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Developmental appearance and age related changes in specific 2-[125I]iodomelatonin binding sites in the suprachiasmatic nuclei of female Syrian hamsters
205
Developmental Brain Research, 73 (1993) 205-212 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00
BRESD 51634
Developmental appearance and age related changes in specific 2-[125I]iodomelatonin binding sites in the suprachiasmatic nuclei of female Syrian hamsters Marilyn J. D u n c a n a,b a n d F r e d e r i c k C. Davis c Department of Anatomy and Neurobiology, University of Missouri Medical School, Columbia, MO 65212 (USA), ~' Department of Anatomy and Neurobiology, Uni~ersity of Kentucky, Chandler Medical Center, Lexington, KY 40536-0084 (USA) and ' Department of Biology, Northeastern Unit'ersity, Boston, MA 02115 (USA) (Accepted 22 December 1992)
Key words." Melatonin; Suprachiasmatic nucleus; Pars tuberalis; Circadian timing system; Pineal
In Syrian hamsters, the circadian timing system is sensitive to melatonin during gestation but is not responsive in the adult. In order to further understand this developmental change in melatonin responsiveness, in vitro autoradiography was used to assess the presence of specific 2-[125I]iodomelatonin binding sites in the suprachiasmatic nuclei of female hamsters of selected embryonic (E) and postnatal (PN) ages (e.g. El3, El4, El5, PNI, PN2, PN12, PN25, PN112-133). Specific 2-[lZSI]iodomelatonin binding sites were seen in the suprachiasmatic nuclei of some of the El4 hamsters and all the perinatal hamsters (El5, PN1 and PN2) but not in older hamsters. In contrast, specific 2-[125I]iodomelatonin binding sites were seen in the pars tuberalis of all hamsters (with the exception of one), regardless of age. The transient expression of specific 2-[1251]iodomelatonin binding sites in the suprachiasmatic nuclei suggests that melatonin may have some special functions restricted to early development. The specific 2-[125I]iodomelatonin binding sites in the embryonic suprachiasmatic nuclei may represent the substrate for maternal melatonin to set the phase of the developing circadian timing system.
INTRODUCTION Circadian rhythms represent a mechanism which enables organisms to be adapted to the daily cycle of alternating light and dark. An important pacemaker which generates mammalian circadian rhythms is the suprachiasmatic nucleus (SCN) of the hypothalamus z6. The SCN exhibits circadian oscillations both in vivo ~ and in vitro 13'22'24. It also receives photic input, via both direct and indirect retinal pathways, which mediates entrainment to the ambient light/dark cycle 27. The SCN begins to function as a circadian pacemaker before birth 7'~'1°'34'35. In the fetus, the phase of this pacemaker is synchronized by the maternal circadian system, rather than directly by the environmental light/dark cycle 7'35. The nature of the maternal signal for setting the phase of the developing circadian pacemaker in offspring is unknown, but some evidence suggests that it may involve the hormone melatonin the
secretory pattern of which is regulated by the circadian system s'lS. In Syrian hamsters, melatonin administration affects the timing of the developing but not the mature circadian system. Injections of melatonin to pregnant Syrian hamsters (Mesocricetus auratus) lead to synchronization of the phases of the circadian rhythms of the pups, as assessed post-natally s. In contrast, administration of melatonin to adult Syrian hamsters is not effective for entraining the circadian timing system I . The prenatal sensitivity to melatonin injections suggests that the pups may respond in utero to maternal melatonin 8 which has been shown to cross the placenta ~7'33. However, it is not known if this entraining effect of melatonin is mediated by a direct effect on the fetus or indirectly through an action by other cyclic maternal systems. A critical test of the hypothesis that melatonin acts directly on the circadian pacemaker within the fetus is to determine whether melatonin
Correspondence." M.J. Duncan, Department of Anatomy and Neurobiology, University of Kentucky, Chandler Medical Center, 800 Rose Street, Lexington. KY 40536-0084, USA. Fax: (1) (606) 258 5946.
206 b i n d i n g sites are p r e s e n t within the fetal Syrian h a m ster SCN. A l t h o u g h specific b i n d i n g sites for 2[~251]iodomelatonin, a p o t e n t and selective r a d i o l i g a n d for m e l a t o n i n r e c e p t o r s ~1">, have b e e n i d e n t i f i e d within the fetal S C N of several species ~<5~'5~, an effect of
m e l a t o n i n on the fetal c i r c a d i a n timing system has only b e e n d e m o n s t r a t e d in Syrian h a m s t e r s s. D e s p i t e the e v i d e n c e for an effect of m e l a t o n i n in the fetus, the site(s) of action for m e l a t o n i n have not b e e n d e m o n s t r a t e d in fetal Syrian hamsters. T h e r e f o r e , the objec-
Fig. t. Coronal sections through the SCN of female Syrian hamsters. Panels A, C and E represent sections stained with thionin. Panels B, D and F represent autoradiograms of adjacent sections incubated with 2-[125I]iodomelatonin (115 pM). A and B represent E15. C and D represent PNI. E and F represent PN119. Sections representing non-specific binding (co-incubated with 1 /xM melatonin and 2-[12sI]iodomelatonin) are not shown; they exhibited labelling that was equivalent to tissue background (barely distinguishable from film background). Therefore, the dark labelling in the autoradiograms represents specific 2-[125I]iodomelatonin binding. SCN, suprachiasmatic nuclei; BSph, basisphenoid bone; PVT; paraventricular thalamic nuclei; CP, choroid plexus; HC, habenular complex. Bar = 1 ram.
2(17
tives o f t h e p r e s e n t s t u d i e s w e r e to i n v e s t i g a t e w h e t h e r 2-[12sI]iodomelatonin
MATERIALS AND METHODS
b i n d i n g sites are p r e s e n t in the
S C N d u r i n g t h e fetal p e r i o d a n d at later ages. T h e pars t u b e r a l i s o f the a d e n o h y p o p h y s i s
w a s also e x a m i n e d
b e c a u s e s p e c i f i c 2 - [ ~ 2 5 I ] i o d o m e l a t o n i n b i n d i n g sites are p r e s e n t in this r e g i o n in m a n y s p e c i e s , i n c l u d i n g a d u l t Syrian h a m s t e r s 9,12'2s'46,54"5~''6°.
Animals and tissue preparation
Female Syrian hamsters (LVG-COB, Charles River Laboratories, Wilmington, MA) were paired with males on the night of ovulation. The following day was considered embryonic day one (El). The day of birth, which corresponded to El6, was termed postnatal day one (PNI). Females of selected embryonic and postnatal ages were used
B
7
Fig. 2. Coronal sections through the pars tuberalis of female Syrian hamsters. Panels A, C and E represent sections stained with thionin. Panels B, D and F represent autoradiograms of adjacent sections incubated with 2-[ 12s"I]iodomelatonin (115 pM). A and B represent El5. (" and D represent PN1. E and F represent PN119. Sections representing non-specific binding (co-incubated with 1 ~,M melatonin) that was equivalent to tissue background (barely distinguishable from film background). Therefore, the dark labelling in the autoradingrams represents specific 2-[12sI]iodomelatonin binding. PT, pars tuberalis: BSph, basisphenoid bone. Bar = 1 mm.
208 in these studies, as described [br each experiment. Breeding pairs and experimental hamsters were exposed to a long day photoperiod (lights on from 08.00 to 22.00 h) and constant temperature (20 ± I°C). Food (Purina 5001) and water were available continuously. Hamsters were sacrificed by decapitation during the latter half of the light phase (approximately 15.00-17.00 h). Adult hamsters were sacrificed on the day after ovulation. In the case of animals two days of age or older, the brain was removed from the cranium and frozen by immersion in 2-methylbutane (approximately -20°C). In the case of younger animals, the whole head was frozen. Three alternating sets of coronal tissue sections (16 ~zm for localization studies and 20/~m for quantitative studies) through the diencephalon were cut on a cryostat, mounted on gelatin-coated slides and stored at -70°C before autoradiography was conducted.
Chemicals All chemicals were obtained from Sigma Chemical Company (St. Louis, MO) with the exception of 2-[125I]iodomelatonin which was synthesized using a method described previously 4~.
Autoradiography Slide-mounted tissue sections were thawed at 25°C in assay buffer (50 mM Tris-HCl with 4 mM CaCI 2 and 0.001% bovine serum albumin) for 15 min. Sections were incubated for 1 h at 25°C with 2-[125I]iodomelatonin ([Free] = 100-250 pM) dissolved in assay buffer in the absence (total binding) or presence (nonspecific binding) of 1 /xM melatonin. Sections were washed twice (10 min each) in ice-cold assay buffer, dipped in ice-cold distilled water for 3-5 s and allowed to dry. X-ray films (SB-5, Eastman Kodak, Rochester, NY) were exposed to the labelled tissue sections for four weeks, unless otherwise stated. One set of tissue sections was not incubated with 2-[IzsI]iodomelatonin but was stained with thionin instead. In order to identify the 2-[12511iodomelatonin labelled structures, the autoradiograms were superimposed over the adjacent thionin-stained section. Also, all of the thionin-stained slides were carefully examined and compared with pictures of rat and mouse brain and pituitary at various ages30-32,37.
