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The area of 2-[125I]iodomelatonin binding in the pars tuberalis of the ground squirrel is decreased during hibernation
Brain Research, 557 (1991) 285-288 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124775P BRES 24775
285
Short Communications
The area of 2-[12Sl]iodomelatonin binding in the pars tuberalis of the ground squirrel is decreased during hibernation Toni L. Stanton 1, Judith A. Siuciak 2, Margarita L. Dubocovich 2 and Diana N. Krause 3 1Department of Physiology, California State University, Long Beach, CA 90840 (U.S.A.), 2Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611 (U.S.A.) and 3Department of Pharmacology, College of Medicine, University of California, Irvine, lrvine, CA 92717 (U.S.A.) (Accepted 30 April 1991)
Key words: Melatonin; 2-[lzsI]iodomelatoninbinding; Ground squirrel; Hibernation; Pars tuberalis; Receptor autoradiography
Quantitative receptor autoradiography was used to analyze 2-[12sI]iodomelatonin binding sites in the brains of golden-mantled ground squirrels (Citellus lateralis). Specific binding appeared to be discretely localized to the pars tuberalis region of the pituitary that surrounds the medial basal hypothalamus. The total area of binding was significantly decreased in brains of hibernating squirrels as compared to those of awake, euthermic animals. These findings support a role for melatonin receptors of the pars tuberalis in seasonal behavior. Melatonin, a major hormone of the pineal gland, appears to be involved in seasonal behavior as well as neural mechanisms that control brain arousal level. Previous studies in ground squirrels 12-14 implicate melatonin in hibernation, a highly organized, complex phenomenon that is controlled by the CNS and appears to be a natural extension of slow wave sleep. Mammalian hibernation is characterized by a dramatic and reversible reduction in the level of physiologic and behavioral functioning, during which body temperature can approach 0 °C. In obligate hibernators, such as ground squirrels, hibernation is seasonal and controlled by endogenous clock mechanisms ~. Animals enter torpor at the appropriate time of year regardless of environmental variables such as light and temperature. In the hibernating ground squirrel, a significant and apparently progressive decline in pineal melatonin levels was observed during the course of a hibernation bout 12' 14, an individual period of deep torpor lasting from 6 to 14 days followed by spontaneous arousal. During the short interbout period of euthermia (body temperature at 37 °C), melatonin levels increased robustly. A highly significant positive correlation was evident between pineal melatonin content and the duration of an animal's hibernation bout 12. These findings suggest that melatonin availability might be one of the factors that determines bout duration. When depleted melatonin stores were replenished by the infusion of melatonin into the lateral
ventricles of hibernating animals, the hibernation bout duration was prolonged 13. To further examine the potential role of melatonin in hibernation, it is necessary to establish the brain substrates for melatonin's actions. 2-[125I]Iodomelatonin has recently been shown to bind specifically and with high affinity to sites exhibiting the same pharmacological characteristics as functional melatonin receptors 3"6. The aim of the present study was to localize and quantify the distribution of specific 2-[125I]iodomelatonin binding sites in the brains of awake (euthermic) and hibernating golden-mantled ground squirrels. A preliminary report of this study has been presented 11. Male and female golden-mantled ground squirrels (Citellus lateralis) from the mountains of northern California were obtained from a licensed trapper. The animals were individually housed in stainless steel cages and provided with food, water and cotton nesting material ad libitum. Euthermic animals were kept in a colony room maintained at 21 _+ 2 °C under light conditions that approximated the natural light:dark cycle. Hibernating animals were maintained in the dark at 5 _+ 0.5 °C in a walk-in cold room in order to approximate conditions of the natural hibernaculum and promote a state of deep hibernation. The course of each animal's hibernation bout cycle was followed using a computercontrolled monitor of nest temperature that was measured via a sensitive YSI thermistor probe permanently
Correspondence: T.L. Stanton, Department of Physiology, California State University, 1250 Bellflower Blvd., Long Beach, CA 90840, U.S.A. Fax: (1) (213) 985-2315.
