Neuroscience Letters, 116 (1990) 81-86 Elsevier Scientific Publishers Ireland Ltd.

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NSL 07058

Autoradiographic localization of D 1 and D 2 dopamine binding sites in the human retina Philippe Denis 1,2, Pierre-Paul Elena 1, Jean-Philippe Nordmann 2, Henri Saraux 2 and Philippe Lapalus 1 1Department of Pharmacology, Facultbde Mbdecine, Nice, (France) and 2Department of Ophthalmology, Hrpital Saint Antoine, Paris (France) (Received 4 January 1990; Revised version received 19 April 1990; Accepted 20 April 1990)

Key words: Dopamine receptor; Autoradiography; Human retina; Human; [J25I]SCH 23982; [125I]Iodosulpride The localization of dopamine binding sites was studied by in vitro autoradiography in the normal human retina using [~2sI]SCH 23982 for DI receptor labelling and [~25Iliodosulpride for D2 receptors. Results demonstrated that both types of binding sites were present in human retina. Binding of [12sl]SCH 23982 to DI dopamine receptor was blocked by I/~M SKF 38393, SCH 23390 (DI specific compounds) whereas bromocriptine and domperidone (D2 specific compounds) were inactive at the same concentration. On the contrary, binding of [~25I]iodosulpride to D2 dopamine receptor was inhibited only by D2 drugs. Precise cellular distribution was given by microautoradiographic techniques and showed that binding sites were exclusively localized to the plexiform layers.

In the last decade, Stoof and Kebabian postulated that two types of dopamine (DA) receptors could be identified from their pharmacological properties as well as the biochemical consequences of their stimulation [25]. Receptors of which the stimulation elicited adenylate cyclase activity have been designated as DI receptors. Conversely, DA receptors which are not stimulatory linked to adenylate cyclase have been termed as D2 receptors. Whereas retinal DA receptors have been identified by conventional in vitro binding assays [10, 18], little is known about their anatomical distribution in humans [26]. In the present work, we undertook autoradiographic localization of DA binding sites with selective radioligands: [I25I]SCH 23982 was used for D1 receptor labelling whereas D2 receptors were characterized by [125I]iodosulpride, a benzamide derivative. [125I]SCH 23982 is an iodinated benzazepine derivative with dopamine D1 receptor characteristics [23]. Despite some interactions with serotonin receptors [14],

Correspondence: P. Denis, Department of Ophthalmology, Saint Antoine Hospital, 184 rue du Faubourg Saint Antoine, 75012, Paris, France. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

82 [125I]SCH 23982 has been presented as a suitable ligand for autoradiographic localization o f DI receptors [1]. Moreover, for D2 receptors, benzamide c o m p o u n d s are actually a m o n g the most selective ligands and are radiolabelled [16, 24]. A m o n g them, [t25I]iodosulpride exhibited the pharmacological profile expected for a drug acting upon D2 receptors and was used successfully for identifying D2 receptors in brain autoradiographic studies [4]. Six freshly enucleated eyeballs from h u m a n donors (ages 55-91 years) were obtained within 6 h after death (mainly due to cardio-respiratory failure). N o n e of these subjects were k n o w n to have neurological diseases, particularly Parkinson disease. Retinas were removed, immersed in Tissue Tek medium, frozen in isopentane cooled ( - 4 0 ° C ) in liquid nitrogen, and stored at - 8 0 ° C . Just before sectioning, tissues were brought to - 20°C and sections (5/~m thick) were cut with a cryostat (Leitz

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a

Fig. 1. Microautoradiograms of [125I]SCH23982 binding in human retina. Bright fields (a, c, e) and dark fields (b, d, f) were obtained in retina after incubation of 150 pM [~25I]SCH23982 alone (a, b), in presence of I ~M SCH 23390 (c, d) or 1 /tM domperidone (e, f). Silver grains visualized in the plexiform layers indicated D~ binding sites since labelling disappeared after incubation with an excess of D~ compound. (opl, outer plexiform layer; ipl, inner plexiform layer.) ( × 500)

