physiopathologie de l'Oeil, U-86, Paris (France), b Laboratoire d'Immunologie et Pathologie, Universit~ de Bordeaux II, Bordeaux (France) and c Service d'Ophtalmologie, H~pital Saint-Antoine, Paris (France) (Accepted 12 January 1993)

Key words: Dopamine; Dopamine D 2 receptor; Rod outer segment; Retina; Rat

The retinal neurotransmitter dopamine (DA), elaborated from intrinsic dopaminergic neurons as amacrine and interplexiform cells, is known to modulate several complex functions mediated by D 1 and D z receptors in the vertebrate retina. In this paper, we characterized and localized DA receptors of the D 2 family on rod outer segments (ROS) of the rat retina by a radioimmunoligand binding assay and by immunohistochemistry. Anti-anti-DA conjugated antibodies (or anti-idiotypic antibodies Ab 2) were used as ligand; BSA-glutaraldehyde-conjugated spiperone, eticlopride (D 2 antagonists) and DA were used as displacers. The linear Scatchard transformation indicated that data were best fit to the one-site model. By using the peroxidase-antiperoxidase technique, an intense labeling was located on rods. These results supported the paracrine action of DA on the photoreceptor cell.

INTRODUCTION

The action of dopamine (DA) as a neurotransmitter in the vertebrate retina and its effects on complex functions at the retinal level is now well documented 58. The morphological support of these actions consists of intrinsic DA neurons including amacrine cells acting in the inner retinal layers, and interplexiform cells (IPcs) which drive a feedback input from the inner to the outer layers of the retina 31'32. During the last decade the action of DA has been emphasized in the outer layers of fish retina because it contains numerous DA IPcs. It has been established that DA acts through a D~ (and possibly D z) mechanism in modulating plasticity of horizontal cell structure and function24'25'5z, while D 2 receptors are involved in retinomotor responses to light, melatonin synthesis, and disc shedding at the level of photoreceptor-pigment epithelium (RPE) complex 12'21'39. The recently observed close relationships between DA processes and photoreceptor cell synaptic terminals can support such an action in fish57.

Using tyrosine hydroxylase (TH) immunocytochemistry DA IPcs have now been evidenced in most mammalian retinas 33, and their sclerally directed processes were demonstrated to reach the outer nuclear layer (ONL) in rat and primate retina 34. In the human retina, this close proximity to photoreceptor cells is especially impressive at the fovea43. Concurrently an action of DA on photoreceptor-RPE complex, through a D 2 mechanism, was gaining support in the mammalian retina. DA receptors have been characterized and classified according to biochemical parameters such as adenylate cyclase23 and pharmacological activity by the use of DA agonists and antagonists through the ligand binding 44. D 1 receptors activate adenylate cyclase activity and K + channels and increase the intracellular accumulation of cyclic AMP (cAMP); D 2 receptors inhibit adenylate cyclase activity, phosphatidyl inositol turnover, and Ca z+ channel activity and decrease the cAMP content 46. D z receptors have been characterized in the retina of some species 44 and human zs'49.

Correspondence: T. Vuvan, Laboratoire d'Immunopathologie de l'Oeil, INSERM U-86, 15, rue de l'Ecole de M6decine, 75270 Paris 06, France.