286 fixed to the floor of the nesting box just beneath the animal's body. Three hibernating animals were sacrificed in March during the mid portion of each animal's hibernation bout. Two euthermic animals were sacrificed in March and two in August. All animals were sacrificed by decapitation between 11:00 and 13:00 h. The brains were r e m o v e d , quick-frozen and stored at - 8 0 °C until time of sectioning.
R e c e p t o r a u t o r a d i o g r a p h y of 2-[~251]iodomelatonin binding was p e r f o r m e d as described previously w. Briefly, serial coronal brain sections (20 /~m) were cut on a cryostat (-15 °C) and t h a w - m o u n t e d onto gelatin-coated slides. Three alternate sets of sections were generated for the d e t e r m i n a t i o n of total binding, non-specific binding and histological staining. A f t e r air-drying for 1-2 min or less, slides were stored at - 7 0 °C for no more than one
O
Fig. 1. Autoradiographic localization of 2-[125I]iodomelatonin binding in the pars tuberalis region of euthermic and hibernating ground squirrels. A-C: from a euthermic ground squirrel, a representative set of 3 serial, coronal sections of the brain cut at the level of the hypothalamus. Autoradiograms show (A) total binding of 100 pM 2-[125I]iodomelatonin and (B) non-specific binding with the addition of 3/~M melatonin. C: histological section stained with thionin. D: autoradiogram of a similar section from a hibernating squirrel that was processed for total binding at the same time and with the same conditions as in (A). Higher magnification of the pars tuberalis in another euthermic squirrel showing (E) total 2-[~25I]iodomelatonin binding and (F) the corresponding histological section. Arrows indicate the pars tuberalis region in A, D, E and E
287 TABLE I
Quantitation of specific 2-[125I]iodomelatonin binding in the pars tuberalis of euthermic and hibernating ground squirrels
Euthermic Hibernating
Total area (mm2)
Average density (fmol/mg protein)
4.09 + 1.52 (n = 4) 0.48 + 0.21" (n = 3)
34.8 + 14.0 16.4 _+ 10.9
*P < 0.01 compared with euthermic animals.
week. Slide-mounted sections were preincubated in 150 mM Tris-HCl buffer (pH 7.4, 22 °C) for 1 h to remove endogenous ligand and then incubated with 75-100 pM 2-[125I]iodomelatonin (sp. act. 1800-2000 Ci/mmol) in Tris-HCl buffer with (non-specific binding) or without (total binding) 3/~M melatonin for 1 h at room temperature. Slides were rinsed in ice-cold Tris-HCl buffer (3 x 5 min each) followed by a rapid rinse in ice-cold distilled water to remove buffer salts. Labeled sections were apposed to Kodak SB5 X-ray film for 3-4 weeks. The film was developed using Kodak D19 developer (4 °C). Tissue sections were stained with thionin for histological examination. Autoradiograms were analysed using a computer-based image analysis system (Imaging Resources Inc., Canada). 14C Standards (ARC, Inc.) were calibrated for use with 125I using the method of Miller and Zahniser s, and protein content in tissue sections was determined from tissue equivalents supplied by the manufacturer of the 14C standards. In all squirrel brains examined, specific binding of 2-[125I]iodomelatonin (75-100 pM) was discretely localized to the basal hypothalamus in the region of the median eminence/pars tuberalis (Fig. 1A,D,E). Previous studies in rat and sheep brain have established that 2-[125I]iodomelatonin binding sites are actually located in the pars tuberalis rather than the median eminence2'17. The pars tuberalis is an area of the adenohypophysis that surrounds the hypophyseal stalk and extends along the basal surface of the median eminence with cells arranged in clusters and cords 9. Examination of the squirrel brains at higher magnification showed a binding distribution pattern characteristic of the pars tuberalis (Fig. 1E,F) and similar to that reported for 2-[125I]iodomelatonin binding in this region in sheep, hamsters, mice, ferrets, and rats 2'4'1°'16'17. Although the squirrel brains were sectioned and examined in their entirety, specific 2[125I]iodomelatonin binding was not apparent in any other brain region. This is in contrast to our studies on mouse brain, in which assay conditions identical to the present study resulted in specific 2-[125I]iodomelatonin binding that could be readily observed in a number of hypothalamic and thalamic structures 1°. In the brains of euthermic animals, specific binding of
