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Localization and Characterization of Melatonin Receptors in Rodent Brain by in Vitro Autoradiography

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Localization and Characterization of Melatonin Receptors in Rodent Brain by in Vitro Autoradiography The Journal of Neuroscience, July 1989, g(7): 2581-2590 Localization and Characterization of Melatonin Receptors in Rodent Brain by in vitro Autoradiography David R. Weaver, Scott A. Rivkees, and Steven M. Reppert Laboratory of Developmental Chronobiology, Children’s Service, Massachusetts General Hospital, and Department of Pediatrics and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02114 Little is known of the neural sites of action for the pineal based on brain lesion studies (Bittman et al., 1979; Rusak, 1980) hormone, melatonin. Thus, we developed an in vitro auto- and experiments using intracerebral melatonin implants (Glass radiographic method using i251-labeled melatonin (I-MEL) to and Lynch, 198 1). study putative melatonin receptors in rodent brain. We first Another effect of melatonin is its ability to influence circadian determined optimal in vitro labeling conditions for autora- rhythmicity (see Underwood and Goldman, 1987, for review). diographic detection of I-MEL binding sites in rat median In mammals, pinealectomy or melatonin implants do not affect eminence, the most intensely labeled area in the rat brain. the period of free-running circadian rhythms (Cheung and We then assessed the pharmacologic and kinetic properties McCormack, 1982) and pinealectomy has only minor effects on of I-MEL binding sites in rat median eminence by quantitative the rate of reentrainment following phase shifts of the lighting autoradiography. These sites have high affinity for I-MEL cycle. However, daily melatonin administration can entrain rats (equilibrium dissociation constant = 43 PM). I-MEL binding (Redman et al., 1983). The suprachiasmatic nuclei (SCN) func- was inhibited by nanomolar concentrations of melatonin or tion as a biological clock in rodents and nonhuman primates, 6-chloromelatonin, but was not inhibited by serotonin, do- regulating a variety of circadian rhythms (Moore, 1983). The pamine, or norepinephrine (100 @I). These results suggest entraining effect of melatonin may be due to an effect on the that I-MEL binding sites identified by in vitroautoradiography SCN, as SCN lesions prevent the entraining effect of melatonin represent specific, high-affinity melatonin receptors. Studies (Cassone et al., 1986). of the distribution of I-MEL binding in rat, Syrian hamster, With the recent discovery that the indole nucleus of melatonin and Djungarian hamster brain confirm that the median em- can be directly iodinated (Vakkuri et al., 1984), a radioligand inence and suprachiasmatic nucleus are major sites of I-MEL (1251-labeled melatonin; I-MEL) of high specific activity (ca. 2000 binding in rodent brain; other brain areas labeled in one or Ci/mmol) is now available for investigating melatonin sites of more of these species were the thalamus (paraventricular, action. The biological activity of iodinated melatonin, both in anteroventral, and reuniens nuclei, nucleus of the stria med- vivo (Weaver et al., 1988~) and in vitro (Dubocovich and Ta- ullaris, and medial part of the lateral habenular nucleus), kahashi, 1987), strongly suggests that this molecule interacts at hypothalamus (dorsomedial nucleus), subiculum, and area biologically relevant sites; localization and characterization of postrema. The presence of putative melatonin receptors in these sites is a critical step in understanding the mechanism of the suprachiasmatic nuclei and median eminence of these melatonin action. rodent species suggests that these brain regions are im- In vitro autoradiographic techniques using I-MEL have re- portant loci for melatonin effects on circadian rhythms and cently been developed to localize putative melatonin receptors reproduction. within individual brain nuclei (VanCEek et al., 1987; Reppert et al., 1988; Weaver et al., 1988~). In the present study, we char- The hormone melatonin (N-acetyl-5methoxytryptamine) is acterized the I-MEL binding site in rat median eminence by produced rhythmically by the vertebrate pineal gland, with el- quantitative receptor autoradiography. To define potential sites evated levels occurring at night. The most well-documented role of melatonin action in species where melatonin effects on phys- of melatonin in mammals is the regulation of photoperiodic iology have been most extensively studied, we also examined responses, especially reproduction (for reviews, see Karsch et the distribution of I-MEL binding sites in brains of adult male al., 1984; Turek et al., 1984; Tamarkin et al., 1985; Underwood rats and Syrian and Djungarian hamsters. and Goldman, 1987). The hypothalamus appears to be the prime site of melatonin action in the modulation of reproduction, Materials and Methods Preparation of I-MEL. Melatonin was iodinated by the method of Vak- kuri et al. (1984) and purified by high-pressure liquid chromatography Received Oct. 21, 1988; revised Dec. 12, 1988; accepted Dec. 16, 1988. (HPLC). Briefly, 10 pg