Distribution and characterization of melatonin receptors in the brain of the Japanese quail, Coturnix japonica

Neuroscience Letters, 150 (1993) 149-152 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00

149

NSL 09284

Distribution and characterization of melatonin receptors in the brain of the Japanese quail, Coturnixjaponica Bruno Cozzi a, Bojidar Stankov b, Carla Viglietti-Panzica c, Simona Capsoni b, Nicoletta Aste c, Valeria Lucini d, F r a n c o Fraschini b and GianCarlo Panzica c aInstitute of Anatomy of Domestic Animals, bChairof Chemotherapy, Department of Pharmacology and Toxicology, University of Milan, Milan (Italy), CDepartmentof Human Anatomy and Physiology, University of Turin, Turin (Italy) and dInstitute of Human Anatomy, University of Milan, Milan (Italy) (Received 25 June 1992; Revised version received 30 September 1992; Accepted 26 October 1992)

Key words: Melatonin; Receptor; Quail; Brain; Distribution; Characteristics; G-protein 2-[~25I]iodomelatonin was used to study the distribution and properties of the melatonin receptor in the Japanese quail brain. High receptor density was detected in the major targets of direct retinal input (optic tectum, nucleus of the optic basal rout, ventrolateral geniculate nucleus), as well as areas representing terminals in the visual pathways (nucleus rotundus, ectostriatum, thalamo-hyperstriatal pathway). Binding was also found in the piriform cortex, the hypophyseal pars tuberalis, the oculomotorius nucleus and the associated Edinger-Westphal nucleus, and in the nuclei of the third, fourth and sixth cranial nerves. A comparison of the receptor pharmacological profile to that of the mammalian brain demonstrated pharmacological identity of the two binding sites. In the saturation experiments, GPTrS decreased the binding affinity, numerical Kd values increasing from 35 pM to --~ 150 pM.

The pineal hormone melatonin is synthesized and released in the peripheral blood on a circadian basis with high circulating levels occurring during the dark phase of the light-dark cycle [11]. Seasonal variations in the duration of the prevailing photoperiod give rise to seasonal changes in melatonin circulating profiles, thus establishing a link between the environmental light conditions and the body physiology. Species in which the photoperiod is crucial for control of the reproductive functions are sensitive to changes in the circulating melatonin levels [11]. The Japanese quail, Coturnix japonica, is a highly photoperiodic bird in which the daylength influences the gonadal status and gamete maturation [8]. In Ayes, timed melatonin administration alters the circadian rhythms, and in some species pinealectomy disrupts or abolishes them [1, 8]. Melatonin interacts with the circadian oscillatory systems of the brain through high affinity receptors localized in a number of brain areas, and their distribution seemingly differs among species (for review see ref. 19). The allocation of the melatonin receptor in avian brain Correspondence: B. Stankov, Chair of Chemotherapy, Department of Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milano, Italy. Fax: (39) (2) 71.86.87.

has not been the subject of comparative investigations. Autoradiographic examinations were performed in Gallus domesticus [13, 15, 20]. One study [24] reported in vitro ligand-receptor binding characteristics of 2-[~25I]iodomelatonin in the quail brain, that were very different from what has been described in the chicken or mammalian brains. The discrepancies could most probably be attributed to methodological factors [19]. On the other hand, no data on the existence and the identity of melatonin receptor signal transduction pathway(s) in the avian brain have been published. The present study investigated the presence, distribution, kinetic parameters, pharmacological profile, and existence of the signal transduction mechanism of the melatonin receptor in the brains of sexually mature Japanese quails, in a series of in vitro autoradiographic and ligand-receptor binding experiments. 2-[~zsI]Iodomelatonin (spec. act. --~ 1800 Ci/mmol) was purchased from Amersham (Aylesbury, Buckinghamshire, UK). Drugs and chemicals were obtained from Sigma Chemical Co. (Saint Louis, MO), unless otherwise stated. 2-Iodomelatonin was purchased from RBI (Natick, MA). 6-Chloromelatonin was a gift from Eli Lilly Laboratories (Indianapolis, IN). The synthesis of 2-bromomelatonin was described elsewhere [5]. Autora-

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Log Ki (M) Rabbit Cortex Fig. 1. Correlation between the affinities of compounds for 2-[~251]iodomelatonin binding sites in rabbit parietal cortex and quail brain. The K~ values were calculated as described previously [18].