TABLE l
Age-related changes m the regional distribution or' specific 2[ z~.lfiodomelatonm bmding sites Age
n
SCN
PT
El3 El4 El5 PN1 PN2 PNI0 PN12 PN25 PN112-PN133
3 6 3 8 4 3 3 3 12
0* 50 100 100 100 100 0 0 0
100 83 100 100 100 t00 100 100 100
* Values shown represent the percentage of animals in each group with specific 2-[12sI]iodomelatonin binding in a given region.
ied. Sections were incubated with one of six radioligand concentrations ranging from 13 to 420 pM. After incubation, washing and drying (as described above), the sections were apposed to X-ray films in cassettes that also contained radioactive standards ([125I]-Microscales; Amersham, Arlington Heights, IL). The exposure times for the X-ray films varied (2-8 weeks) among tissue sections exposed to different 2-[12sI]iodomelatonin concentrations. RESULTS
Experiment 1. Localization o f specific 2-[125I]iodome latonin binding sites in the S C N S p e c i f i c 2 - [ t 2 5 I ] i o d o m e l a t o n i n sites w e r e d e t e c t e d in t h e f e t a l S C N ( T a b l e I; Fig. 1). A l l o f t h e h a m s t e r s sacrificed on E15
had
specific 2-[125I]iodomelatonin
b i n d i n g sites in t h e S C N , c o m p a r e d
Data and statistical analyses
hamsters sacrificed on El4
For saturation studies, the amount of 2-[~zsI]iodomelatonin labelling in discrete regions was quantitated by computer-assisted densitometry (Imaging Research Inc., St. Catharines, Ontario, Canada) of the autoradiograms. Saturation analysis was conducted with the E B D A / L I G A N D computer program 2s's4. The values for specific binding, which were first calculated from the standard curve as dpm/mg plastic, were converted to fmol/mg protein as described previously 29. Differences in binding parameters between age groups were assessed with Bonferroni's t-test and were considered significant if P < 0.05.
ficed on El3.
Experiment 1. Localization of specific 2-[1251]iodomelatonin binding sites in the SCN This experiment investigated whether specific 2-[12sI]iodomelatonin binding sites are present in the SCN and/or pars tuberalis of embryonic, neonatal, prepubertal and adult hamsters. Brains from hamsters of various embryonic (E) or postnatal (PN) ages (El3, El4, El5, PNI, PN2, PNI2, PN25, and PNl12-133; n = 3-12 each) were examined.
Experiment 2. Saturation analysis of specific 2-[I251]iodomelatonin binding sites PN1 and PNll2 hamsters Saturation studies were conducted in order to determine whether the specific 2-[125Iliodomelatonin binding sites in the SCN and pars tuberalis represented high affinity melatonin binding sites and to further characterize them biochemicatly. Brain sections from neonatal (PNI; n = 4) rather than fetal hamsters were used because the neonatal SCN was neuroanatomically more distinct and therefore a greater number of sections of this structure could be identified and collected. Sections from adult brains (PN112; n = 6) were also stud-
to h a l f o f t h e
a n d n o n e o f t h o s e sacri-
Specific 2-[t25I]iodomelatonin
binding
sites w e r e also p r e s e n t in t h e S C N o f n e o n a t a l h a m sters (PN1
or PN2) but not older hamsters
(PN12,
P N 2 5 , P N 1 1 2 - P N 1 3 3 ) ( T a b l e I; Fig. 1). In c o n t r a s t to the SCN, specific 2-[12sI]iodomelatonin
b i n d i n g sites
w e r e p r e s e n t in t h e p a r s t u b e r a l i s at all a g e s e x a m i n e d ( T a b l e I; Fig. 2). T h e p a r a v e n t r i c u l a r t h a l a m i c n u c l e i a n d h a b e n u l a r c o m p l e x also e x h i b i t e d s p e c i f i c 2[ 1 2 5 I ] i o d o m e l a t o n i n b i n d i n g sites in m o s t h a m s t e r s , w i t h the exception of the El3 hamsters. In the fetuses and neonates,
specific 2-[tzsI]iodomelatonin
b i n d i n g sites
w e r e also p r e s e n t in t h e p a r s distalis. T i s s u e s e c t i o n s o f this r e g i o n w e r e n o t o b t a i n e d f r o m o l d e r h a m s t e r s .
Experiment 2. Saturation analysis o f specific 2[1251]iodomelatonin binding sites in PN1 and P N l t 2 hamsters S a t u r a t i o n s t u d i e s i n d i c a t e d t h a t t h e r e w e r e signific a n t l y ( P = 0.01) f e w e r s p e c i f i c 2 - [ 1 2 5 I ] i o d o m e l a t o n i n b i n d i n g sites in t h e p a r s t u b e r a l i s o f a d u l t s (Bm~ x = 3.37 + 0.29 f m o l / m g
p r o t e i n ; n = 6) t h a n in t h e p a r s t u b e r -
alis o f n e o n a t e s (Bma x = 4.98 + 0.10 f m o l / m g
protein;
n = 4) (Fig. 3). T h e r e w a s n o d i f f e r e n c e in t h e affinity
209
5
.2O
.15 '"',, ,, ,,,
°i: c~
30
1 ,'
[]
0 0 I00 200 300 400 2-[ ~251 ]-IOOOMELATONIN (pM)
i
i
i
1
2
3
,,
4
i
5
~
L
6
BOUND
Fig. 3. Saturation analysis of specific 2-[ 125I]iodomelatonin binding to the pars tuberalis of female Syrian hamsters. Values shown represent the m e a n _+S.E.M. Left, saturation isotherms of PNI (e: n = 4 ) or PN112 ( [] ; n = 6); right, Rosenthal (Scatchard) plots.
of the specific 2-[~25I]iodomelatonin binding sites between these ages (adults, K d = 14.0 + 2.9 pM; neonates, K d = 19.9 ± 2.9 pM; P = 0.21). Saturation studies (n = 4) also showed that the specific 2-[125I]iodomelatonin binding sites in the neonatal S e N were of high affinity ( K d = 65.3 ± 21.8 pM) but lower density ( B m a x = 1.53 ± 0.24 f m o l / m g protein) as compared with the specific 2-[~2sI]iodomelatonin binding sites in the neonatal pars tuberalis (see Fig. 4). Specific 2-[~2sI]iodomelatonin binding to the S e N was not detectable in any of the sections obtained from the adult hamsters. The limit of detectability of the autoradiography was approximately 0.15 f m o l / m g protein, based on the lowest detectable standard on the standard curve. DISCUSSION These studies demonstrated that high affinity, specific 2-[12sI]iodomelatonin binding sites are present in the SCN of fetal Syrian hamsters. These sites appear early in the development of the SCN, by E14-E15, thus shortly after the cessation of mitosis which is
2.0 020 15
~>~ i
1.0
x T
.015
c,_
g
x
.010
\.
0.5 ( ,,:'F pO0~
O.C ' ' ' .... 0 I00 200 300 400 2-[ ' 2~I]-IODOMELATONIN(pM)
,
, ~,~
,
0.0 0.5 1.0 1.5 2.0 BOUND(FMOLES/MGPROTEIN)
Fig. 4. Saturation analysis of specific 2-[12S• I]iodomelatonin binding to the SCN of PN1 hamsters. Values shown represent the mean _+S.E.M. (n = 4). Left, saturation isotherm; right, Rosenthal (Scatchard) plot.