melatonin (1 &PI in 100% ethanol; Sigma, St. We thank Ignacio Provencio for expert technical assistance and Dr. Greg Crosby Louis, MO) and Na1*5I (2 mCi; specific activity, 1775-2 113 Ci/mmol; for use of his image-analysis system. This work was supported by NRSA HD- 06976 (D.R.W.), the Pediatric Career Scientist Training Program Award under Amersham, Arlington Heights, IL) were combined in an Eppendorftube PHS Grant UlO-HD-00850 (S.A.R.), and HD-14427 (S.M.R.). S.M.R. is an Es- coated with Iodogen (10 pg, 1,3,4,6 tetrachloro-3a,6a-diphenyl-glycol- tablished Investigator of the American Heart Association. Portions of this work uril; Pierce Chemical Co., Rockford, IL). The reaction was terminated have been previously reported (Weaver et al., 1988a, b). after 60 set by addition of 200 ~1 chloroform. The organic phase was Correspondence should be addressed to David R. Weaver, Ph.D., at the above evaporated to drynessunder nitrogen and resuspended in 100% ethanol. address. The reaction product was then injected onto an HPLC column (Ul- Copyright 0 1989 Society for Neuroscience 0270-6474/89/072581-10$02.00/O trasphere 5 Km, 4.6 mm x 15 cm; Beckman, San Ramon, CA) and 2582 Weaver et al. l Melatonin Receptors in Rodent Brain eluted with a linear gradient of isopropanol (1040% over 30 min at 1 values were calibrated using a photographic step tablet (Kodak #3, ml/min) in distilled water. Fractions were collected every 30 sec. Frac- Rochester, NY). For quantitative assessment, optical density values of tions (4) eluting at ca. 30% isopropanol (retention time, 20 min) con- individual brain regions were converted to radioactivity by comparison tained a neak of radioactivitv composed primarily of I-MEL. Under with radioactive standards (20-pm-thick lzsI microscales, batch #4, these conditions, tritiated meiatonin (2-aminoethyl-3H, 43.9 Ci/mmol; Amersham) included on each film. A single set of 125I microscales was New England Nuclear, Boston, MA) eluted at 20% isopropanol (reten- used for this series of experiments, and the amount of radioactivity in tion time, ca. 10 min) and was clearly separated from I-MEL. Each the standards was calculated to compensate for decay of the isotope for fraction was extracted with 5 vol of chloroform, and the chloroform each set of films. was washed with 1 ~010.45 M sodium borate buffer (pH 9.7). The organic While the standard curve relating optical density and radioactivity fractions were combined and dried under nitrogen. I-MEL was dissolved could be described as linear for each film (r z 0.90), linear plots poorly in 100% ethanol and diluted in buffer. Iodination yield (counts I-MEL estimated the level of radioactivity at some optical density values (see recovered/counts NarZSI initially) was approximately 10%. Fig. 1). We therefore used a computer-generated best-fit multiparameter Thin-laver chromatoaranhv IO.25 mm silica gel (F-254). EM reagents (cubic) equation (BRAIN program GU: BMFP), which provided a good division, Brinkman Ins‘trumen’ts, Inc., Westbu-v, NY] using ethyl-ace- fit throughout the optical density range examined (see Fig. 1). tate as the mobile phase (Weaver et al., 1988~) was used to test purity Radioreceptor assay. Rat brains were removed rapidly, and brain of I-MEL prior to autoradiographic labeling experiments. The radio- regions were dissected with the aid of a dissecting microscope. For SCN activity migrated as a single peak that comigrated with nonradioactive and median eminence, 40 animals were used. Samples of cortex and iodomelatonin standard. I-MEL was >95% pure and remained stable hypothalamus (a dissection including both SCN and median eminence) for at least 2 months after preparation. were obtained from 8 other rats. Tissue was pooled by brain region, Animals. Male Sprague-Dawley rats (28-35 d old) were obtained from sonicated (Kontes microultrosonic cell disruptor, tune = 3, power = 8, Charles River Breeding Laboratories (Kingston, NY) and Zivic-Miller 10 set) in Tris buffer (50 mM, pH 7.4, 4°C) and centrifuged (50,000 x Laboratories (Allison Park, PA); no differences in distribution or inten- g, 20 min, 4°C). Pellets (crude membrane preparation) were washed sity of I-MEL binding were noted between animals from the 2 suppliers. twice, resuspended in Tris buffer, frozen, and stored at -70°C for up Male Syrian (golden) hamsters (Mesocricetus aurutus, LAKLVG SYR, to 3 weeks before use. 48-54 d old) were obtained from Charles River Breeding Laboratories For assay, aliquots of membrane preparation (100 rg protein) were (Wilmington, MA), and 60-d-old male Djungarian (Siberian) hamsters incubated with I-MEL (50 DM) in a final volume of 200 vl at 25°C for (Phodopus sungorus) were from our colony, established in 1985 with 30 min. The incubation‘was terminated by filtration through prewetted animals provided by Drs. D. A. Damassa, M. H. Stetson, and G. N. Whatman GF/B filters, followed by a rapid rinse with 10 ml ice-cold Wade. Rats were housed in a light-dark cycle of 12 hr light: 12 hr dark buffer. Counts bound to protein were assessed by counting the filters in (12L: 12D), Syrian hamsters were housed in 14L: 1OD, and Djungarian a gamma counter.
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