2-[125I]iodomelatonin in the pars tuberalis extended rostrally through about 2 mm of brain. Considerable variation in binding density was seen among the cell dusters from section to section which precluded saturation analysis. The brains were studied using concentrations of ligand which produced maximal binding in previous studies4'7'16; and, in the squirrels, resulted in an average binding density of 35 fmol/mg protein (Table I). Binding in spring and summer euthermic animals appeared similar. Brains taken from hibernating animals also showed discrete 2-[125I]iodomelatonin binding in the pars tuberalis region (Fig. 1D). However in all 3 animals, binding was much less extensive than in the euthermic animals. The extent of specific binding in the euthermic and hibernating squirrel brains was quantitated by measuring the total area of specific binding for each animal (Table I). The area of specific binding was significantly decreased in the brains of hibernating animals. While there was also a tendency for the average binding density to be lower in these animals, this was not significant (Table I). Receptor autoradiography of brains from euthermic and hibernating ground squirrels indicate that the pars tuberalis region of the pituitary is the primary site of high affinity 2-[125I]iodomelatonin binding. Although not well understood, the function of this region is thought to be involved in neuroendocrine regulation. It contains predominantly secretory cells, e.g. thyrotropes and gonadotropes, and exhibits photoperiod-dependent changes in morphology9. It has been proposed that this is a site where melatonin mediates its photoperiodic effects on the neuroendocrine system2,15-17. Melatonin binding sites located in the pars tuberalis may be relevant for melatonin's ability to induce gonadal regression which, in the case of ground squirrels, is a prerequisite for successful hibernation 5. Interestingly, in studies of several other seasonally responsive species, i.e. sheep 2, hamsters 15 and ferrets 16, it has also been reported that the pars tuberalis, but no brain areas, displayed specific high affinity 2-[125I]iodomelatonin binding sites. At this point, however, we cannot rule out the possible existence of lower affinity melatonin binding sites which may be difficult to detect using the standard washing procedures employed for receptor autoradiography4,7. Preliminary evidence from euthermic squirrels indicates that, in some animals, a relatively low density of specific binding can be detected in several brain areas including the suprachiasmatic nuclei. The latter region is thought to generate circadian rhythmicity and exhibits 2-[125I]iodomelatonin binding in a number of mammals including rats and mice 6'7A°. Studies to increase the detection of binding in the squirrel brain are currently in progress.
288 In the pars tuberalis, the area exhibiting specific binding of 2-[125I]iodomelatonin was less extensive in hibernating squirrels as c o m p a r e d to euthermic, awake animals. This finding may reflect either a loss of receptors or a shift in the conformation of the receptors, e.g. from an active high-affinity state to an inactive low-affinity state. The cells which express 2-[125I]iodomelatonin
tory cells, which contain g o n a d o t r o p i n - or thyrotropinlike immunoreactivity, a p p e a r e d less active with secretory granules showing signs of focal cytoplasmic degeneration 9. The current study suggests that during hibernation, the pars tuberalis also decreases its receptivity to melatonin.
binding sites may undergo a general change in their activity state and ultrastructure similar to what was r e p o r t e d recently for cells in the pars tuberalis of hibernating hedgehogs 9. In the hibernating state, secre-
This study was supported in part by California State University, Long Beach (T.L.S.), Grant MH42922 (M.L.D.) and postdoctoral fellowships NS-07140 and MH09997 (J.A.S.). We thank Dr. F. Leslie, University of California, lrvine, for use of the quantitative image analysis system.