58 D 2 receptors were localized by ligand autoradiography on photoreceptor cells of several mammalian species including primates 3'8"53'61, and DA and agonists have been demonstrated to reduce a light sensitive pool of cAMP in mouse photoreceptor cells 9. On the other hand, there are some indirect evidences for DA acting in the control of daily rhythms in the photoreceptor-RPE complex in mammals 5'~'27'41 Moreover, recent data on human diseases such as Parkinsonism, in which the retinal DA content is decreased 2°, suggest a circadian control of human retinal activities by a DA mechanism 51. Interactions between DA and melatonin were also observed in the mammalian retina since DA and apomorphine inhibit melatonin synthesis and release in rat and rabbit 35, and melatonin increases the rhythm of phagocytosis of shedded discs in rats 5s. However, if the rhythmic regulation of disc shedding is generally attributed to DA, melatonin and cAMP in lower vertebrates, supporting evidences are still needed to confirm an analogous regulation in mammals 6. This would be of special interest in the human because the dysfunction of such a mechanism could be involved in the etiology of human diseases such as age-related macuiopathy. In order to support a direct influence of DA on photoreceptor cells in the rat retina, we have attempted to demonstrate the existence of DA receptors on these cells by a new approach. Anti-idiotypic antibodies to DA (Ab 2) have been used to characterize and localize D 2 receptors by radioimmunoligand binding assay and by immunohistochemistry. MATERIALS AND METHODS Preparation of the Ab e antibody The Ab 2 antibody was prepared as described elsewhere29. Briefly, the Ab~ antibody was raised in rabbits by alternate immunizations against both purified rabbit polyclonal and mouse monoclonal antibodies (Ab 1) directed against conjugated DA [DA covalently coupled by glutaraldehyde (G) to bovine serum albumine (BSA) as protein carrier as describedVAg]. The anti-isotypic and anti-allotypic antibodies were removed by affinity chromatography. The ability of the internal image Ab 2 of conjugated DA to recognize DA receptors was demonstrated by [3H]DA displacement and competition experiments in ELISA, between Ab 2 and different conjugates for the binding to DA receptor-enriched membranes. They have been already tested for (i) the recognition of DA receptors by immunohistochemistry in rat striatal sections; (ii) immunoblotting using mouse neuroblastoma membranes29; and (iii) in vivo activity 3°. Rod outer segment purification The retina was dissected from the eyes of 120 albino Wistar rats (200-300 g) and the rod outer segments (ROS) were purified as described 3. Protein content was determined as reported 2 with bovine lgG as standards. Binding experiments" Radioiodination: 10/xg of Ab 2 were iodinated by the chloramineT method with 250 ~Ci of Na~25I (Amersham, UK) resulting in the ~25I-Ab2, specific activity 10.5 /xCi//xg.

Radioligand binding assays were performed in triplicate by incubating 400 /~1 of purified ROS suspension (125 /xg/ml) in binding buffer (50 mM Tris-HCl, 120 mM NaCI, 1 mM MgCIe, 1 mM CaCI e, 5 mM KCI, 5.7 mM ascorbic acid, pH 7.4) with 100/zl of t251-Ab2 at concentrations ranging from 0.01 to 1 nM for saturation experiments, in the presence or absence of 10 ~M (-)-apomorphine (Research Biochemicals Inc., USA) to determine non-specific binding and total binding, respectively. The mixture was incubated for 30 min at 30°C. The reaction was stopped by soaking the tubes in ice. ROS suspensions were then rapidly filtered under vacuum through G F / C filters (Whatman) pretreated with 0.3% polyethyleneimine, and washed with 3 × 4 ml of cold 50 mM Tris-HCl pH 7.4. Filters were counted in a multiwell gamma counter (Kontron, France) with a counting efficiency of 64.4%. The non-specific binding due to filters was determined by incubating ROS-free binding buffer with 125I-Ab2, and counting the filters as described above. For competition experiments, ROS suspensions in binding buffer were incubated for 30 rain at 30°C with 0.12 nM 12Sl-Abz and with each of the following displacers: spiperone-, eticlopride-(Research Biochemicals Inc., USA) and DA-G-BSA conjugates of decreasing concentrations ranging from 10 4 to 10 it M.

Immunohistochemistry For immunohistochemistry, 6 Wistar rats were used, two of which were kept in complete darkness for 10 days before sacrifice. They were fixed by transcardiac perfusion of a mixture of 4% paraformaldehyde and 0.02% picric acid in 0.1 M phosphate buffer, pH 7.4, under barbital anesthesia. After enucleation, the eyes were opened at the limbus, post-fixed for 2 h and rinsed overnight in buffer. The eye cup was then cut in 3 tzm sections at -20°C 1, and the sections collected on gelatinized slides were incubated as follows. The primary antiserum (Ab 2) was diluted 1/200 in PBS containing 0.3% Triton X-100 and 10% normal sheep serum. Immunoreactivity was demonstrated by the streptavidine-biotine-peroxidase method, using biotinylated donkey anti-rabbit 1/I00 as a second antibody. The demonstration of peroxidase activity was enhanced by adding 0.04% ammonium nickel sulfate to diamino benzidine-hydrogen peroxide reagent. Ab2-free buffer or non-immune serum or Ab 2 preabsorbed with the polyclonal and monoclonal antibodies Ab 1 were used as controls.