diographic film (X-OMAT S) was from Eastman Kodak (Rochester, NY). Brains were obtained from sexually active adult male quails kept under controlled photoperiod of 16 h of light and 8 h of darkness (LD 16:8). They were killed around the middle of the light phase. Brains were rapidly removed and frozen by immersion in cold isopenthane at - 30°C (autoradiographic studies) or snap-frozen in liquid nitrogen (in vitro ligand-receptor binding studies). Slide-mounted sections were processed as described previously [17]. Following development, the autoradiograms were recorded by means of an image-processing system [10] and the areas identified according to an anatomical atlas of the chick brain [6], adjusted to the quail brain [9]. The isolation of crude membrane preparations, the binding conditions and the calculations and statistical analysis were as described elsewhere [18]. When GTPrS was used, its final concentration was 100 HM, chosen on the basis of two pilot dose-response experiments. Kinetic studies were performed with a fixed concentration of radioactive melatonin ~ 60 pM). Incubation time varied from 1 to 240 min. In the displacement experiments, 1 /IM 2-iodomelatonin was added 60 min after the beginning of the reaction. Saturation experiments were generated in the range of the labelled ligand, 8.6-263 pM. The competition experiments employed various drugs (range 1E-~~-1E-4 M). The binding characteristics and the presence of signal-transduction pathway in the quail brain were compared to those described in the cortex of the rabbit (Oryctolagus cuniculus) [5, 16, 18].

Association of the labelled ligand to the crude membrane preparations of the whole brain occurred rapidly at 37°C, reaching equilibrium at about 60 min, remaining stable for up to 240 min. Binding was reversible, as seen by dissociation kinetic curves of 2-[~25I]iodomelatonin following addition of excess non-radioactive 2-iodomelatonin (1/aM). The calculated kinetic dissociation constant was 20 pM. The specific binding in whole brain homogenates represented 85-97% of the total binding, increased linearly with increasing concentrations of the labelled ligand (8.6-65 pM) and reached saturation at ~ 120 pM ligand concentration. The calculated Kd was between 20 and 40 pM. The non-hydrolyzable GTP analogue, GTPrS (100 /.tM) produced a shift in the apparent affinity, the numerical Kd values going up to ~ 95-153 pM, with a negligible decrease in the Bmaxvalues. A summary of the results from the kinetic and saturation studies is reported in Table I. The competition experiments demonstrated that binding was highly specific. Only N-acetyl-5-methoxytryptamines, such as 2-iodomelatonin, 2-bromomelatonin, 2methylmelatonin, 6-chioromelatonin and melatonin effectively competed with 2-[J25I]iodomelatonin. Prazosin, norepinephrine, dopamine, serotonin and GABA were all extremely weak inhibitors (IC50 values > 10,000 nM). Comparative pharmacological profiles (quail brain vs. rabbit parietal cortex) are shown in Fig. 1. High receptor density was identified in several brain areas pertaining to the visual perception pathways [12]. In particular, the optic tectum, the basal optic nuclei, and ventrolateral geniculate nuclei were intensely labeled. Melatonin receptors were also identified within the ventral nuclei of the supraoptic decussation. In several avian species [2, 3], these nuclei represent a specific retino-recipient region, which has been recognized as a homologue of the mammalian suprachiasmatic nuclei (for a

TABLE I CHARACTERISTICS OF THE BINDING PARAMETERS IN THE WHOLE Q U A I L BRAIN M E M B R A N E PREPARATIONS IN PRESENCE OR ABSENCE OF 100 p M GTPyS Data are the means of three independent determinations. The dissociation constants derived from saturation studies (Kd2) represent the resuits, obtained by using non-linear fitting strategies. Kd~, mean Kd values (pM) obtained from the kinetic experiments; Kd2, mean Kd values (pM) from the saturation studies; Hill, Hill coefficients. Bmaxis reported in fmol/mg protein. NT, not tested.