1 Beckman, A.L. and Stanton, T.L., Properties of the CNS during the state of hibernation. In A.L. Beckman (Ed.), The Neural Basis of Behavior, Spectrum, New York, 1982, pp. 19-45. 2 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. 3 Dubocovich, M.L. and Takahashi, J.S., Use of 2[125I]iodomelatonin to characterize melatonin binding sites in chicken retina, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 3916-3920. 4 Duncan, M.J., Takahashi, J.S. and Dubocovich, M.L., Characteristics and autoradiographic localization of 2[125I]iodomelatonin binding sites in Djungarian hamster brain, Endocrinology, 125 (1989) 1011-1018. 5 Jansk~, L., Pineal, gonads and hibernation, Pineal Res. Rev., 4 (1986) 141-181. 6 Krause, D.N. and Dubocovich, M.L., Regulatory sites in the melatonin system of mammals, Trends Neurosci., 13 (1990) 464-470. 7 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 (1990) 2110-2115. 8 Miller, J. and Zahniser, N., The use of x4C labeled standards for the calibration of 1251labeled ligands in quantitative autoradiography, Neurosci. Lett., 81 (1987) 345-350. 9 Riinen, B., Hewing, M. and Wittkowski, W., Seasonal ultrastructural changes of the hypophyseal pars tuberalis in the hedgehog (Erinaceus europaeus L.), Acta Anat., 133 (1988)
217-223. 10 Siuciak, J.A., Fang, J.-M. and Dubocovich, M.L., Autoradiographic localization of 2-[125I]iodomelatonin binding sites in the brains of C3H/HeN and C57BL/6J strains of mice, Eur. J. Pharmacol., 180 (1990) 387-390. 11 Stanton, T.L., Siuciak, J.A., Krause, D.N. and Dubocovich, M.L., Autoradiographic localization of 2-[125I]iodomelatonin binding sites in the brain of ground squirrels (Citellus lateralis), Soc. Neurosci. Abstr., 16 (1990) 773. 12 Stanton, T.L., Craft, C.M. and Reiter, R.J., Decreases in pineal melatonin content during the hibernation bout in the goldenmantled ground squirrel, Life Sci., 35 (1984) 1461-1467. 13 Stanton, T.L., Daley III, J.C. and Salzman, S.K., Prolongation of hibernation bout duration by continuous intracerebroventricular infusion of melatonin in hibernating ground squirrels, Brain Research, 413 (1987) 350-355. 14 Stanton, T.L., Craft, C.M. and Reiter, R.J., Pineal melatonin: circadian rhythm and variations during the hibernation cycle in the ground squirrel, Spermophilus lateralis, J. Exp. Zool., 239 (1986) 247-254. 15 Van~ek J. and Jansk~, L., Short days induce changes in specific melatonin binding in hamster median eminence and anterior pituitary, Brain Research, 477 (1989) 387-390. 16 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) 2607-2609. 17 Williams, L.M. and Morgan, P.J., Demonstration of melatoninbinding sites on the pars tuberalis of the rat, J. Endocrinol., 119 (1988) R1-R3.