complete by E l 3 ~. Results of tract-tracing studies have shown that retinohypothalamic projections reach the SCN during PN4-15 ~s. Therefore, the appearance of the specific 2-[l>I]iodomelatonin binding sites in the SCN precedes the time of innervation of the SCN by the retinohypothalamic tract ~. The ontogeny of specific 2-[125I]iodomelatonin binding sites in the SCN coincides with the time that circadian oscillations can be entrained by the maternal system 7. Previous studies have shown that mclatonin injections to the mother will set the phase of the fetal circadian timing system s'4'~. As few as four daily injections during E12-E15 are sufficient to set the phase s, and preliminary results indicate that a single injection on El5 is equally effective 4'). The potential effectiveness of single injections of melatonin earlier during gestation has not yet been investigated. The present results suggest that the entraining effect of exogenous melatonin administration is mediated by a direct effect of melatonin on the fetal SCN. These findings support the hypothesis that circulating melatonin levels in the pregnant hamster participate in the maternal entrainment of the developing circadian timing system s . Furthermore, the present findings that specific 2[J251]iodomelatonin binding sites are present in the SCN during PN 1-PN 10 would predict that the capacity for maternal entrainment by melatonin may continue into the early postnatal period. Melatonin may be transferred from lactating mothers to pups during the time before the neonatal pineal is secreting melatonin 41"4~. Recent studies have shown that 5- to 10-day-old suckling rat pups have day-night fluctuations in circulating melatonin levels, and that these fluctuations depend upon the maternal pineal gland 4s. The specific 2-[125I]iodomelatonin binding sites in the perinatal SCN decreased to undetectable levels before adulthood, This loss of specific 2-[]251]iodome latonin binding sites was apparently not mediated by pubertal hormonal changes because it occurcd by PN 12, i.e. well before the expected onset of puberty. The absence of specific 2-[leSl]iodomelatonin binding sites in the SCN of the eighteen adult female Syrian hamsters in the present studies are in contrast to previous studies localizing specific 2-[~-'5I]iodomelatonin binding sites to the SCN of adult male rats > 20.44,47,>7,5,, and adult Siberian male hamsters ~zSe'5~'. Although it is possible that the sex difference contributes to this discrepancy, there are also some striking species differences concerning responses to melatonin. In contrast to adult hamsters, the timing of the circadian system of adult rats can be entrained by melatonin administrationS4e: this effect in rats depends on thc intact SCN s. Melatonin has also been shown to reset circadian rhythms
210 measured in rat SCN explants in vitro:*. In Siberian hamsters, but not Syrian hamsters, the inhibitory effect of melatonin on the reproductive system requires the intact SCN 2'3"a~. Therefore, the specific 2-[12Sl]iodo melatonin binding sites in the SCN of adult rats or Siberian hamsters are probably involved in mediating the biological effect of melatonin. In adult Syrian hamsters, the apparent absence of specific 2-[~25I]iodome latonin binding sites in the SCN is consistent with previous findings in this species that melatonin injections do not entrain the adult circadian timing system ~ and that SCN lesions do not prevent melatonin induction of gonadal quiescence -~. The findings of the present studies concerning adult female Syrian hamsters are in contrast not only to findings in rats and Siberian hamsters but also to some studies localizing these binding sites in adult male Syrian hamsters s<~'°. However, there have also been reports that specific 2-[~25I]iodomelatonin binding sites are absent from the SCN of male Syrian hamsters 4~''55. The results of present and previous studies, taken together, suggest that specific 2-[J2sI]iodometatonin binding sites in the SCN are less abundant in adult hamsters relative to adult rats 4<55"s6'6°. It may be that potential effects of differences in sex, age, time of day of sacrifice, or subtle variations in assay procedures are more pronounced in studies of specific 2-[J25I]iodome latonin binding sites in the SCN of hamsters than in rats. Unlike the specific 2-[~25I]iodomelatonin binding sites in the SCN, the specific 2-[12-~I]iodomelatonin binding sites in the pars tuberalis of female Syrian hamsters were present by E l 3 and persisted throughout development. The affinity of the specific 2[~2sI]iodomelatonin binding sites in the pars tuberalis (K o approximately 20 pM) was the same at P N l l 2 as at PN1 but the density of these sites was 32% lower in the P N l l 2 hamsters. This finding suggests that some reduction of specific 2-[1251]iodomelatonin binding sites during postnatal development may be a general phenomenon. Indeed, rats exhibit age-related decreases in the density of specific 2-[~2Sl]iodomelatonin binding sites in the anterior pituitary gland 45 and in the anterior cerebral artery and the caudal artery 21, but not in the SCN or the area postrema z~. The complete loss of detectable specific 2-[~2sI]iodomelatonin binding sites from the SCN of hamsters in the present study, suggests a unique developmental role for melatonin in the SCN that is different from other areas. The time of appearance of specific 2-[~2sI]iodome latonin binding sites in Syrian hamsters is similar to that of Siberian hamsters and rats 4'ss. Siberian hamsters have been studied because photoperiodie infor-
mation perceived during the prenatal period aflecls the postnatal time of onset of puberty 14"";'4U':~tl'q3, and maternal circulating levels of melatonin arc inw)lved in this phenomenon u'5°'5~. In this species, specific 2[leSl]iodomelatonin binding sites were localized in the SCN, the pars tuberalis and other regions in embryos obtained four days before the expected day of birth 4. Furthermore, melatonin receptors in Siberian hamsters appear to be functional during the prenatal period, based on demonstrations that melatonin inhibited forskolin-stimulated cyclic A M P release from explants of the median e m i n e n c e / p a r s tuberatis in vitro 4. The presence of specific 2-[12sI]iodomelatonin binding sites in fetal Siberian hamsters suggested that melatonin may act directly on the fetus to influence postnatal reproductive development a. In rats, specific 2-[~e51] iodomelatonin binding sites have been localized to the SCN of embryos sacrificed on gestational day 18, i.e. three-four days before the expected day of birth 5s. The biological significance of the specific 2-[~25I]iodomela tonin binding sites in rat fetuses is not clear. In conclusion, these results report specific 2[12sI]iodomelatonin binding sites in female Syrian hamsters and additionally assess their presence during embryonic, neonatal, and prepubertal development and in the adult. These studies showed that specific 2[~25I]iodomelatonin binding sites appear during the fetal period; they are present as early as three days before birth (El3) in the pars tuberalis and by o n e - t w o days before birth (E14-E15) in the SCN. The biological significance of the specific 2-[~esl]iodomelatonin binding sites in the pars tuberalis of the fetus or hamsters of other ages is as yet unknown. The continued presence of these sites in this region throughout life suggests that they are important. The presence of specific 2-[125I]iodomelatonin binding sites in the fetal SCN suggests that the developing circadian timing system can respond directly to melatonin at a time when it is not receiving light information. Acknowledgements. The authors thank Lih Sia Mann, Karen Heller
and Kathrin Jaeck for technical assistance with experiments and Dr. Willis K. Paull for assistance with identification of fetal ueuroanatomical structures. We are grateful to Dr. Bruce Maley and Mary Gail Engle for photography. This work was supported by USPHS Grants DK-42056 (to MJ.D.) and HD-18686 (to F.C.D.) and by an award from the University of Missouri Medical School Research Council (to M.J.D.). Portions of this work were previously presented in abstract form: Soc. Neurosci. Abstr., 16 (19901 773 and Soc. Neurosci. Abstr., 17 (1991) 674. REFERENCES I Armstrong, S.M. and Redman, J., Melatonin administration: effects on rodent circadian rhythms. In: S. Clark (Ed.), Photoperiodism, Melatonin and the Pineal, Pitman, London, 1085; pp. 188-202. 2 Bartness, T.J., Goldman, B.D. and Bittman, E.L., SCN lesions