RESULTS

Radioimmunoligand binding assays The binding of 125I-Ab2 on rat ROS is shown in Fig. 1. DA binding sites were saturable with increasing amounts of labeled antibodies from 0.01 to 1 nM. The non-specific binding was determined in the presence of 10 /zM ( - ) - a p o m o r p h i n e . The specific binding curve transformed by the Scatchard method (Fig. 2) indicated that the data were best fit to the one-site model. The parameters of binding were K d = 0.12 -+ 0.02 nM and Bmax = 347.3 + 21.5 fmol/mg. The binding of lzsI-Ab2 was displaceable by increasing concentrations of antagonist conjugates spiperoneG-BSA and eticlopride-G-BSA in the presence of ( - ) - a p o m o r p h i n e (Fig. 3). ICs0 determined for each conjugate was 3.16 + 0.21 nM, 4.63 + 0.38 nM, and 316.4 +_ 23.6 nM for spiperone-G-BSA, eticlopride-GBSA and DA-G-BSA, respectively. According to these results, DA and antagonists conjugates were able to displace 125I-Ab2 from its binding sites on rat ROS.

59

"•

100

800

TB

- ~

• a

spiperone-G-BSA eticlopride-G-BSA

80" g 600 ,c,

~ "o <,-

-O ¢-

NSB

60'

O 400 40'

SB

<, 2O

200

0 0

, 0.2

0.0

, 0.4

i 0,6

-11

w 0.8

1.0

1251-Ab2 (nM) Fig. l. Saturation experiments were performed by incubating rat ROS with an increasing amount (0.01-1 nM) of x25I-Abz in the absence and presence of 10 ~M (-)-apomo~hine to determine the total binding (TB) and the non-specific binding (NSB), respectively. Each point represents the mean of three determinations. SB = specific binding.

-10

-9

-B

-7

-6

-5

-4

log [M] conjugates Fig. 3. Inhibition of 12SI-Abz binding on rat ROS by increasing concentrations of D 2 antagonists glutaraldehyde-BSA conjugates: spiperone-G-BSA, eticlopride-G-BSA and DA-G-BSA. Specific binding of labeled antibodies in the absence of D 2 antagonist conjugates was given an arbitrary value of 100%. The decrease of binding in the presence of D 2 antagonist conjugates was expressed relative to this arbitrary value. 1Cs0 values of spiperone-G-BSA, eticlopride-GBSA and DA-G-BSA were 3.16+0.21 nM, 4.63+0.38 nM and 356.4 _+23.6 nM, respectively.

The affinity of these conjugates was classified as follows:

spiperone-G-BSA > eticlopride-G-BSA >> DA-G-BSA Immunocytochemistry All the retinal layers contained more or less heavy labeling, except the outer nuclear layer (ONL). The most intense labeling was observed at the margin between ROS and R P E and was associated with the tips of ROS as can be easily observed in some detached places of the retina (Fig. 42). The inner and outer segments of the photoreceptor cell were only weakly labeled in light-adapted retinas, while the entire inner

B/F o3

0.21

0

100

200

300

400

B (fmol/mg)

Fig. 2. The Scatchard transformation of the specific binding curve in Fig. 1. The parameters of binding were K d = 0.12+_0.02 nM and Bmax = 347.3_+21.5 fmol/mg.

segments (RIS) was more heavily immunoreactive in dark-adapted retinas (Fig. 46). An increased density was located at the junction between RIS and ROS, and a band of intense labeling was associated with the outer limiting m e m b r a n e (Fig. 42). Neither the photoreceptor somata nor their synaptic endings exhibited a significant immunoreactivity. A moderate labeling was observed in the OPL. Some more intensely labeled somata with processes in the O P L were observed at the scleral margin of the I N L (Fig. 4~). The entire I N L was slightly immunoreactive but cell bodies and primary dendrites at its vitreal margin were delineated by a heavier immunoreactivity associated with their cell m e m b r a n e (Fig. 43). The whole I P L exhibited a moderate immunoreactivity, slightly stronger in sublamina 1. Scattered ovoid cell bodies showed a membrane-associated ring of immunoreactivity in the IPL. Almost all the cell bodies were labeled in the ganglion cell layer (GCL). The majority were labeled in their whole somata and primary dendrites (Fig. 46), while others exhibited only a ring of immunoreactivity like those in the IPL. Immunolabeling was abolished in the three controls (see Material and methods) (Fig. 45). Depending on lighting conditions, blood capillaries running along both margins of the I N L together with their anastomosis crossing the I P L were either unlabeled (dark-adaptation) or heavily labeled (light-adaptation) (Fig. 46), while the larger vessels in the G C L were unstained.