83 1720, West Germany), mounted onto gelatin-coated glass slides and stored at - 20°C until use. In a previous investigation, we determined biochemical parameters of D~ and Dz binding sites in the rabbit retina [8]. In the current study, similar experimental conditions were used to label these receptor sites in human retina. These conditions gave an optimal, reproducible labelling of DA binding sites and provided a maximal specific-to-non-specific ratio. For the D1 binding sites, sections were incubated in a 5 mM Tris-HCl buffer (pH 7.4) containing 120 mM NaCI, 5 mM KC1, 2 mM CaC12, 1 mM MgCI2 and 150 pM [125I]SCH 23982 (New England Nuclear, 2200 Ci/mmol). Non-specific binding was determined by 1/tM SCH 23390. After a 75 min incubation at room temperature, sections were washed for 10 min in cold buffer and dipped in distilled water. For the D2 binding sites, sections were incubated in a 50 mM Tris-HCl buffer (pH 7.4) containing 120 mM NaCI, 5 mM KC1, 1 mM CaC12, 1 mM MgC12, 5.7 mM ascorbic acid and 200 pM [125I]iodosulpride (Amersham, 2200 Ci/mmol). Non-specific binding was determined by 1/~M domperidone. After a 20 min incubation at room temperature, sections were rinsed for 4 min (2 × 2 min) in cold buffer and dipped in distilled water. Dried sections were apposed to emulsion coated-coverslip (Kodak AR-10) for 21 days. After exposure, films were developed and sections were stained with hematoxylin and eosin. All compounds were from Sigma (MO, U.S.A.) except domperidone (Janssen, France), sulpiride (Delagrange, France), bromocriptine (Sandoz, France), SKF 38393, SCH 23390 (Research Biochemicals Incorporation, MA, U.S.A.). Microautoradiographic experiments performed with both ligands revealed that human retina were heavily labelled by [125I]SCH 23982 and [125I]iodosulpride (Figs. 1 and 2, panels a, b), indicating that both D1 and DE binding sites were present in this tissue. Displacement studies showed that SKF 38393 (Dl agonist) or SCH 23390 (D1 antagonist) (Fig. 1, panels c, d) were able to displace [125I]SCH 23982 from binding sites while bromocriptine (Dz agonist) or domperidone (D 2 antagonist) (Fig. 1, panels e, f) had no effect on the labelling at the same concentration (1 /~M). Moreover, [125I]iodosulpride binding was completely inhibited by D2 compounds and unaffected by Dl ones (Fig. 2). The ability of a variety of selective compounds to displace [125I]SCH 23982 and [125I]iodosulpride binding then indicated a high degree of pharmacological specificity associated with D1 and D2 binding sites. Though some reports previously documented on the existence of retinal DA receptors in several species [ 10, 18], our work extends these findings to the human tissue. Furthermore, since each retina was independently labelled with [125I]SCH 23982 and [125I]iodosulpride, our data clearly showed that human retina possesses both D1 and D 2 receptors. Microautoradiographic studies allowed to localize precisely [125I]SCH 23982 and [J25I]iodosulpride binding sites in the inner and outer plexiform layers (Figs. 1 and 2, panels a, b). Some of our findings were partly in agreement with a previous autoradiographic study performed with [3H]spiperone [26] showing D2 binding sites in the plexiform layers but also in the two nuclear layers. The reason of this discrepancy between sites labelled by the tritiated compound and [125I]iodosulpride is not yet well established. The difficulty, using [3H]spiperone to localize D2 binding sites, is that this

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Fig. 2. Microautoradiograms of [1251]iodosulpridebinding in human retina. Bright fields (a, c, e) and dark fields (b, d, f) were obtained in retina after incubation of 200 pM [125I]iodosulpridealone (a, b), in presence of 1 ItM SCH 23390 (c, d) or 1 /IM domperidone (e, f). High densities of silver grains are observed in the plexiform layers. (opl, outer plexiform layer; ipl, inner plexiform layer.) ( × 500).

ligand specifically labels other classes o f binding sites in addition to D2 receptors (particularly, 5-HT2 or non-transmitter sites, the so-called 'spirodecanone' sites) [20]. Based on the finding that these n o n - D A sites were found to be negligible in the retina, most o f the [3H]spiperone binding sites were attributed to D2 d o p a m i n e receptors. The only possibility to explain the distribution discrepancy is to consider a loss o f binding sites due to age differences between the subjects or to the time elapsed between death and autopsy. N u m e r o u s evidences support a neurotransmitter role for D A in the m a m m a l i a n retina. D A has been shown to be the p r e d o m i n a n t retinal catechotamine [1 1]. If species-related differences in the distribution o f retinal dopaminergic neurons have been observed, it is now well accepted that, in mammals, D A cells belong to a subpopula-