211 block responses to systemic melatonin infusions in Siberian hamsters, Am. J. Physiol., 260 (1991) R102-R112. 3 Bittman, E.L., Crandell, R.G. and Lehman, M.N., Influences of the paraventricular and suprachiasmatic nuclei and olfactory bulbs on melatonin responses in the golden hamster, Biol. Reproduction, 411 (1989) 118-126. 4 Carlson, L.L., Weaver, D.R. and Reppert, S.M., Melatonin receptors and signal transduction during development in Siberian hamsters (Phodopus sungorus), Dee. Brain Res., 59 (1991) 83-88. 5 Cassone, V.M., Chesworth, M.J. and Armstrong, S.M., Dose-dependent entrainment of rat circadian rhythms by daily injection of melatonin, J. Biol. Rhythms, 1 (1986) 219-229. 6 Davis, F.C., Boada, R. and LeDeaux, J., Neurogenesis of the hamster suprachiasmatic nucleus, Brain Res., 519 (1990) 192-199. 7 Davis, F.C. and Gorski, R.A., Development of hamster circadian rhythms: Role of the maternal suprachiasmatic nucleus, J. Comp. Physiol. A, 162 (1988) 601-610. 8 Davis, F.C. and Mannion, J., Entrainment of hamster pup circadian rhythms by prenatal melatonin injections to the mother, Am. J. Physiol., 255 (1988) R439 R448. 9 De Reviers, M.M., Ravault, J.P., Tillet, Y. and Pelletier, J., Melatonin binding sites in the sheep pars tuberalis, Neurosci. Lett., 100 (1989) 89-93. 10 Deguchi, T., Ontogenesis of a biological clock for serotonin acetyl coenzyme A N-acetyltransferase in pineal gland of rat, Proe. Natl. Acad. Sci. USA, 72 (1975) 2814-2818. 11 Dubocovich, M.L. and Takahashi, J.S., Use of 2-[125I]iodomelatonin to characterize melatonin binding sites in chicken retina, Proc. Natl. Acad. Sci. USA, 84 (1987) 3916-3920. 12 Duncan, M.J., Takahashi, J.S. and Dubocovich, M.L., Characteristics and autoradiographic localization of 2-[12Sl]iodomelatonin binding sites in Djungarian hamster brain, Endocrinolo©', 125 (1989) 1011-1118. 13 Earnest, D.J. and Sladek, C.D., Circadian rhythms of vasopressin release from individual rat suprachiasmatic explants in vitro. Brain Res., 382 11986) 129-133. 14 Horton, T.H., Ray, S.L. and Stetson, M.H., Maternal transfer of photoperiodic information in Siberian hamsters. III. Melatonin injections program postnatal reproductive development expressed in constant light, Biol. Reproduction, 41 (1989) 34 39. 15 Illnerova, H., Hoffmann, K. and Vanecek, J., Adjustment of pineal melatonin and N-acetyltransferase rhythms to change from long to short photoperod in the Djungarian hamster, Phodopus sungorus, NeuroendocrinologT, 38 (1984) 226-231. 16 Inouye, S.T. and Kawamura, H., Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus, ,°roe. Natl. Acad. Sci. USA, 76 (19791 5962-5966. 17 Klein, D.C., Evidence for the placental transfer of 3H-acetylmelatonin, Nature, 237 119721 117-118. 18 Laitinen, J.T., Castren, E., Vakkuri, O. and Saavedra, J.M., Diurnal rhythm of melatonin binding in the rat suprachiasmatic nucleus, Endocrinology,'3', 124 (19891 1585-1587. 19 Laitinen, J.T., Flugge, G. and Saavedra, J.M., Characterization of melatonin receptors in the rat area postrema: Modulation of affinity with cations and guanine nucleotides, Neuroendoerinology, 51 (19901 619-624. 211 Laitinen, J.T. and Saavedra, J.M., Characterization of melatonin receptors in the rat suprachiasmatic nuclei: Modulation of affinity with cations and guanine nucleotides, Endocrinology, 126 (19911) 2110-2115. 21 Laitinen, J.T., Viswanathan, M., Vakkuri, O. and Saavedra, J.M., Differential regulation of the rat melatonin receptors: Selective age-associated decline and lack of melatonin-induced changes, Endocrinolog3', 130 (1992) 2139-2144. 22 Margraf, R.R., Zlomanczuk, P., Liskin, L.A. and Lynch, G.R., Circadian differences in neuronal activity of the suprachiasmatic nucleus in brain slices prepared form photo-responsive and photo-non-responsive Djungarian hamsters, Brain Res., 544 ( 1991 ) 42-48. 23 Maywood, E.S., Buttery, R.C., Vance, G.H.S., Herbert, J. and Hastings, M.H., Gonadal responses of the male Syrian hamster to
programmed infusions of melatonin are sensitive to signal duration and frequency but not to signal phase nor to lesions of the suprachiasmatic nuclei, Biol. Reproduction, 43 (199//) 174-182. 24 McArthur, A.J., Gillette, M,U. and Prosser, R.A., Melatonin directly resets the rat supraehiasmatic circadian clock in vitro, Brain Res., 565 (19911 158-161. 25 McPherson, G.A., A practical computer-based approach to the analysis of radioligand binding experiments, Comp. Prog. Biomed., 17(19921 107 115. 26 Meijer, J.H. and Rietveld, W.J., Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Ph3siol. Rer., 69 (1989) 671-707. 27 Moore, R.Y. and Lenn, N.Y., A retinohypothalamic projection in the rat, ,L Comp. Neurol., 146 (19721 1-14. 28 Morgan, P.J., Williams, L.M., Davidson, G.. Lawson, W. and Howell, E., Melatonin receptors on ovinc pars tuberalis: Characterization and autoradiographical localization, J. Neuroendoerinol., 1 (1989) 1 4. 29 Nazarali, A.J., Gutkind, J.S. and Saavedra, J.M., Calibration of 125I-polymer standards with t25I-brain paste standards for use in quantitative receptor autoradiography, .1. Neurosci. Methods, 30 ( 19891 247-253. 30 Paull, W.K. A light and eleetron study of the derelopment ~f the neurohypophysis of the fetal rat, 1'473, Ph.D. Dissertation, Univcr sity of Southern California. 31 Paxinos, G.. Tork, I., Tecott, L.H. and Valentino, K.L. Atlas ~l the Deceloping Rat Brain, Academic Press, San Diego. 1991. 32 Paxinos, G. and Watson, C. The Rat Bruin in Stereotaxic Coordinates, Academic Press, San Diego, 1986. 33 Reppert, S.M., Chez, R.A., Anderson, A. and Klein, D.C., Maternal-fetal transfer of melatonin in the non-human primate, Pediatric Res., 13 (1979) 788-791. 34 Reppert, S.M. and Schwartz, W.J., The suprachiasmatic nuclei of the fetal rat: Characterization of a functional circadian clock using laC-labeled deoxyglucose, .I. Neurosei, 4 (19841 1677 1682. 35 Reppert, S.M., Weaver, D.R. and Rivkccs, S.A.. Prenatal ftn]ction and entrainment of a circadian clock. In: S.M. Rcppcrt (Ed.) Research in Perinatal Medicine. lqd. L~L Decelopment of Circadian Rhythmicity and Photoperiodism in Mamma£, Perinatology Press, 1989, pp. 25-44. 36 Reppert, S.M., Weaver, D.R.. Rivkees, S.A. and Slopa, E.(}., Putative melatonin receptors in a human biological clock, Scienee, 242 11988) 78-81. 37 Schambra, U.B., Lauder, J.M. and Silver, J., Atlas of the Prenatal Mouse Brain, Academic Press, San Diego, 1992. 325 pp. 38 Speh, J.C. and Moore, R.Y., Synaptogenesis and retinohypothalamic tract (RHT) development in the hamster suprachiasmatic nucleus (SCN), Soc. Neurosei. Ahstr., 16 (19911) 6112. (Abstract) 39 Stetson, M.H., Elliot, J.A. and Goldman, B.D., Maternal transfer of photoperiodic information influences the photoperiodic response of prepubertal l)jungarian hamster (Phodopus sungoru.~ stmgorus), Biol. Reprod., 34 11986)664 669. 40 Stetson, M.H., Ray, S.L., Creyaufmiller, N. and Hortou, T.tI.. Maternal transfer of photoperk)dic information in Siberian hamsters. II. The nature of the maternal signal, time of signal transfer, and the effect of the maternal signal on peripubcrtal reproductive development in the absencc of photoperiodic input. Biol. Reprod., 40 (19891 458-465. 41 Tamarkin, L., Reppert, S.M., Orloff, D.J., Klein, D.('., Yclhm, S.M. and Goldman, B.D., Ontogeny of the pineal melatonin rhythm in the Syrian (Mesocricetus aurutliS) and Siberian (Phodopus sungorus) hamsters and in the rat, Endoerimdogy, 1117 (1980) 1(161 11164. 42 Thomas, E.M.V. and Armstrong, S.M., Mclatonin administration entrains female rat activity rhythms in constant darkness but not in constant light, Am. ,L Physiol., 255 (1988) R237 R242. 43 Vakkuri, O., Leppaluota, J. and Vuoltcenaho, O., Development and validation of a melatonin radioimmunoassay using radioiodihated melatonin as tracer, Aetu Emloermol. (CopenhagenL 106 (1984) 152-157. 44 Vanecek, J., Melatonin binding sites, ,I. Neurochem., 51 (1988) 1436-1440.
212 45 Vanecek, J., The melatonin receptors in rat ontogenesis, Neuroen&~crinology, 48 (1988) 201-203. 46 Vanecek, J. and Jansky, L., Short days induce cbanges in specific melatonin binding in hamster median eminence and anterior pituitary, Brain Res., 477 (1989) 387-39(I. 47 Vanecek, J., Pavlik, A. and lllnerova, H., Hypothalamic melatonin receptor sites revealed by autoradiography, Brain Res., 435 (1987) 359 362. 48 Velazquez, E., Esquifino, A.I., Zueco, J.A., Albusac, J.M.R. and Blazquez, E., Evidence that circadian variations of circulating melatonin levels in fetal and suckling rats are dependent on maternal melatonin transfer, Neuroendocrmolo~', 55 (1992) 32132~. 49 Viswanathan, N. and Davis, F.C., Melatonin sets the phase of the developing circadian rhythm in Syrian hamsters, Soc. Res. Biol. Rhythms Abstr.,(1992) 95. (Abstract) 50 Weaver, D.R., Keohan, J.T. and Reppert, S.M., Definition of a prenatal sensitive period for maternal-fetal communication of daylength, Am. Y. Physiol., 253 (1987) E701-E704. 51 Weaver, D.R., Namboodiri, M.A.A. and Reppert, S.M., Iodinated melatonin mimics melatonin action and reveals discrete binding sites in fetal brain, FEBS Lett., I (1988) 123-127. 52 Weaver, D.R., Provencio, I., Carlson, L.L. and Reppert, S.M., Melatonin receptors and signal transduction in photorefractory Siberia n hamsters ( Phodopus sungorus ), Endocrinology', 128 ( 1991 ) 1086-1092. 53 Weaver, D.R. and Reppert, S.M., Maternal melatonin communi-
54
55
56
57
58
59
6(I
cates daylength to the fetus in l))ungarian hamster. ::mtocrimd o:,'3', 119 (1986) 2861-2863. Weaver, D.R. and Reppert, S.M.. Melatonin receptors are present in the ferret pars tuberalis and pars distalis, but not in brain, Endocrinology, 127 (1990) 26(/7-2609. Weaver, D.R., Rivkees, S.A. and Reppert, S.M., Autoradiographic localization of iodomelatonin binding sites in hypothalamus of three rodent species, Abstracts of the 70th Annual Meeting of the Endocrine Society, 1988, p. 265. Weaver, D.R., Rivkees, S.A. and Reppert, S.M., Localization and characterization of melatonin receptors in rodent brain by m vitro autoradiography, J. Neurosci., 9 (1989) 2581-2590. Williams, L.M., Melatonin-binding sites in the rat brain and pituitary mapped by in-vitro autoradiography, J. Mol. Endoerinol., 3 (1989) 71-75. Williams, L.M., Martinoli, M.G., Titchener, L.T. and Pelletier, G., The ontogeny of central melatonin binding sites in the rat, Endocrinolog.,y, 128 (1991) 2083-2090. Williams, L.M. and Morgan, P.J., Demonstration of melatoninbinding sites on the pars tuberalis of the rat, .Z Endocrinol., 119 (1988) R1-R3. Williams, L.M., Morgan, P.J., Hastings, M.H., Lawson, W., Davidson, G. and Howell, H.E., Melatonin receptor sites in the Syrian hamster brain and pituitary. Localization and characterization using [125I]iodomelatonin, J. Neuroendocrinol., I (1989) 315320.