60 DISCUSSION In

receptor

binding

studies,

labeled agonist (or antagonist)

authors

often

use

as ligand and unlabeled

a

agonist (or antagonist) as displacer. In this paper, we have characterized D 2 receptors on rat ROS by using 125I-anti-DA anti-idiotypic antibodies (or internal image Ab 2 of D A ) 29 as ligand instead of labeled D A

RPE ROS RIS

r

~

~!iii!~i

!i!~¸' i!!! i~i!~i~

5

Fig. 4. (1) General view of the pattern of labeling obtained with the anti-anti-DA antibody in a dark-adapted rat retina. Note the intense labeling at the interface between RPE and ROS. At places where the RPE is detached it is easy to observe that the labeling is associated with ROS tips. RIS are also labeled. A faint labeling occurs in the ONL and IPL. Cell bodies in the INL are delineated by anti-anti-DA immunoreactivity in the amacrine (arrows) and horizontal (double arrow) cell sublayers. Blood vessels are unstained (arrowheads). Bar = 50/xm. (2) A more enlarged view of the photoreceptor inner and outer segments. Note the intense labeling associated with ROS tips and the weaker labeling of RIS. A more intense staining is observed at the junction between ROS and RIS (arrowheads). Bar = 10/~m. (3) An enlargement of the margin between INL and IPL showing a cell in the amacrine cell sublayer whose soma and primary dendrites bear anti-anti-DA immunoreactivity. Bar = 10/~m. (4) Pattern of anti-anti-DA immunoreactivity observed in the retina of a rat adapted to normal 12 h / 1 2 h lighting cycle. Note the heavy labeling of ROS and IPL and the absence of labeling from RIS. Blood vessels are strongly stained (arrowheads). Compare with figures (1) and (6) obtained from dark-adapted rats. (5) Control retina: no labeling is observed. Bar = 50 g m for 4, 5, and 6. (6) Pattern of anti-anti-DA labeling in a clark-adapted rat retina. Note the staining of RIS and cells in the ganglion cell layer. Blood vessels (arrowheads) are unstained as in Fig. (1). Nomarski optics for 4, 5, and 6.

61 alone. Anti-idiotypic antibody has been used to study several membrane receptors 5° because it allows to avoid the isolation and purification of the receptor itself. Briefly, ligand is used to elicit first anti-ligand (or idiotypic) antibodies (Ab0; these Ab I were then used as immunogens to raise anti-idiotypic Ab 2. An antispiroperidol and an anti-haloperidol/anti-idiotype antibody have been used to study D A receptors on striatal membranes ~6'45. By using 125I-Ab2 in the binding on ROS suspensions we have tried to displace the labeled ligand by DA and some D 2 antagonists like eticlopride and spiperone without success in spite of the high affinity constants of these antagonists 47 and even with a concentration of several hundred times higher than that of labeled Ab 2. This statement could be explained by the steric hindrance resulting from the high relative molecular mass of immunoglobulin antibody molecule (150,000) comparing to that of DA, eticlopride and spiperone (189.6, 377.31 and 431.94, respectively). We have thus used the protein carrier BSA ( M r = 66,000) to couple with DA and its antagonists in order to augment their relative molecular mass resulting in DAG-BSA, spiperone-G-BSA and eticlopride-G-BSA conjugates which finally manage to displace the labeled Ab 2 . Our results have shown that D A receptors on rat ROS were saturable by increasing amounts of labeled Ab 2. The specific binding of these labeled Ab 2 fits to the one-site model according to the Scatchard linear transformation. The displacement of 125I-Ab2 binding on rat ROS by DA-G-BSA have proved that the binding was DA-specific. Since the two antagonists eticlopride and spiperone are D 2 selective, their inhibition of the labeled Ab 2 binding have demonstrated that the binding site on rat ROS by the internal image of DA was identified as the D 2 receptors. Three subtypes (D2,D3,D 4) of D 2 receptors have now been cloned with a high structure homology between human D 4 / and D 2, D 3 receptor genes and these D 4 genes are the rat D 4 analogs 38'47'55. It can be questioned whether specific functions involve specific subtypes. In rodent retina the D 4 subtype seems to specifically modulate cAMP levels in rods while D 2 would act at the ganglion cell level 1°'54. D 3 subtype was not yet localized in the retina, but polymerase chain reaction we have used in separated experiments indicates that it does not reach a sufficient amount to be considered as occurring in the rat retina (unpublished data). In any case, it is likely that the Ab 2 recognizes both D 2 and D 4 subtypes in the retina, as demonstrated by the wide distribution of immunoreactivity. Nevertheless, while D 4 receptor has relatively lower