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tion of amacrine cells with cell bodies located in the inner nuclear layer (INL) and sending processes to the inner plexiform layer (IPL) [9]. In retinas of teleosts and monkeys, another class of dopaminergic cells, called interplexiform cells, have been identified [6, 13]. These neurons realize synapses with amacrine cells in the IPL and with horizontal and bipolar cells in the outer plexiform layer (OPL). In rat and monkey retinas, tyrosine hydroxylase-containing neurons have recently been shown to have thin processes extending beyond the ONL [19]. In a previous study, we localized and characterized DA receptors in rat and rabbit retinas [8]; in both species, D1 binding sites are found in the OPL, INL and IPL, while D2 sites are restricted to the plexiform layers. The retinal function of DA has been extensively investigated. In the IPL, D1 dopamine antagonists have been shown to modulate ON-ganglion cells electrophysiological activity [17]. It has also been demonstrated that DA can reduce sensitivity of horizontal cells to light in goldfish retina [12] and modulate their receptive field size in turtle retina (through both Dl and D2 receptors [21]. Released DA would bind to horizontal cells postsynaptic D1 receptors, with an increase of cyclic AMP and subsequently electrophysiological changes in electric synapses between these cells [3]. Similarly, in human retina, distribution of some of the DI binding sites to the OPL may be related to horizontal cells. D2 receptors are thought to be involved in light-induced modulation of a DA release from rabbit retina [7]. Another site of action for the D2 receptor is the photoreceptor. In lower vertebrates, dopamine has been recently demonstrated to mimic the effects of light on retinomotor responses [5], to modulate serotonin N-acetyltransferase circadian activity (a key enzyme in the melatonin synthesis) in Xenopus retina [15] and to regulate the daily rhythm of rod outer segments shedding by the retinal pigment epithelium [22]. The exact cellular source of the dopamine binding sites described in this study cannot be undoubtedly identified, but some of the D2 binding sites found in OPL may be located on the photoreceptor cell and involved in the disc shedding regulation (since retinomotor movements only exist in lower vertebrates). Our results also support a functional role for DA in vision of humans. Clinically, a DA deficiency such as Parkinson's disease seemed to be responsible for impaired visual performances, including delayed visual evoked potentials [2]. In summary, our findings demonstrate that human retina contains both DI and D2 binding sites, exclusively located in the two plexiform layers and the autoradiographic study discloses an interesting correlation between the distribution of DA binding sites, DA-containing neurons (whose processes are mainly located in the plexiform layers), and DA target cells. 1 Aiso, M., Shigematsu, K., Kebabian, J.W., Potter, W.Z., Cruciani, R.A. and Saavedra, J.M., Dopamine DI receptor in rat brain: a quantitative autoradiographic study with p25I]-SCH 23982, Brain Res., 408 (1987) 281 285. 2 Bodis-Wollner, I., Yahr, D., Mylin, L. and Thornton, Dopaminergic deficiency and delayed visual evoked potentials in humans, Ann. Neurol., 11 (1982) 478483. 3 Bodis-Wollner, I. and Piccolino, M., Dopaminergic Mechanisms in Vision, Alan Liss, New York, 1988. 4 Bouthenet, M.L., Martres, M.P., Sales, N. and Schwartz, J.C., A detailed mapping of dopamine D-2 receptors in rat central nervous system by autoradiography with [~25I]-iodosulpride, Neuroscience, 20 (1987) 117 155.