Developmental Brain Research, 73 (1993) 205-212 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00
BRESD 51634
Developmental appearance and age related changes in specific 2-[125I]iodomelatonin binding sites in the suprachiasmatic nuclei of female Syrian hamsters Marilyn J. D u n c a n a,b a n d F r e d e r i c k C. Davis c Department of Anatomy and Neurobiology, University of Missouri Medical School, Columbia, MO 65212 (USA), ~' Department of Anatomy and Neurobiology, Uni~ersity of Kentucky, Chandler Medical Center, Lexington, KY 40536-0084 (USA) and ' Department of Biology, Northeastern Unit'ersity, Boston, MA 02115 (USA) (Accepted 22 December 1992)
Key words." Melatonin; Suprachiasmatic nucleus; Pars tuberalis; Circadian timing system; Pineal
In Syrian hamsters, the circadian timing system is sensitive to melatonin during gestation but is not responsive in the adult. In order to further understand this developmental change in melatonin responsiveness, in vitro autoradiography was used to assess the presence of specific 2-[125I]iodomelatonin binding sites in the suprachiasmatic nuclei of female hamsters of selected embryonic (E) and postnatal (PN) ages (e.g. El3, El4, El5, PNI, PN2, PN12, PN25, PN112-133). Specific 2-[lZSI]iodomelatonin binding sites were seen in the suprachiasmatic nuclei of some of the El4 hamsters and all the perinatal hamsters (El5, PN1 and PN2) but not in older hamsters. In contrast, specific 2-[125I]iodomelatonin binding sites were seen in the pars tuberalis of all hamsters (with the exception of one), regardless of age. The transient expression of specific 2-[1251]iodomelatonin binding sites in the suprachiasmatic nuclei suggests that melatonin may have some special functions restricted to early development. The specific 2-[125I]iodomelatonin binding sites in the embryonic suprachiasmatic nuclei may represent the substrate for maternal melatonin to set the phase of the developing circadian timing system.
INTRODUCTION Circadian rhythms represent a mechanism which enables organisms to be adapted to the daily cycle of alternating light and dark. An important pacemaker which generates mammalian circadian rhythms is the suprachiasmatic nucleus (SCN) of the hypothalamus z6. The SCN exhibits circadian oscillations both in vivo ~ and in vitro 13'22'24. It also receives photic input, via both direct and indirect retinal pathways, which mediates entrainment to the ambient light/dark cycle 27. The SCN begins to function as a circadian pacemaker before birth 7'~'1°'34'35. In the fetus, the phase of this pacemaker is synchronized by the maternal circadian system, rather than directly by the environmental light/dark cycle 7'35. The nature of the maternal signal for setting the phase of the developing circadian pacemaker in offspring is unknown, but some evidence suggests that it may involve the hormone melatonin the
secretory pattern of which is regulated by the circadian system s'lS. In Syrian hamsters, melatonin administration affects the timing of the developing but not the mature circadian system. Injections of melatonin to pregnant Syrian hamsters (Mesocricetus auratus) lead to synchronization of the phases of the circadian rhythms of the pups, as assessed post-natally s. In contrast, administration of melatonin to adult Syrian hamsters is not effective for entraining the circadian timing system I . The prenatal sensitivity to melatonin injections suggests that the pups may respond in utero to maternal melatonin 8 which has been shown to cross the placenta ~7'33. However, it is not known if this entraining effect of melatonin is mediated by a direct effect on the fetus or indirectly through an action by other cyclic maternal systems. A critical test of the hypothesis that melatonin acts directly on the circadian pacemaker within the fetus is to determine whether melatonin
Correspondence." M.J. Duncan, Department of Anatomy and Neurobiology, University of Kentucky, Chandler Medical Center, 800 Rose Street, Lexington. KY 40536-0084, USA. Fax: (1) (606) 258 5946.
206 b i n d i n g sites are p r e s e n t within the fetal Syrian h a m ster SCN. A l t h o u g h specific b i n d i n g sites for 2[~251]iodomelatonin, a p o t e n t and selective r a d i o l i g a n d for m e l a t o n i n r e c e p t o r s ~1">, have b e e n i d e n t i f i e d within the fetal S C N of several species ~<5~'5~, an effect of
m e l a t o n i n on the fetal c i r c a d i a n timing system has only b e e n d e m o n s t r a t e d in Syrian h a m s t e r s s. D e s p i t e the e v i d e n c e for an effect of m e l a t o n i n in the fetus, the site(s) of action for m e l a t o n i n have not b e e n d e m o n s t r a t e d in fetal Syrian hamsters. T h e r e f o r e , the objec-
Fig. t. Coronal sections through the SCN of female Syrian hamsters. Panels A, C and E represent sections stained with thionin. Panels B, D and F represent autoradiograms of adjacent sections incubated with 2-[125I]iodomelatonin (115 pM). A and B represent E15. C and D represent PNI. E and F represent PN119. Sections representing non-specific binding (co-incubated with 1 /xM melatonin and 2-[12sI]iodomelatonin) are not shown; they exhibited labelling that was equivalent to tissue background (barely distinguishable from film background). Therefore, the dark labelling in the autoradiograms represents specific 2-[125I]iodomelatonin binding. SCN, suprachiasmatic nuclei; BSph, basisphenoid bone; PVT; paraventricular thalamic nuclei; CP, choroid plexus; HC, habenular complex. Bar = 1 ram.
2(17
tives o f t h e p r e s e n t s t u d i e s w e r e to i n v e s t i g a t e w h e t h e r 2-[12sI]iodomelatonin
MATERIALS AND METHODS
b i n d i n g sites are p r e s e n t in the
S C N d u r i n g t h e fetal p e r i o d a n d at later ages. T h e pars t u b e r a l i s o f the a d e n o h y p o p h y s i s
w a s also e x a m i n e d
b e c a u s e s p e c i f i c 2 - [ ~ 2 5 I ] i o d o m e l a t o n i n b i n d i n g sites are p r e s e n t in this r e g i o n in m a n y s p e c i e s , i n c l u d i n g a d u l t Syrian h a m s t e r s 9,12'2s'46,54"5~''6°.
Animals and tissue preparation
Female Syrian hamsters (LVG-COB, Charles River Laboratories, Wilmington, MA) were paired with males on the night of ovulation. The following day was considered embryonic day one (El). The day of birth, which corresponded to El6, was termed postnatal day one (PNI). Females of selected embryonic and postnatal ages were used
B
7
Fig. 2. Coronal sections through the pars tuberalis of female Syrian hamsters. Panels A, C and E represent sections stained with thionin. Panels B, D and F represent autoradiograms of adjacent sections incubated with 2-[ 12s"I]iodomelatonin (115 pM). A and B represent El5. (" and D represent PN1. E and F represent PN119. Sections representing non-specific binding (co-incubated with 1 ~,M melatonin) that was equivalent to tissue background (barely distinguishable from film background). Therefore, the dark labelling in the autoradingrams represents specific 2-[12sI]iodomelatonin binding. PT, pars tuberalis: BSph, basisphenoid bone. Bar = 1 mm.
208 in these studies, as described [br each experiment. Breeding pairs and experimental hamsters were exposed to a long day photoperiod (lights on from 08.00 to 22.00 h) and constant temperature (20 ± I°C). Food (Purina 5001) and water were available continuously. Hamsters were sacrificed by decapitation during the latter half of the light phase (approximately 15.00-17.00 h). Adult hamsters were sacrificed on the day after ovulation. In the case of animals two days of age or older, the brain was removed from the cranium and frozen by immersion in 2-methylbutane (approximately -20°C). In the case of younger animals, the whole head was frozen. Three alternating sets of coronal tissue sections (16 ~zm for localization studies and 20/~m for quantitative studies) through the diencephalon were cut on a cryostat, mounted on gelatin-coated slides and stored at -70°C before autoradiography was conducted.
Chemicals All chemicals were obtained from Sigma Chemical Company (St. Louis, MO) with the exception of 2-[125I]iodomelatonin which was synthesized using a method described previously 4~.
Autoradiography Slide-mounted tissue sections were thawed at 25°C in assay buffer (50 mM Tris-HCl with 4 mM CaCI 2 and 0.001% bovine serum albumin) for 15 min. Sections were incubated for 1 h at 25°C with 2-[125I]iodomelatonin ([Free] = 100-250 pM) dissolved in assay buffer in the absence (total binding) or presence (nonspecific binding) of 1 /xM melatonin. Sections were washed twice (10 min each) in ice-cold assay buffer, dipped in ice-cold distilled water for 3-5 s and allowed to dry. X-ray films (SB-5, Eastman Kodak, Rochester, NY) were exposed to the labelled tissue sections for four weeks, unless otherwise stated. One set of tissue sections was not incubated with 2-[IzsI]iodomelatonin but was stained with thionin instead. In order to identify the 2-[12511iodomelatonin labelled structures, the autoradiograms were superimposed over the adjacent thionin-stained section. Also, all of the thionin-stained slides were carefully examined and compared with pictures of rat and mouse brain and pituitary at various ages30-32,37.