binding affinities than D 2 and D 3 receptors for most of the agonists and antagonists, its binding affinities for clozapine was higher than D 2 receptor 55'3s. Thus the O 2 receptor demonstrated on the ROS in this paper could possibly be assimilated to the D 4 receptor until confirmed by the displacement test with clozapine. In both light- and dark-adapted retinas, a diffuse labeling is found in the IPL which is consistent with the wide interaction of DA cells with amacrine and bipolar cells in this layer. No clear sublayering (except in sublamina 1) is observed in spite of the disposition of DA cell processes in sublayers 1, 3, and 532. The same observation was made in ligand autoradiographic localization ~7. The largest amacrine cells in the INL, and those scattered in the IPL, delineated by a ring of immunoreactivity may represent DA cells bearing autoreceptors. It is probably also the case for the ringbearing cells in the GCL which may be displaced D A amacrine cells, the other labeled cells in the GCL being ganglion cells. The spontaneous and light-evoked firing of ganglion cells is known to be modified by a DA mechanism 22 which is mediated, at least in the kitten retina, by D 2 receptors 42. Recent in situ hybridization experiments have confirmed the existence of O 2 receptors on ganglion cells in rodents 1°'54. The three types of cells enclosed in the INL (amacrine, bipolar and horizontal cells) are candidates for bearing DA receptors. The All amacrine cells of the rod pathway are encircled by D A processes which provide synapses onto their somata 56, while bipolar and horizontal cells are presumed to be the main targets of sclerally directed processes of DA IPcs 14. A diffuse labeling was also observed in the INL and OPL in rat and human retina with ligand autoradiography and was related to the input of D A IPcs onto horizontal cell somata and dendrites. At present there is only a few arguments supporting a role of DA on retinal blood flow 6°. However, retinal capillaries are lying along the two margins of the INL which also contains the great majority of DA cell processes, and close contacts between DA varicosities and capillary walls have been observed in rat, cat and monkey retina ~8'26. Although it is fitting to be very careful with the interpretation of immunoreactivity on blood vessels because of their general tendency to bind immunoglobulins, it is likely that the immunoreactivity observed was specific because blood vessels were unlabeled in the controls and differently labeled according to the lighting conditions. The fixation by perfusion eliminates red blood cells with their pseudo-peroxidase activity. In spite of a penetration of processes of D A IPcs into the ONL, this layer is devoid of labeling. It seems likely that DA released at this level must reach by

62 diffusion the receptors situated outside the outer limiting membrane on RIS and ROS. The heavy labeling observed at the margin between ROS and RPE is consistent with the positive radioimmunoligand binding and is thus confirmed as ROS-associated. However, it cannot be excluded that fragments of RPE apical processes remained attached on ROS tips. D 2 receptor m R N A have been recently localized in photoreceptor cells but not in the RPE in the human retina ~3, although DA binding sites were observed in the RPE of cat 4. Nevertheless the help of electron microscopy would be needed to clarify the problem of this localization. It is known that dark-adaptation up-regulates the expression of DA receptors in the mammalian retina, as a result of the dark-induced decrease in DA release 15'36"37. The heavier labeling observed on RIS and ROS of the dark-adapted animals confirms this finding and supports the involvement of these receptors in cyclic events of photoreceptor cells. The wide distribution of D e receptor family within the retina better supports the De-mediated action of DA as neurohormonal. It is in agreement with the small number of DA synapses observed in the outer layers of the retina and confirms the general statement that De-mediated actions are in majority non-synaptic in the C N S 48. In lower vertebrates where clear evidences exist for a De-mediated regulation of photoreceptor cell metabolism and cyclic events, it is currently assessed that DA can reach the photoreceptors by diffusion from interplexiform cell processes situated in the OPL, at least in fish t2. In amphibians, in which DA IPcs are not observed, DA released from amacrine cells at a still longer distance, can also reach the D 2 receptors on rods 21. It seems thus likely that the same mechanism can occur in the mammalian retina. The demonstration of DA receptors on rods by ligand autoradiography and in situ hybridization in a number of mammalian retinas together with the present data obtained by radioimmunoligand binding and immunohistochemistry become the support of a paracrine action of DA on photoreceptor cell function in mammals. It would be of major interest to confirm such interaction in the human retina because a DA dysregulation could be involved in photoreceptor pathology. Acknowledgements. The authors thank Genevieve Prenant for excellent technical assistance. Part of this work (M.G.) was supported by the Fondation pour la Recherche M6dicale and the Universit~ de Bordeaux I1. ABBREVIATIONS RPE ROS

retinal pigment epithelium rod outer segments

RIS ONL OPL INL IPL GCL

rod inner segments outer nuclear layer outer plexiform layer inner nuclear layer inner plexiform layer ganglion cell layer