285
Short Communications
The area of 2-[12Sl]iodomelatonin binding in the pars tuberalis of the ground squirrel is decreased during hibernation Toni L. Stanton 1, Judith A. Siuciak 2, Margarita L. Dubocovich 2 and Diana N. Krause 3 1Department of Physiology, California State University, Long Beach, CA 90840 (U.S.A.), 2Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611 (U.S.A.) and 3Department of Pharmacology, College of Medicine, University of California, Irvine, lrvine, CA 92717 (U.S.A.) (Accepted 30 April 1991)
Key words: Melatonin; 2-[lzsI]iodomelatoninbinding; Ground squirrel; Hibernation; Pars tuberalis; Receptor autoradiography
Quantitative receptor autoradiography was used to analyze 2-[12sI]iodomelatonin binding sites in the brains of golden-mantled ground squirrels (Citellus lateralis). Specific binding appeared to be discretely localized to the pars tuberalis region of the pituitary that surrounds the medial basal hypothalamus. The total area of binding was significantly decreased in brains of hibernating squirrels as compared to those of awake, euthermic animals. These findings support a role for melatonin receptors of the pars tuberalis in seasonal behavior. Melatonin, a major hormone of the pineal gland, appears to be involved in seasonal behavior as well as neural mechanisms that control brain arousal level. Previous studies in ground squirrels 12-14 implicate melatonin in hibernation, a highly organized, complex phenomenon that is controlled by the CNS and appears to be a natural extension of slow wave sleep. Mammalian hibernation is characterized by a dramatic and reversible reduction in the level of physiologic and behavioral functioning, during which body temperature can approach 0 °C. In obligate hibernators, such as ground squirrels, hibernation is seasonal and controlled by endogenous clock mechanisms ~. Animals enter torpor at the appropriate time of year regardless of environmental variables such as light and temperature. In the hibernating ground squirrel, a significant and apparently progressive decline in pineal melatonin levels was observed during the course of a hibernation bout 12' 14, an individual period of deep torpor lasting from 6 to 14 days followed by spontaneous arousal. During the short interbout period of euthermia (body temperature at 37 °C), melatonin levels increased robustly. A highly significant positive correlation was evident between pineal melatonin content and the duration of an animal's hibernation bout 12. These findings suggest that melatonin availability might be one of the factors that determines bout duration. When depleted melatonin stores were replenished by the infusion of melatonin into the lateral
ventricles of hibernating animals, the hibernation bout duration was prolonged 13. To further examine the potential role of melatonin in hibernation, it is necessary to establish the brain substrates for melatonin's actions. 2-[125I]Iodomelatonin has recently been shown to bind specifically and with high affinity to sites exhibiting the same pharmacological characteristics as functional melatonin receptors 3"6. The aim of the present study was to localize and quantify the distribution of specific 2-[125I]iodomelatonin binding sites in the brains of awake (euthermic) and hibernating golden-mantled ground squirrels. A preliminary report of this study has been presented 11. Male and female golden-mantled ground squirrels (Citellus lateralis) from the mountains of northern California were obtained from a licensed trapper. The animals were individually housed in stainless steel cages and provided with food, water and cotton nesting material ad libitum. Euthermic animals were kept in a colony room maintained at 21 _+ 2 °C under light conditions that approximated the natural light:dark cycle. Hibernating animals were maintained in the dark at 5 _+ 0.5 °C in a walk-in cold room in order to approximate conditions of the natural hibernaculum and promote a state of deep hibernation. The course of each animal's hibernation bout cycle was followed using a computercontrolled monitor of nest temperature that was measured via a sensitive YSI thermistor probe permanently
Correspondence: T.L. Stanton, Department of Physiology, California State University, 1250 Bellflower Blvd., Long Beach, CA 90840, U.S.A. Fax: (1) (213) 985-2315.
286 fixed to the floor of the nesting box just beneath the animal's body. Three hibernating animals were sacrificed in March during the mid portion of each animal's hibernation bout. Two euthermic animals were sacrificed in March and two in August. All animals were sacrificed by decapitation between 11:00 and 13:00 h. The brains were r e m o v e d , quick-frozen and stored at - 8 0 °C until time of sectioning.