TABLE l
Age-related changes m the regional distribution or' specific 2[ z~.lfiodomelatonm bmding sites Age
n
SCN
PT
El3 El4 El5 PN1 PN2 PNI0 PN12 PN25 PN112-PN133
3 6 3 8 4 3 3 3 12
0* 50 100 100 100 100 0 0 0
100 83 100 100 100 t00 100 100 100
* Values shown represent the percentage of animals in each group with specific 2-[12sI]iodomelatonin binding in a given region.
ied. Sections were incubated with one of six radioligand concentrations ranging from 13 to 420 pM. After incubation, washing and drying (as described above), the sections were apposed to X-ray films in cassettes that also contained radioactive standards ([125I]-Microscales; Amersham, Arlington Heights, IL). The exposure times for the X-ray films varied (2-8 weeks) among tissue sections exposed to different 2-[12sI]iodomelatonin concentrations. RESULTS
Experiment 1. Localization o f specific 2-[125I]iodome latonin binding sites in the S C N S p e c i f i c 2 - [ t 2 5 I ] i o d o m e l a t o n i n sites w e r e d e t e c t e d in t h e f e t a l S C N ( T a b l e I; Fig. 1). A l l o f t h e h a m s t e r s sacrificed on E15
had
specific 2-[125I]iodomelatonin
b i n d i n g sites in t h e S C N , c o m p a r e d
Data and statistical analyses
hamsters sacrificed on El4
For saturation studies, the amount of 2-[~zsI]iodomelatonin labelling in discrete regions was quantitated by computer-assisted densitometry (Imaging Research Inc., St. Catharines, Ontario, Canada) of the autoradiograms. Saturation analysis was conducted with the E B D A / L I G A N D computer program 2s's4. The values for specific binding, which were first calculated from the standard curve as dpm/mg plastic, were converted to fmol/mg protein as described previously 29. Differences in binding parameters between age groups were assessed with Bonferroni's t-test and were considered significant if P < 0.05.
ficed on El3.
Experiment 1. Localization of specific 2-[1251]iodomelatonin binding sites in the SCN This experiment investigated whether specific 2-[12sI]iodomelatonin binding sites are present in the SCN and/or pars tuberalis of embryonic, neonatal, prepubertal and adult hamsters. Brains from hamsters of various embryonic (E) or postnatal (PN) ages (El3, El4, El5, PNI, PN2, PNI2, PN25, and PNl12-133; n = 3-12 each) were examined.
Experiment 2. Saturation analysis of specific 2-[I251]iodomelatonin binding sites PN1 and PNll2 hamsters Saturation studies were conducted in order to determine whether the specific 2-[125Iliodomelatonin binding sites in the SCN and pars tuberalis represented high affinity melatonin binding sites and to further characterize them biochemicatly. Brain sections from neonatal (PNI; n = 4) rather than fetal hamsters were used because the neonatal SCN was neuroanatomically more distinct and therefore a greater number of sections of this structure could be identified and collected. Sections from adult brains (PN112; n = 6) were also stud-
to h a l f o f t h e
a n d n o n e o f t h o s e sacri-
Specific 2-[t25I]iodomelatonin
binding
sites w e r e also p r e s e n t in t h e S C N o f n e o n a t a l h a m sters (PN1
or PN2) but not older hamsters
(PN12,
P N 2 5 , P N 1 1 2 - P N 1 3 3 ) ( T a b l e I; Fig. 1). In c o n t r a s t to the SCN, specific 2-[12sI]iodomelatonin
b i n d i n g sites
w e r e p r e s e n t in t h e p a r s t u b e r a l i s at all a g e s e x a m i n e d ( T a b l e I; Fig. 2). T h e p a r a v e n t r i c u l a r t h a l a m i c n u c l e i a n d h a b e n u l a r c o m p l e x also e x h i b i t e d s p e c i f i c 2[ 1 2 5 I ] i o d o m e l a t o n i n b i n d i n g sites in m o s t h a m s t e r s , w i t h the exception of the El3 hamsters. In the fetuses and neonates,
specific 2-[tzsI]iodomelatonin
b i n d i n g sites
w e r e also p r e s e n t in t h e p a r s distalis. T i s s u e s e c t i o n s o f this r e g i o n w e r e n o t o b t a i n e d f r o m o l d e r h a m s t e r s .
Experiment 2. Saturation analysis o f specific 2[1251]iodomelatonin binding sites in PN1 and P N l t 2 hamsters S a t u r a t i o n s t u d i e s i n d i c a t e d t h a t t h e r e w e r e signific a n t l y ( P = 0.01) f e w e r s p e c i f i c 2 - [ 1 2 5 I ] i o d o m e l a t o n i n b i n d i n g sites in t h e p a r s t u b e r a l i s o f a d u l t s (Bm~ x = 3.37 + 0.29 f m o l / m g
p r o t e i n ; n = 6) t h a n in t h e p a r s t u b e r -
alis o f n e o n a t e s (Bma x = 4.98 + 0.10 f m o l / m g
protein;
n = 4) (Fig. 3). T h e r e w a s n o d i f f e r e n c e in t h e affinity
209
5
.2O
.15 '"',, ,, ,,,
°i: c~
30
1 ,'
[]
0 0 I00 200 300 400 2-[ ~251 ]-IOOOMELATONIN (pM)
i
i
i
1
2
3
,,
4
i
5
~
L
6
BOUND
Fig. 3. Saturation analysis of specific 2-[ 125I]iodomelatonin binding to the pars tuberalis of female Syrian hamsters. Values shown represent the m e a n _+S.E.M. Left, saturation isotherms of PNI (e: n = 4 ) or PN112 ( [] ; n = 6); right, Rosenthal (Scatchard) plots.
of the specific 2-[~25I]iodomelatonin binding sites between these ages (adults, K d = 14.0 + 2.9 pM; neonates, K d = 19.9 ± 2.9 pM; P = 0.21). Saturation studies (n = 4) also showed that the specific 2-[125I]iodomelatonin binding sites in the neonatal S e N were of high affinity ( K d = 65.3 ± 21.8 pM) but lower density ( B m a x = 1.53 ± 0.24 f m o l / m g protein) as compared with the specific 2-[~2sI]iodomelatonin binding sites in the neonatal pars tuberalis (see Fig. 4). Specific 2-[~2sI]iodomelatonin binding to the S e N was not detectable in any of the sections obtained from the adult hamsters. The limit of detectability of the autoradiography was approximately 0.15 f m o l / m g protein, based on the lowest detectable standard on the standard curve. DISCUSSION These studies demonstrated that high affinity, specific 2-[12sI]iodomelatonin binding sites are present in the SCN of fetal Syrian hamsters. These sites appear early in the development of the SCN, by E14-E15, thus shortly after the cessation of mitosis which is
2.0 020 15
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1.0
x T
.015
c,_
g
x
.010
\.
0.5 ( ,,:'F pO0~
O.C ' ' ' .... 0 I00 200 300 400 2-[ ' 2~I]-IODOMELATONIN(pM)
,
, ~,~
,
0.0 0.5 1.0 1.5 2.0 BOUND(FMOLES/MGPROTEIN)
Fig. 4. Saturation analysis of specific 2-[12S• I]iodomelatonin binding to the SCN of PN1 hamsters. Values shown represent the mean _+S.E.M. (n = 4). Left, saturation isotherm; right, Rosenthal (Scatchard) plot.