REFERENCES

1 Barthel, L.K. and Raymond, P.A., Improved method for obtaining 3 ~ m cryosections for immunocytochemistry, Z Histochem. Cytochem., 38 (1990) 1383-1388. 2 Bradford, M.M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem., 72 (1976) 248-254. 3 Brann, M.R. and Young, W.S. III, Dopamine receptors are located on rods in bovine retina, Neurosci. Lett., 69 (1986) 221226. 4 Bruinink, A., Dawis, S., Niemeyer, G. and Lichtensteiger, W., Catecholamine binding sites in cat retinal pigment epithelium and choroid, Exp. Eye Res., 43 (1986) 147-151. 5 Bubenik, G.A. and Purtill, R.A., The role of melatonin and dopamine in retinal physiology, Can. J. Physiol. Pharmacol., 58 (1980) 1457-1462. 6 Cahill, G.M., Grace, M.S. and Besharse, J.C., Rhythmic regulation of retinal melatonin: metabolic pathways, neurochemical mechanisms, and the ocular circadian clock, Cell. MoL Neurobiol., 11 (1991) 529-560. 7 Chagnaud, J-L., Mons, N., Tuffet, S., Grandin-Vazeille, X. and Geffard, M., Monoclonal antibodies against glutaraldehyde-conjugated dopamine, J. Neurochem., 49 (1987) 487-494. 8 Cohen, A.I., Mouse photoreceptors contain D 2 receptors that inhibit adenylate cyclase, Incest. Ophthalmol. Vis. Sci., Suppl. 30 (1989) 319. 9 Cohen, A.I. and Blazynski, C., Dopamine and its agonists reduce a light-sensitive pool of cyclic AMP in mouse photoreceptors, V/s. Neurosci., 4 (1990) 43-52. 10 Cohen, A.I., O'Malley, K. and Meador-Woodruff, J., Mouse photoreceptors contain D-4 receptors coupled to adenylate cyclase, Invest. Ophthalmol. l/is. Sci., 33 (1992) 1404. 11 Dawis, S.M. and Niemeyer, G., Dopamine influences the light peak in the perfused mammalian eye, Invest. Ophthalmol. Vis. Sci., 27 (1986) 330-335. 12 Dearry, A. and Burnside, B., Dopaminergic regulation of cone retinomotor movement in isolated teleost retina, I: Induction of cone contraction is mediated by D z receptors, J. Neurochem., 46 (1986) 1006-1021. 13 Dearry, A., Falardeau, P., Shores, C. and Caron, M.G., D 2 dopamine receptors in the human retina. Cloning of cDNA and localization of messenger RNA, Cell. MoL Neurobiol., 11 (1991) 437-454. 14 Dowling, J.E., Ehinger, B. and Floren, I., Fluorescence and electron microscopical observations on the amine accumulating neurons of the Cebus monkey retina, J. Comp. Neurol., 192 (1980) 665-686. 15 Dubocovich, M.L., Lucas, R.C. and Takahashi, J.S., Light-dependent regulation of dopamine receptors in mammalian retina, Brain Res., 335 (1985) 321-325. 16 Elazar, Z., Kanety, H., Schreiber, M. and Fuchs, S., Antiidiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor, Life Sci., 42 (1988) 1987-1993. 17 Elena, P.P., Denis, P., Kosina-Boix, M. and Lapalus, P., Dopamine receptors in rabbit and rat eye: characterization and localisation of DA 1 and DA 2 binding sites, Current Eye Res., 8 (1989) 75-83. 18 Favard, C., Simon, A., Vigny, A. and Nguyen-Legros, J., Ultrastructural evidence for a close relationship between dopamine cell processes and blood capillary walls in Macaca monkey and rat retina, Brain Res., 523 (1990) 127-133.