86 5 Dearry, A. and Burnside, B., Dopaminergic regulation of cone retinomotor movement in isolated teleost retinas. I. Induction of cone contraction is mediated by D2 receptors, J. Neurochem., 46 (1986) 1006--1021. 6 Dowling, J.E., The interplexiform cell system. I. Synapses of the dopamine neurons of the goldfish retina, Proc. Roy. Soc. Lond. Ser. B, 201 (1978) 7 26. 7 Dubocovich, M.L. and Weiner, N., Modulation of the stimulation-evoked release of [3H] dopamine through activation of dopamine autoreceptors of the D-2 subtype in the isolated rabbit retina. In G.N. Woodruff, M. Kohsaka, Y. Tsukada and T. Shomori (Eds.), Advances in Biosciences, Vol. 37, Pergamon, Oxford, 198l, pp. 273 278. 8 Elena, P.P., Denis, P., Kosina-Boix, M. and Lapalus, P., Dopamine receptors in rabbit and rat eye: characterization and localization of DA-1 and DA-2 binding sites. Curr. Eye Res., 8 (1989) 75-83. 9 Frederick, J.M., Rayborn, M.E, Laties, A.M., Lam, D.M.K. and Hollyfield, J.G., Dopaminergic neurons in the human retina, J. Comp. Neurol., 210 (1982) 65 79. 10 Gredat, O., Parkinson, D. and Nielsen, M., Binding of [3H]SCH 23390 to dopamine D-1 receptors in rat retina in vitro, Eur. J. Pharmacol., 137 (1987) 241-245. 11 Haggendal, J. and Malmfors, T., Evidence of dopamine-containing neurons in the retina of the rabbit, Acta Physiol. Scand., 59 (1963) 295-296. 12 Hedden, W.L. and Dowling, J.E., The interplexiform cell. II. Effects of dopamine on goldfish retinal neurons. Proc. R. Soc. Lond. Ser. B., 201 (1978) 27 55. 13 Holmgren, I., Synaptic organization of the dopaminergic neurons in the retina of the Cynomolgus monkey, Invest. Ophthalmol. Vis. Sci., 22 (1982) 8-24. 14 Hoyer, D. and KarpL A., [~25I]SCH 23982, a 'selective' D-1 receptor antagonist, labels with high affinity 5-HT lc sites in pig choroid plexus, Eur. J. Pharmacol., 150 (1988) 181 184. 15 Iuvone, P.M., Evidence for a D2 dopamine receptor in frog retina that decreases cyclic AMP accumulation and serotonin N-acetyttransferase activity, Life Sci., 38 (1986) 331-342. 16 Janowsky, A., De Paulis, T., Clanton, J.A., Smith, H.E., Ebert, M.H. and Kessler, R.M., [~251]Iodopride: a specific high affinity radioligand for labelling dopamine D-2 receptors, Eur. J. Pharmacol,, 150 (1988) 203 205. 17 Jensen, R.J. and Daw, N.W., Effects of dopamine antagonists on receptive fields of brisk cells and directionally selective ceils in the rabbit retina, J. Neurosci., 4 (1984) 2972-2985. 18 McGonigle, P., Wax, M.B. and Molinoff, P.B., Characterization of binding sites of 3H-Spiroperidol in human retina. Invest. Ophthalmol. Vis. Sci., 29 (1988) 687 694. 19 N'Guyen-Legros, J., Simon, A. and Moussafi, F., Dopaminergic terminals from interplexiform cells reach the outer nuclear layer in rat and monkey retinas, ARVO Abstr., 4 (1989) 120. 20 Patacios, J.M., Niehoff, D.L. and Kuhar, M.J., [3H]Spiperone binding sites in brain: autoradiographic localization of multiple receptors, Brain Res., 213 (1981) 277 289. 21 Piccolino, M., Demontis, G., Witkovsky, P., Strettoi, E., Cappagli, G.C., Porceddu, M.L., De Montis, M.G., Pepitoni, S., Biggio, G., Meller, E. and Bohmaker, K., Involvement of DI and D2 dopamine receptors in the control of horizontal cell electrical coupling in the turtle retina. Eur. J, Neurosci., 1 (1989) 247 257. 22 Pierce, M,E. and Besharse, J.C., Melatonin and dopamine interactions in the regulation of rhythmic photoreceptor metabolism. In P.J. O'Brien and D.C. Klein (Eds.), Pineal and Retinal Relationships, Academic Press, 1986, pp. 219 237. 23 Sidhu, A., Van Oene, J.C., Dandridge, P., Kaiser, C. and Kebabian, J.W., [t25I]SCH 23982: the tigand of choice for identifying the D-1 dopamine receptor, Eur. J. Pharmacol., 128 (1986) 213 220. 24 Singhaniyom, W., Tsai, Y.F., Briicke, T., McLellan, C.A., Cohen, R.M., Kung, H.F. and Chiueh, C.C., Blockade of in vivo binding of t25-1abelled 3-iodobenzamide (IBZM) to dopamine receptors by D2 antagonist and agonist. Brain Res., 453 (1988) 393 396. 25 Stool J.C. and Kebabian, J.W., Two dopamine receptors: biochemistry, physiology and pharmacology, Life Sci., 35 (1984) 281 296. 26 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., 374 (1986) 75-92.

Autoradiographic localization of D1 and D2 dopamine binding sites in the human retina.

The localization of dopamine binding sites was studied by in vitro autoradiography in the normal human retina using [125I]SCH 23982 for D1 receptor la...
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