complete by E l 3 ~. Results of tract-tracing studies have shown that retinohypothalamic projections reach the SCN during PN4-15 ~s. Therefore, the appearance of the specific 2-[l>I]iodomelatonin binding sites in the SCN precedes the time of innervation of the SCN by the retinohypothalamic tract ~. The ontogeny of specific 2-[125I]iodomelatonin binding sites in the SCN coincides with the time that circadian oscillations can be entrained by the maternal system 7. Previous studies have shown that mclatonin injections to the mother will set the phase of the fetal circadian timing system s'4'~. As few as four daily injections during E12-E15 are sufficient to set the phase s, and preliminary results indicate that a single injection on El5 is equally effective 4'). The potential effectiveness of single injections of melatonin earlier during gestation has not yet been investigated. The present results suggest that the entraining effect of exogenous melatonin administration is mediated by a direct effect of melatonin on the fetal SCN. These findings support the hypothesis that circulating melatonin levels in the pregnant hamster participate in the maternal entrainment of the developing circadian timing system s . Furthermore, the present findings that specific 2[J251]iodomelatonin binding sites are present in the SCN during PN 1-PN 10 would predict that the capacity for maternal entrainment by melatonin may continue into the early postnatal period. Melatonin may be transferred from lactating mothers to pups during the time before the neonatal pineal is secreting melatonin 41"4~. Recent studies have shown that 5- to 10-day-old suckling rat pups have day-night fluctuations in circulating melatonin levels, and that these fluctuations depend upon the maternal pineal gland 4s. The specific 2-[125I]iodomelatonin binding sites in the perinatal SCN decreased to undetectable levels before adulthood, This loss of specific 2-[]251]iodome latonin binding sites was apparently not mediated by pubertal hormonal changes because it occurcd by PN 12, i.e. well before the expected onset of puberty. The absence of specific 2-[leSl]iodomelatonin binding sites in the SCN of the eighteen adult female Syrian hamsters in the present studies are in contrast to previous studies localizing specific 2-[~-'5I]iodomelatonin binding sites to the SCN of adult male rats > 20.44,47,>7,5,, and adult Siberian male hamsters ~zSe'5~'. Although it is possible that the sex difference contributes to this discrepancy, there are also some striking species differences concerning responses to melatonin. In contrast to adult hamsters, the timing of the circadian system of adult rats can be entrained by melatonin administrationS4e: this effect in rats depends on thc intact SCN s. Melatonin has also been shown to reset circadian rhythms
210 measured in rat SCN explants in vitro:*. In Siberian hamsters, but not Syrian hamsters, the inhibitory effect of melatonin on the reproductive system requires the intact SCN 2'3"a~. Therefore, the specific 2-[12Sl]iodo melatonin binding sites in the SCN of adult rats or Siberian hamsters are probably involved in mediating the biological effect of melatonin. In adult Syrian hamsters, the apparent absence of specific 2-[~25I]iodome latonin binding sites in the SCN is consistent with previous findings in this species that melatonin injections do not entrain the adult circadian timing system ~ and that SCN lesions do not prevent melatonin induction of gonadal quiescence -~. The findings of the present studies concerning adult female Syrian hamsters are in contrast not only to findings in rats and Siberian hamsters but also to some studies localizing these binding sites in adult male Syrian hamsters s<~'°. However, there have also been reports that specific 2-[~25I]iodomelatonin binding sites are absent from the SCN of male Syrian hamsters 4~''55. The results of present and previous studies, taken together, suggest that specific 2-[J2sI]iodometatonin binding sites in the SCN are less abundant in adult hamsters relative to adult rats 4<55"s6'6°. It may be that potential effects of differences in sex, age, time of day of sacrifice, or subtle variations in assay procedures are more pronounced in studies of specific 2-[J25I]iodome latonin binding sites in the SCN of hamsters than in rats. Unlike the specific 2-[~25I]iodomelatonin binding sites in the SCN, the specific 2-[12-~I]iodomelatonin binding sites in the pars tuberalis of female Syrian hamsters were present by E l 3 and persisted throughout development. The affinity of the specific 2[~2sI]iodomelatonin binding sites in the pars tuberalis (K o approximately 20 pM) was the same at P N l l 2 as at PN1 but the density of these sites was 32% lower in the P N l l 2 hamsters. This finding suggests that some reduction of specific 2-[1251]iodomelatonin binding sites during postnatal development may be a general phenomenon. Indeed, rats exhibit age-related decreases in the density of specific 2-[~2Sl]iodomelatonin binding sites in the anterior pituitary gland 45 and in the anterior cerebral artery and the caudal artery 21, but not in the SCN or the area postrema z~. The complete loss of detectable specific 2-[~2sI]iodomelatonin binding sites from the SCN of hamsters in the present study, suggests a unique developmental role for melatonin in the SCN that is different from other areas. The time of appearance of specific 2-[~2sI]iodome latonin binding sites in Syrian hamsters is similar to that of Siberian hamsters and rats 4'ss. Siberian hamsters have been studied because photoperiodie infor-
mation perceived during the prenatal period aflecls the postnatal time of onset of puberty 14"";'4U':~tl'q3, and maternal circulating levels of melatonin arc inw)lved in this phenomenon u'5°'5~. In this species, specific 2[leSl]iodomelatonin binding sites were localized in the SCN, the pars tuberalis and other regions in embryos obtained four days before the expected day of birth 4. Furthermore, melatonin receptors in Siberian hamsters appear to be functional during the prenatal period, based on demonstrations that melatonin inhibited forskolin-stimulated cyclic A M P release from explants of the median e m i n e n c e / p a r s tuberatis in vitro 4. The presence of specific 2-[12sI]iodomelatonin binding sites in fetal Siberian hamsters suggested that melatonin may act directly on the fetus to influence postnatal reproductive development a. In rats, specific 2-[~e51] iodomelatonin binding sites have been localized to the SCN of embryos sacrificed on gestational day 18, i.e. three-four days before the expected day of birth 5s. The biological significance of the specific 2-[~25I]iodomela tonin binding sites in rat fetuses is not clear. In conclusion, these results report specific 2[12sI]iodomelatonin binding sites in female Syrian hamsters and additionally assess their presence during embryonic, neonatal, and prepubertal development and in the adult. These studies showed that specific 2[~25I]iodomelatonin binding sites appear during the fetal period; they are present as early as three days before birth (El3) in the pars tuberalis and by o n e - t w o days before birth (E14-E15) in the SCN. The biological significance of the specific 2-[~esl]iodomelatonin binding sites in the pars tuberalis of the fetus or hamsters of other ages is as yet unknown. The continued presence of these sites in this region throughout life suggests that they are important. The presence of specific 2-[125I]iodomelatonin binding sites in the fetal SCN suggests that the developing circadian timing system can respond directly to melatonin at a time when it is not receiving light information. Acknowledgements. The authors thank Lih Sia Mann, Karen Heller
and Kathrin Jaeck for technical assistance with experiments and Dr. Willis K. Paull for assistance with identification of fetal ueuroanatomical structures. We are grateful to Dr. Bruce Maley and Mary Gail Engle for photography. This work was supported by USPHS Grants DK-42056 (to MJ.D.) and HD-18686 (to F.C.D.) and by an award from the University of Missouri Medical School Research Council (to M.J.D.). Portions of this work were previously presented in abstract form: Soc. Neurosci. Abstr., 16 (19901 773 and Soc. Neurosci. Abstr., 17 (1991) 674. REFERENCES I Armstrong, S.M. and Redman, J., Melatonin administration: effects on rodent circadian rhythms. In: S. Clark (Ed.), Photoperiodism, Melatonin and the Pineal, Pitman, London, 1085; pp. 188-202. 2 Bartness, T.J., Goldman, B.D. and Bittman, E.L., SCN lesions