63 19 Geffard, M., Buijs, R.M., Seguela, P., Pool, C.W. and LeMoal, M., First demonstration of highly specific and sensitive antibodies against dopamine, Brain Res., 294 (1984) 161-165. 20 Harnois, C. and Di Paolo, T., Decreased dopamine in the retinas of patients with Parkinson's disease, Invest. Ophthalmol. Vis. Sci., 31 (1990) 2473-2475. 21 luvone, M.P., Evidence for a D 2 dopamine receptor in frog retina that decreases cyclic AMP accumulation and serotonin Nacetyltransferase activity, Life Sci., 38 (1986) 331-342. 22 Jensen, R.J. and Daw, N.W., Effects of dopamine and its antagonists on the receptive field properties of ganglion cells in the rabbit retina, Neuroscience, 17 (1986) 837-856. 23 Kebabian, J.W. and Caine, D.B., Multiple receptors for dopamine, Nature, 277 (1979) 93-96. 24 Kirsch, M., Wagner, H.J. and Djamgoz, M.B.A., Dopamine and plasticity of horizontal cell function in the teleost retina Regulation of a spectral mechanism through D l receptors, Vision Res., 31 (1991) 401-412. 25 Kohler, K., Kolbinger, W., Kurz-Isler, G. and Weiler, R., Endogenous dopamine and cyclic events in the fish retina. II. Correlation of retinomotor movement, spinule formation and connexon density of gap junctions with dopamine activity during light/dark cycles, Vis. Neurosci., 5 (1990) 417-428 26 Kolb, H., Cuenca, N., Wang, H.H. and Dekorver, L., The synaptic organization of the dopaminergic amacrine cell in the cat retina, J. NeurocytoL, 19 (1990) 343-366. 27 Mariani, A.P., Neff, N.H. and Hadjiconstantinou, M., 1-methyl4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) treatment decreases dopamine and increases lipofuscin in mouse retina, Neurosci. Lett., 72 (1986) 221-226. 28 McGonigle, P., Wax, M.B. and Molinoff, P.B., Characterization of binding sites for [3H]spiroperidol in human retina in vitro, Invest. Ophthalmol. Vis. Sci., 29 (1988) 687-694. 29 Mons, N., Dubourg, P., Messier, C., Chiavaroli, C., Calas, A. and Geffard, M., Polyclonal anti-idiotypic antibodies as internal images of dopamine. Applications for biochemical and morphological studies of DA receptors in the rat brain, J. Hirnforsch., 32 (1991) 617-625. 30 Mrabet, O., Messier, C., Mons, N., Destrade, C. and Geffard, M., Locomotor bias produced by intra-accumbens and intracaudate injection of polyclonal dopamine anti-idiotypic antibodies, J. Hirnforsch., 32 (1991) 627-633. 31 Negishi, K., Teranishi, T. and Kato, S., The dopamine system of the teleost fish retina. In N. Osborne and G. Chader (Eds) Progress in Retinal Research, Vol. 9, Pergamon, Oxford, 1990, pp. 1-48.

32 Nguyen-Legros, J., Morphology and distribution of catecholamine neurons in mammalian retina. In N. Osborne and G. Chader (Eds.), Progress in Retinal Research, Vol. 7, Pergamon, Oxford, 1988, pp. 113-147, 33 Nguyen-Legros, J., Les cellules interplexiformes de la r&ine des mammif~res, Ann. Sci. Nat. Zool., 12 (1991) 71-88. 34 Nguyen-Legros, J., Moussafi, F. and Simon, A., Sclerally directed processes of dopaminergic interplexiform cells reach the outer nuclear layer in rat and monkey retina, I/is. Neurosci., 4 (1990) 547-553. 35 Nowak, J.Z., Melatonin inhibits [3H]-dopamine release from the rabbit retina evoked by light, potassium and electrical stimulation, Med. Sci. Res., 16 (1989) 1073-1075. 36 Nowak, J.Z., Zawilska, J., Sek, B. and Schorderet, M., Light modulates dopamine-regulated Walsh inhibitor activity and dopamine-dependent cyclic AMP accumulation in the rabbit retina, J. Pharmacol. Pharm., 42 (1990) 457-471. 37 Nowak, J.Z., Sek, B. and Schorderet, M., Dark-induced supersensitivity of dopamine D 1 and D 2 receptors in rat retina, Neuroreport, 2 (1991) 429-432. 38 O'Malley, K.L., Harmon, S., Tang, L. and Todd, R.D., The rat