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Fig. 2. Distribution of the specific2-[125I]iodomelatoninbinding in coronal sectionsof the quail brain. The non-specificbindingwas verylow, homogeneousand equal to the background. CPi, cortex piriformis;E, ectostriatum; EW, Edinger-Westphalnucleus; GLv, nucleus geniculatus lateralis, pars ventralis; HA, hyperstriatum accessorium; HV, hyperstriatum ventrale; nVI, nucleus of the sixth cranial nerve; nBOR, nucleus opticus basalis; nDSV, nucleus decussatio supraoptica ventralis; ROT, nucleus rotundus; OM, nucleus nervi oculomotorii;OS, nucleusolivarissuperior; PPC, nucleusprincipalsprecommisuralis;PT, nucleus pretectalis; Pt, hypophysealpars tuberalis; ROT, nucleus rotundus; TeO, tectumopticum. Bar = 5 mm.

comprehensive argumentation see refs. 2, 7, 9, 14), the site of an endogenous pacemaker. Binding was also strong in brain areas related to processing of visual stimuli not directly connected to the retina [4, 21-23], such as the nucleus rotundus, the ectostriatum, and the thalamohyperstriatal complex. Worth noting here is that melatonin receptors were identified in the nuclei of the third, fourth and sixth cranial nerves, and the EdingerWestphal nuclei, involved the control of eye movements (Fig. 2). The piriform cortex and the pars tuberalis of the adenohypophysis were clearly labelled, as well. The data obtained in the in vitro ligand-receptor binding experiments plainly demonstrated that the kinetic parameters and the pharmacological profile of the binding site are suggestive of a functional melatonin receptor. The apparent Kd and 2-iodomelatonin K~ values, calculated from the kinetic, saturation and competition experiments, were in the low picomolar range, and the apparent melatonin Ki was around 1 nM. Moreover, co-incubation with GTPrS resulted in a decreased affinity, confirming that similarly to other brain areas in diverse mammalian species the high-affinity melatonin receptor is linked to a G-protein as a first step of the signaltransduction pathway. The 2-[~25I]iodomelatonin binding properties in Coturnix described by another group [24] differed signifi-

cantly. The apparent KdS were reported to be between 500 and 700 pM, with a melatonin Ki of 14 nM, values that would question the physiological significance of the binding site taken the picomolar peripheral blood melatonin levels. The incubation conditions in those experiments were quite different, i.e. low (4°C) incubation temperature. It has been repeatedly demonstrated that low incubation temperatures disturb the interaction of the receptor with its G-protein and lead to erroneous determination of the apparent affinity constants [19]. Our comparison of the receptor's pharmacological profile in the quail brain vs. that in the rabbit cortex clearly demonstrated the pharmacological identity of the two binding sites (see Fig. 1). Collectively, the data reported herein show that the distribution of the melatonin receptor in the quail brain is similar to that described for chicken brain [13, 15], and possibly a passerine species (unpublished data cited elsewhere [13]), pointing out to the existence of a typical avian distribution pattern. The present study suggests that melatonin could participate in the control of the visual perception and processing in Coturnix, since melatonin receptors are located in sensory areas related to sight. Though generally similar, the binding distribution pattern in the quail brain presents some remarkable differences from what has been described in the other avian species. A noteworthy finding was the presence of melatonin receptors in the nuclei of the cranial nerves responsible for coordination of eye movements. A potential rationale for presence of melatonin receptors in these nuclei might be the possibility that the animal acquires adaptive advantages, when shifting the eye position, so that light hits discrete retinal areas. In Coturnix, pinealectomy does not abolish the circadian rhythmicity and the persistent high nocturnal peripheral blood melatonin levels in pinealectomized quails are of retinal origin. The quail is sensitive to retinal melatonin rhythms, since both enucleation and pinealectomy result in arhythmic activity. This work was supported in part by C N R Grants 203.04.12 (B.C.), 038.233 (B.S.) and 92.01056 (G.C.P.) and by M.U.R.S.T, Rome, grants to B.C. and to G.C.E and C.V.E 1 Barrett,R.K. and Underwood,H., The superiorcervicalgangliaare not necessaryfor entrainmentor persistenceof the pinealmelatonin rhythm in Japanese quail, Brain Res., 569 (1992) 249-254. 2 Cassone, V.M. and Moore, R.Y., Retinohypothalamicprojection and suprachiasmaticnucleusof the house sparrow, Passerdomesticas, J. Comp. Neurol., 266 (1988) 171-182. 3 Cooper, M.L., Pickard, G.E. and Silver, R., Retinohypothalamic pathwayin the dovedemonstratedby anterogradeHRP, Brain Res. Bull., 10 (1983) 715-718.

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