211 block responses to systemic melatonin infusions in Siberian hamsters, Am. J. Physiol., 260 (1991) R102-R112. 3 Bittman, E.L., Crandell, R.G. and Lehman, M.N., Influences of the paraventricular and suprachiasmatic nuclei and olfactory bulbs on melatonin responses in the golden hamster, Biol. Reproduction, 411 (1989) 118-126. 4 Carlson, L.L., Weaver, D.R. and Reppert, S.M., Melatonin receptors and signal transduction during development in Siberian hamsters (Phodopus sungorus), Dee. Brain Res., 59 (1991) 83-88. 5 Cassone, V.M., Chesworth, M.J. and Armstrong, S.M., Dose-dependent entrainment of rat circadian rhythms by daily injection of melatonin, J. Biol. Rhythms, 1 (1986) 219-229. 6 Davis, F.C., Boada, R. and LeDeaux, J., Neurogenesis of the hamster suprachiasmatic nucleus, Brain Res., 519 (1990) 192-199. 7 Davis, F.C. and Gorski, R.A., Development of hamster circadian rhythms: Role of the maternal suprachiasmatic nucleus, J. Comp. Physiol. A, 162 (1988) 601-610. 8 Davis, F.C. and Mannion, J., Entrainment of hamster pup circadian rhythms by prenatal melatonin injections to the mother, Am. J. Physiol., 255 (1988) R439 R448. 9 De Reviers, M.M., Ravault, J.P., Tillet, Y. and Pelletier, J., Melatonin binding sites in the sheep pars tuberalis, Neurosci. Lett., 100 (1989) 89-93. 10 Deguchi, T., Ontogenesis of a biological clock for serotonin acetyl coenzyme A N-acetyltransferase in pineal gland of rat, Proe. Natl. Acad. Sci. USA, 72 (1975) 2814-2818. 11 Dubocovich, M.L. and Takahashi, J.S., Use of 2-[125I]iodomelatonin to characterize melatonin binding sites in chicken retina, Proc. Natl. Acad. Sci. USA, 84 (1987) 3916-3920. 12 Duncan, M.J., Takahashi, J.S. and Dubocovich, M.L., Characteristics and autoradiographic localization of 2-[12Sl]iodomelatonin binding sites in Djungarian hamster brain, Endocrinolo©', 125 (1989) 1011-1118. 13 Earnest, D.J. and Sladek, C.D., Circadian rhythms of vasopressin release from individual rat suprachiasmatic explants in vitro. Brain Res., 382 11986) 129-133. 14 Horton, T.H., Ray, S.L. and Stetson, M.H., Maternal transfer of photoperiodic information in Siberian hamsters. III. Melatonin injections program postnatal reproductive development expressed in constant light, Biol. Reproduction, 41 (1989) 34 39. 15 Illnerova, H., Hoffmann, K. and Vanecek, J., Adjustment of pineal melatonin and N-acetyltransferase rhythms to change from long to short photoperod in the Djungarian hamster, Phodopus sungorus, NeuroendocrinologT, 38 (1984) 226-231. 16 Inouye, S.T. and Kawamura, H., Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus, ,°roe. Natl. Acad. Sci. USA, 76 (19791 5962-5966. 17 Klein, D.C., Evidence for the placental transfer of 3H-acetylmelatonin, Nature, 237 119721 117-118. 18 Laitinen, J.T., Castren, E., Vakkuri, O. and Saavedra, J.M., Diurnal rhythm of melatonin binding in the rat suprachiasmatic nucleus, Endocrinology,'3', 124 (19891 1585-1587. 19 Laitinen, J.T., Flugge, G. and Saavedra, J.M., Characterization of melatonin receptors in the rat area postrema: Modulation of affinity with cations and guanine nucleotides, Neuroendoerinology, 51 (19901 619-624. 211 Laitinen, J.T. and Saavedra, J.M., Characterization of melatonin receptors in the rat suprachiasmatic nuclei: Modulation of affinity with cations and guanine nucleotides, Endocrinology, 126 (19911) 2110-2115. 21 Laitinen, J.T., Viswanathan, M., Vakkuri, O. and Saavedra, J.M., Differential regulation of the rat melatonin receptors: Selective age-associated decline and lack of melatonin-induced changes, Endocrinolog3', 130 (1992) 2139-2144. 22 Margraf, R.R., Zlomanczuk, P., Liskin, L.A. and Lynch, G.R., Circadian differences in neuronal activity of the suprachiasmatic nucleus in brain slices prepared form photo-responsive and photo-non-responsive Djungarian hamsters, Brain Res., 544 ( 1991 ) 42-48. 23 Maywood, E.S., Buttery, R.C., Vance, G.H.S., Herbert, J. and Hastings, M.H., Gonadal responses of the male Syrian hamster to
programmed infusions of melatonin are sensitive to signal duration and frequency but not to signal phase nor to lesions of the suprachiasmatic nuclei, Biol. Reproduction, 43 (199//) 174-182. 24 McArthur, A.J., Gillette, M,U. and Prosser, R.A., Melatonin directly resets the rat supraehiasmatic circadian clock in vitro, Brain Res., 565 (19911 158-161. 25 McPherson, G.A., A practical computer-based approach to the analysis of radioligand binding experiments, Comp. Prog. Biomed., 17(19921 107 115. 26 Meijer, J.H. and Rietveld, W.J., Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Ph3siol. Rer., 69 (1989) 671-707. 27 Moore, R.Y. and Lenn, N.Y., A retinohypothalamic projection in the rat, ,L Comp. Neurol., 146 (19721 1-14. 28 Morgan, P.J., Williams, L.M., Davidson, G.. Lawson, W. and Howell, E., Melatonin receptors on ovinc pars tuberalis: Characterization and autoradiographical localization, J. Neuroendoerinol., 1 (1989) 1 4. 29 Nazarali, A.J., Gutkind, J.S. and Saavedra, J.M., Calibration of 125I-polymer standards with t25I-brain paste standards for use in quantitative receptor autoradiography, .1. Neurosci. Methods, 30 ( 19891 247-253. 30 Paull, W.K. A light and eleetron study of the derelopment ~f the neurohypophysis of the fetal rat, 1'473, Ph.D. Dissertation, Univcr sity of Southern California. 31 Paxinos, G.. Tork, I., Tecott, L.H. and Valentino, K.L. Atlas ~l the Deceloping Rat Brain, Academic Press, San Diego. 1991. 32 Paxinos, G. and Watson, C. The Rat Bruin in Stereotaxic Coordinates, Academic Press, San Diego, 1986. 33 Reppert, S.M., Chez, R.A., Anderson, A. and Klein, D.C., Maternal-fetal transfer of melatonin in the non-human primate, Pediatric Res., 13 (1979) 788-791. 34 Reppert, S.M. and Schwartz, W.J., The suprachiasmatic nuclei of the fetal rat: Characterization of a functional circadian clock using laC-labeled deoxyglucose, .I. Neurosei, 4 (19841 1677 1682. 35 Reppert, S.M., Weaver, D.R. and Rivkccs, S.A.. Prenatal ftn]ction and entrainment of a circadian clock. In: S.M. Rcppcrt (Ed.) Research in Perinatal Medicine. lqd. L~L Decelopment of Circadian Rhythmicity and Photoperiodism in Mamma£, Perinatology Press, 1989, pp. 25-44. 36 Reppert, S.M., Weaver, D.R.. Rivkees, S.A. and Slopa, E.(}., Putative melatonin receptors in a human biological clock, Scienee, 242 11988) 78-81. 37 Schambra, U.B., Lauder, J.M. and Silver, J., Atlas of the Prenatal Mouse Brain, Academic Press, San Diego, 1992. 325 pp. 38 Speh, J.C. and Moore, R.Y., Synaptogenesis and retinohypothalamic tract (RHT) development in the hamster suprachiasmatic nucleus (SCN), Soc. Neurosei. Ahstr., 16 (19911) 6112. (Abstract) 39 Stetson, M.H., Elliot, J.A. and Goldman, B.D., Maternal transfer of photoperiodic information influences the photoperiodic response of prepubertal l)jungarian hamster (Phodopus sungoru.~ stmgorus), Biol. Reprod., 34 11986)664 669. 40 Stetson, M.H., Ray, S.L., Creyaufmiller, N. and Hortou, T.tI.. Maternal transfer of photoperk)dic information in Siberian hamsters. II. The nature of the maternal signal, time of signal transfer, and the effect of the maternal signal on peripubcrtal reproductive development in the absencc of photoperiodic input. Biol. Reprod., 40 (19891 458-465. 41 Tamarkin, L., Reppert, S.M., Orloff, D.J., Klein, D.('., Yclhm, S.M. and Goldman, B.D., Ontogeny of the pineal melatonin rhythm in the Syrian (Mesocricetus aurutliS) and Siberian (Phodopus sungorus) hamsters and in the rat, Endoerimdogy, 1117 (1980) 1(161 11164. 42 Thomas, E.M.V. and Armstrong, S.M., Mclatonin administration entrains female rat activity rhythms in constant darkness but not in constant light, Am. ,L Physiol., 255 (1988) R237 R242. 43 Vakkuri, O., Leppaluota, J. and Vuoltcenaho, O., Development and validation of a melatonin radioimmunoassay using radioiodihated melatonin as tracer, Aetu Emloermol. (CopenhagenL 106 (1984) 152-157. 44 Vanecek, J., Melatonin binding sites, ,I. Neurochem., 51 (1988) 1436-1440.
212 45 Vanecek, J., The melatonin receptors in rat ontogenesis, Neuroen&~crinology, 48 (1988) 201-203. 46 Vanecek, J. and Jansky, L., Short days induce cbanges in specific melatonin binding in hamster median eminence and anterior pituitary, Brain Res., 477 (1989) 387-39(I. 47 Vanecek, J., Pavlik, A. and lllnerova, H., Hypothalamic melatonin receptor sites revealed by autoradiography, Brain Res., 435 (1987) 359 362. 48 Velazquez, E., Esquifino, A.I., Zueco, J.A., Albusac, J.M.R. and Blazquez, E., Evidence that circadian variations of circulating melatonin levels in fetal and suckling rats are dependent on maternal melatonin transfer, Neuroendocrmolo~', 55 (1992) 32132~. 49 Viswanathan, N. and Davis, F.C., Melatonin sets the phase of the developing circadian rhythm in Syrian hamsters, Soc. Res. Biol. Rhythms Abstr.,(1992) 95. (Abstract) 50 Weaver, D.R., Keohan, J.T. and Reppert, S.M., Definition of a prenatal sensitive period for maternal-fetal communication of daylength, Am. Y. Physiol., 253 (1987) E701-E704. 51 Weaver, D.R., Namboodiri, M.A.A. and Reppert, S.M., Iodinated melatonin mimics melatonin action and reveals discrete binding sites in fetal brain, FEBS Lett., I (1988) 123-127. 52 Weaver, D.R., Provencio, I., Carlson, L.L. and Reppert, S.M., Melatonin receptors and signal transduction in photorefractory Siberia n hamsters ( Phodopus sungorus ), Endocrinology', 128 ( 1991 ) 1086-1092. 53 Weaver, D.R. and Reppert, S.M., Maternal melatonin communi-
54
55
56
57
58
59
6(I
cates daylength to the fetus in l))ungarian hamster. ::mtocrimd o:,'3', 119 (1986) 2861-2863. Weaver, D.R. and Reppert, S.M.. Melatonin receptors are present in the ferret pars tuberalis and pars distalis, but not in brain, Endocrinology, 127 (1990) 26(/7-2609. Weaver, D.R., Rivkees, S.A. and Reppert, S.M., Autoradiographic localization of iodomelatonin binding sites in hypothalamus of three rodent species, Abstracts of the 70th Annual Meeting of the Endocrine Society, 1988, p. 265. Weaver, D.R., Rivkees, S.A. and Reppert, S.M., Localization and characterization of melatonin receptors in rodent brain by m vitro autoradiography, J. Neurosci., 9 (1989) 2581-2590. Williams, L.M., Melatonin-binding sites in the rat brain and pituitary mapped by in-vitro autoradiography, J. Mol. Endoerinol., 3 (1989) 71-75. Williams, L.M., Martinoli, M.G., Titchener, L.T. and Pelletier, G., The ontogeny of central melatonin binding sites in the rat, Endocrinolog.,y, 128 (1991) 2083-2090. Williams, L.M. and Morgan, P.J., Demonstration of melatoninbinding sites on the pars tuberalis of the rat, .Z Endocrinol., 119 (1988) R1-R3. Williams, L.M., Morgan, P.J., Hastings, M.H., Lawson, W., Davidson, G. and Howell, H.E., Melatonin receptor sites in the Syrian hamster brain and pituitary. Localization and characterization using [125I]iodomelatonin, J. Neuroendocrinol., I (1989) 315320.