dopamine D4 receptor: sequence, gene structure, and demonstration of expression in the cardiovascular system, New Biol., 4 (1992) 137-146. 39 Pierce, M.E. and Besharse, J.C., Melatonin and dopamine interaction in the regulation of rhythmic photoreceptor metabolism. In P.J. O'Brien and D.C. Klein (Eds) Pineal and Retinal Relationships, Academic Press, London, 1986, pp. 219-237. 40 Qu, Z.X., Fertel, R., Neff, N.H. and Hadjiconstantinou, M., Pharmacological characterization of rat retinal dopamine receptors, J. Pharmacol. Exp. Ther., 248 (1989) 621-625. 41 Rem6, C., Wirz-Justice, A., Rhyner, A. and Hofmann, S., Circadian rhythm in the light response of rat retinal disk shedding and autophagy, Brain Res., 369 (1986) 356-360. 42 Robbins, J., Wakakura, K. and Ikeda, H., Noradrenaline action on cat retinal ganglion cells is mediated by dopamine (D 2) receptors, Brain Res., 438 (1988) 52-60. 43 Savy, C., Simon, A. and Nguyen-Legros, J., Spatial geometry of the dopamine innervation in the avascular area of the human fovea, His. Nearosci., 7 (1991) 4487-4498. 44 Schorderet, M. and Nowak, J.Z., Retinal dopamine D~ and D 2 receptors: characterization by binding or pharmacological studies and physiological functions, Cell Mol. Neurobiol., 10 (1990) 303305. 45 Schreiber, M., Fogelfeld, L., Souroujon, M.C., Kohen, F. and Fuchs, S., Antibodies to spiroperidol and their antiidiotypes as probes for studying dopamine receptors, Life Sci., 33 (1983) 1519-1526. 46 Sibley, D.R. and Monsma, F.J. Jr., Molecular biology of dopamine receptors, Trends Pharmacol. Sci., 13 (1992) 61-69. 47 Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L. and Schwartz, J.-C., Molecular cloning and characterization of a novel dopamine receptor (D 3) as a target to neuroleptics, Nature, 347 (1990) 146-151. 48 Stoof, J.C,, Localization and pharmacology of some DA receptor complexes in the striatum and pituitary gland: synaptic and non synaptic communication, Acta Morphol. Neerl-Scand., 26 (1989) 115-130. 49 Stormann, T.M., Gdula, D.C., Weiner, D.M., and Brann, M.R., Molecular cloning and expression of a dopamine D 2 receptor from human retina, Mol. Pharmacol., 37 (1990) 1-6. 50 Strosberg, A.D., Interaction of anti-idiotypic antibodies with membrane receptors: practical considerations. In J.J. Langone (Ed.), Antibodies, Antigens, and Molecular Mimicry, Methods in Enzymology, Vol. 178, Academic Press, San Diego, 1989, pp. 179-191. 51 Struck, L.K., Rodnitzky, R.L. and Dobson, J.K., Circadian fluctuations of contrast sensitivity in Parkinson's disease, Neurology, 40 (1990) 467-470. 52 Teranishi, T., Negishi, K. and Kato, S., Regulatory effect of dopamine on spatial properties of horizontal cells in carp retina, J. Neurosci., 4 (1984) 1271-1280. 53 Tran, V.T. and Dickman, M., Differential localization of dopamine D I and D 2 receptors in rat retina, Invest. Ophthalmol. Vis. Sci., 33 (1992) 1620-1626. 54 Tran, V.T. and Wu, L.Y., In situ localization of subtypes of D 2 dopamine receptors in the rat retina, Invest. Ophthalrnol. Vis. Sci., 33 (1992) 1404. 55 Van Tol, H.H.M., Bunzow, J.R., Guan, H.C., Sunahara, R.K. Seeman, P., Niznik, H.B. and Civelli, O., Cloning of the gene for a human dopamine D 4 receptor with high affinity for the antipsychotic clozapine, Nature, 350 (1991) 610-614. 56 Voigt, T. and W/issle, H., Dopaminergic innervation of All amacrine cells in mammalian retina, J. Neurosci., 7 (1987) 41154128. 57 Wagner, H.J. and Wulle, I., Dopaminergie interplexiform cells contact photoreceptor terminals in catfish retina, Cell Tissue Res., 261 (1990) 359-365.

64 58 White, M.P. and Fisher, L.J., Effects of exogenous melatonin on circadian disc shedding in the albino rat retina, Vision Res., 29 (1989) 167-179. 59 Witkovsky, P. and Dearry, A., Functional roles of dopamine in the vertebrate retina. In N. Osborne and G. Chader (Eds.), Progress in Retinal Research, Vol. 11, Pergamon, Oxford, 1991, pp. 247-292. 60 Yan, H.Y. and Chiou, G.C.T., Effects of L-Timolol, haloperidol

and domperidone on rabbit retinal blood flow measured with laser Doppler method, Ophthalmol. Res., 19 (1987) 45-48. 61 Zarbin, M.A., Wamsley, J.K., Palacios, J.M. and Kuhar, M.J., Autoradiographic localization of high affinity GABA, benzodiazepine, dopaminergic, adrenergic and muscarinic cholinergic receptors in the rat, monkey and human retina, Brain Res., 3374 (1986) 75-92.