R e c e p t o r a u t o r a d i o g r a p h y of 2-[~251]iodomelatonin binding was p e r f o r m e d as described previously w. Briefly, serial coronal brain sections (20 /~m) were cut on a cryostat (-15 °C) and t h a w - m o u n t e d onto gelatin-coated slides. Three alternate sets of sections were generated for the d e t e r m i n a t i o n of total binding, non-specific binding and histological staining. A f t e r air-drying for 1-2 min or less, slides were stored at - 7 0 °C for no more than one
O
Fig. 1. Autoradiographic localization of 2-[125I]iodomelatonin binding in the pars tuberalis region of euthermic and hibernating ground squirrels. A-C: from a euthermic ground squirrel, a representative set of 3 serial, coronal sections of the brain cut at the level of the hypothalamus. Autoradiograms show (A) total binding of 100 pM 2-[125I]iodomelatonin and (B) non-specific binding with the addition of 3/~M melatonin. C: histological section stained with thionin. D: autoradiogram of a similar section from a hibernating squirrel that was processed for total binding at the same time and with the same conditions as in (A). Higher magnification of the pars tuberalis in another euthermic squirrel showing (E) total 2-[~25I]iodomelatonin binding and (F) the corresponding histological section. Arrows indicate the pars tuberalis region in A, D, E and E
287 TABLE I
Quantitation of specific 2-[125I]iodomelatonin binding in the pars tuberalis of euthermic and hibernating ground squirrels
Euthermic Hibernating
Total area (mm2)
Average density (fmol/mg protein)
4.09 + 1.52 (n = 4) 0.48 + 0.21" (n = 3)
34.8 + 14.0 16.4 _+ 10.9
*P < 0.01 compared with euthermic animals.
week. Slide-mounted sections were preincubated in 150 mM Tris-HCl buffer (pH 7.4, 22 °C) for 1 h to remove endogenous ligand and then incubated with 75-100 pM 2-[125I]iodomelatonin (sp. act. 1800-2000 Ci/mmol) in Tris-HCl buffer with (non-specific binding) or without (total binding) 3/~M melatonin for 1 h at room temperature. Slides were rinsed in ice-cold Tris-HCl buffer (3 x 5 min each) followed by a rapid rinse in ice-cold distilled water to remove buffer salts. Labeled sections were apposed to Kodak SB5 X-ray film for 3-4 weeks. The film was developed using Kodak D19 developer (4 °C). Tissue sections were stained with thionin for histological examination. Autoradiograms were analysed using a computer-based image analysis system (Imaging Resources Inc., Canada). 14C Standards (ARC, Inc.) were calibrated for use with 125I using the method of Miller and Zahniser s, and protein content in tissue sections was determined from tissue equivalents supplied by the manufacturer of the 14C standards. In all squirrel brains examined, specific binding of 2-[125I]iodomelatonin (75-100 pM) was discretely localized to the basal hypothalamus in the region of the median eminence/pars tuberalis (Fig. 1A,D,E). Previous studies in rat and sheep brain have established that 2-[125I]iodomelatonin binding sites are actually located in the pars tuberalis rather than the median eminence2'17. The pars tuberalis is an area of the adenohypophysis that surrounds the hypophyseal stalk and extends along the basal surface of the median eminence with cells arranged in clusters and cords 9. Examination of the squirrel brains at higher magnification showed a binding distribution pattern characteristic of the pars tuberalis (Fig. 1E,F) and similar to that reported for 2-[125I]iodomelatonin binding in this region in sheep, hamsters, mice, ferrets, and rats 2'4'1°'16'17. Although the squirrel brains were sectioned and examined in their entirety, specific 2[125I]iodomelatonin binding was not apparent in any other brain region. This is in contrast to our studies on mouse brain, in which assay conditions identical to the present study resulted in specific 2-[125I]iodomelatonin binding that could be readily observed in a number of hypothalamic and thalamic structures 1°. In the brains of euthermic animals, specific binding of
2-[125I]iodomelatonin in the pars tuberalis extended rostrally through about 2 mm of brain. Considerable variation in binding density was seen among the cell dusters from section to section which precluded saturation analysis. The brains were studied using concentrations of ligand which produced maximal binding in previous studies4'7'16; and, in the squirrels, resulted in an average binding density of 35 fmol/mg protein (Table I). Binding in spring and summer euthermic animals appeared similar. Brains taken from hibernating animals also showed discrete 2-[125I]iodomelatonin binding in the pars tuberalis region (Fig. 1D). However in all 3 animals, binding was much less extensive than in the euthermic animals. The extent of specific binding in the euthermic and hibernating squirrel brains was quantitated by measuring the total area of specific binding for each animal (Table I). The area of specific binding was significantly decreased in the brains of hibernating animals. While there was also a tendency for the average binding density to be lower in these animals, this was not significant (Table I). Receptor autoradiography of brains from euthermic and hibernating ground squirrels indicate that the pars tuberalis region of the pituitary is the primary site of high affinity 2-[125I]iodomelatonin binding. Although not well understood, the function of this region is thought to be involved in neuroendocrine regulation. It contains predominantly secretory cells, e.g. thyrotropes and gonadotropes, and exhibits photoperiod-dependent changes in morphology9. It has been proposed that this is a site where melatonin mediates its photoperiodic effects on the neuroendocrine system2,15-17. Melatonin binding sites located in the pars tuberalis may be relevant for melatonin's ability to induce gonadal regression which, in the case of ground squirrels, is a prerequisite for successful hibernation 5. Interestingly, in studies of several other seasonally responsive species, i.e. sheep 2, hamsters 15 and ferrets 16, it has also been reported that the pars tuberalis, but no brain areas, displayed specific high affinity 2-[125I]iodomelatonin binding sites. At this point, however, we cannot rule out the possible existence of lower affinity melatonin binding sites which may be difficult to detect using the standard washing procedures employed for receptor autoradiography4,7. Preliminary evidence from euthermic squirrels indicates that, in some animals, a relatively low density of specific binding can be detected in several brain areas including the suprachiasmatic nuclei. The latter region is thought to generate circadian rhythmicity and exhibits 2-[125I]iodomelatonin binding in a number of mammals including rats and mice 6'7A°. Studies to increase the detection of binding in the squirrel brain are currently in progress.
288 In the pars tuberalis, the area exhibiting specific binding of 2-[125I]iodomelatonin was less extensive in hibernating squirrels as c o m p a r e d to euthermic, awake animals. This finding may reflect either a loss of receptors or a shift in the conformation of the receptors, e.g. from an active high-affinity state to an inactive low-affinity state. The cells which express 2-[125I]iodomelatonin
tory cells, which contain g o n a d o t r o p i n - or thyrotropinlike immunoreactivity, a p p e a r e d less active with secretory granules showing signs of focal cytoplasmic degeneration 9. The current study suggests that during hibernation, the pars tuberalis also decreases its receptivity to melatonin.
binding sites may undergo a general change in their activity state and ultrastructure similar to what was r e p o r t e d recently for cells in the pars tuberalis of hibernating hedgehogs 9. In the hibernating state, secre-
This study was supported in part by California State University, Long Beach (T.L.S.), Grant MH42922 (M.L.D.) and postdoctoral fellowships NS-07140 and MH09997 (J.A.S.). We thank Dr. F. Leslie, University of California, lrvine, for use of the quantitative image analysis system.
1 Beckman, A.L. and Stanton, T.L., Properties of the CNS during the state of hibernation. In A.L. Beckman (Ed.), The Neural Basis of Behavior, Spectrum, New York, 1982, pp. 19-45. 2 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. 3 Dubocovich, M.L. and Takahashi, J.S., Use of 2[125I]iodomelatonin to characterize melatonin binding sites in chicken retina, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 3916-3920. 4 Duncan, M.J., Takahashi, J.S. and Dubocovich, M.L., Characteristics and autoradiographic localization of 2[125I]iodomelatonin binding sites in Djungarian hamster brain, Endocrinology, 125 (1989) 1011-1018. 5 Jansk~, L., Pineal, gonads and hibernation, Pineal Res. Rev., 4 (1986) 141-181. 6 Krause, D.N. and Dubocovich, M.L., Regulatory sites in the melatonin system of mammals, Trends Neurosci., 13 (1990) 464-470. 7 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 (1990) 2110-2115. 8 Miller, J. and Zahniser, N., The use of x4C labeled standards for the calibration of 1251labeled ligands in quantitative autoradiography, Neurosci. Lett., 81 (1987) 345-350. 9 Riinen, B., Hewing, M. and Wittkowski, W., Seasonal ultrastructural changes of the hypophyseal pars tuberalis in the hedgehog (Erinaceus europaeus L.), Acta Anat., 133 (1988)
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