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Serotonin receptor concentration during the estrous cycle of the rat
Brain Research, 187 (1980) 221-225 © Elsevier/North-HollandBiomedicalPress
221
Serotonin receptor concentration during the estrous cycle of the rat
A. BIEGON, H. BERCOVITZand D. SAMUEL Dept. of Isotope Research, Weizmann Institute of Science, Rehovot (Israel)
(Accepted November 29th, 1979) Key words: serotoninreceptor -- estrous cycle
Changes in receptor density are known to modify the responsiveness of the tissue to transmitter agonists and antagonists. An increase in receptor levels causes supersensitivity, while a reduction in receptor levels produces subsensitivity. Thus, changes in receptor levels could be part of the regulation of neurotransmission in the CNS. However, all of the published data on receptor supersensitivity or subsensitivity have been derived from experiments involving surgical or pharmacological intervention, e.g. denervation, transmitter depletion or chronic treatment with receptor agonists or antagonistsz,5,15. So far, there is no report of changes in receptor levels in the brains of intact animals under physiological conditions. We wish to present data demonstrating that serotonin receptor levels undergo regular changes during the estrous cycle of the rat. We have measured the binding of labeled serotonin [aH]5HT, (New England Nuclear, 28.2 Ci/mmol)to several brain regions from intact, freely cycling female rats. Males were included in the study in order to compare our binding data with those previously reported 6,15,20. Adult (12-14week-old) male and female Wistar rats from a local breeding colony were kept under controlled lighting (12 h dark/12 h light) and temperature (24 :~ 1 °C), with food and water available ad lib. Females underwent daily vaginal smears and only those exhibiting at least 3 consecutive regular 4-day cycles were included in the study. Animals were sacrificed by decapitation between 12 and 14 h. The brains were quickly removed and dissected in the cold. Brain regions (basal forebrain, hippocampus, cortex and caudate) from 2-3 animals were pooled and homogenized in 50 mM Tris.HC1 buffer, pH 7.6, using a polytron. The homogenate was spun at 30,000 × g for 10 min, washed and spun again. The pellet was resuspended in 20 ml buffer and incubated for 10 min at 37 °C in a shaker bath; followed by centrifugation at 30,000 × g for 10 min. The pellet was then homogenized in 50 mM Tris.HC1 buffer, pH 7.4, containing 100/~M pargyline and 0.02 ~ ascorbic acid, and incubated at 37 °C for 10 mln. Samples (1 ml) of this homogenate were added to test tubes containing the appropriate concentrations of label in 0.5 ml, and 0.5 ml of buffer with or without cold serotonin to a final concentration of 1 #M. After 10 min at 37 °C, the tubes were transferred to an ice-water bath and the contents filtered through GF/B glass fiber
222
_'5f 0
20
'-'
15
I
°=
o2 I
PE~
J
EI g rn
~:~o
O
BASAL F~EBRAIN HIPPOCAMPUS
/111 CORTEX
CAUDATE
Fig. 1. Specificbinding of [aH]5HT (5 nM) to several brain regions from female rats at different stages of the estrous cycle. D1, diestrous 1 ; D2, diestrous 2 ; PE, proestrous ; E, estrous. Results are means :E S.E.M. of 2-6 determinations. * Significantly lower than estrous and diestrous (Anova: F = 7.92, P < 0.002; Duncan's test: P < 0.05). filters, followed by 2 × 5 ml washes with ice-cold buffer. The filters were then transferred to vials and shaken overnight with 10 ml of toluene-Triton scintillation fluid, and counted in a Packard Tri-Carb scintillation counter, at 30~o efficiency. Protein was determined according to Lowry et al. 1°. In one series of experiments, we measured the specific binding of serotonin (5 nM) to several brain regions from cycling female rats (Fig. 1). The results were subjected to an analysis of variance followed by Duncan's test for multiple comparisons. In the basal forebrain (including the hypothalamus, septum and preoptic area) we found a clear and significant difference between the binding on diestrous-1 and 2 and proestrous-estrous. The binding on proestrous and estrous was about 40 °/o lower than on diestrous. In the hippocampus, a similar direction was observed, which, however, did not reach statistical significance. There was no effect of the cycle on serotonin binding in the cortex and caudate. A reduction in binding could be the result of changes in receptor affinity and/or concentration. In order to investigate these possibilities, we have performed complete saturation experiments on the basal forebrain region. Fig. 2 illustrates the binding curves and Scatchard plots obtained in one experiment for males, and females in diestrous-1 compared to proestrous. Similar comparisons were made for the other stages of the cycle. On the basis of the first series of experiments we have decided to pool the (very similar) Ka and Bmax values from diestrous-I and 2; and f r o m proestrous and estrous females. The results are summarized in Table I. Serotonin is thought to be strongly involved in the regulation of sexual behavior. Many reports have demonstrated an inhibitory role for serotonin on sexual behavior in both males and females in several species a,la,xs. It has also been shown to inhibit ovulation in the female 16. In this context, our results are not surprising: since serotonin is inhibitory for both sexual behavior and ovulation, one would expect it to be down-regulated when sexual behavior and ovulation occur - - namely, on the days
223 [SH]-SHT Bound(cpm xlO-3) 0
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[3HI 5 HT, nM Fig. 2. Binding o f [SH]5HT to basal forebrain from: (a) male, (b) female rats. The top drawings are the Scatchard plots of the binding data. ~', males; PE, proestrous females; DI, diestrous-1 females.
of proestrous and estrous. Interestingly, serotonin levels have been reported to go downY, 9 on proestrous, while serotonin uptake increases1, lz. Thus the reduction in serotonin receptor levels seems to be one of a number of mechanisms activated on proestrous day so as to decrease serotoninergic transmission. The mechanism responsible for the changes in receptor levels reported here is not known. However, the basal forebrain, containing the hypothalamus and septalpreoptic area, is considered to be the main target of sex steroids in the brain14; and steroidal sex hormones are known to exert a large part of their effects through an TABLE I
Binding characteristics o f serotonin to basal forebrain from males and females at different stages o f the estrous cycle
Results are the means + S.E.M. o f 4-8 experiments. Males (n = 5)
Proestrousandestrous
Diestrous I and 2 (n = 4)
(n = 8)
Ka (nM) 2.6 -t- 0.5 Bm==x (pm/rng protein) 0.18 -t- 0.02
1.9 -4- 0.6 0.11 ± 0.01"
2.2 -t- 0.3 0.17 -t- 0.009
* Significantly lower than other groups. (Anova: F = 6.72, P < 0.01 ; D u n c a n ' s test: P < 0.05).
224 interaction with gene expression a n d regulation of protein synthesis 4. Since m o n o a m i n e receptors are believed to be m e m b r a n a l proteins, it is possible that sex h o r m o n e s are involved in their synthesis or degradation. Estradiol in particular has been shown to influence the levels of progesterone receptors in the brain 11 testosterone receptors in the chick oviduct 19 a n d n o r e p i n e p h r i n e receptors Iboth a and /¢) in the uterusS, ~v. F u r t h e r experiments with gonadectomized animals and sex steroid replacement are needed in order to determine the possible role of this h o r m o n e in the regulation of m o n o a m i n e receptor levels in the brain.
l Biegon, A. and Samuel, D., Effect of sex and endocrine state on neurotransmitter uptake, submitted for publication. 2 Burt, D. R., Creese, I. and Snyder, S. H., Dopamine receptor binding enhancement accompanies lesion induced behavioral supersensitivity, Science, 197 (1977) 596--598. 3 Carter, S. C. and Davis, J. M., Biogenic amines, reproductive hormones and female sexual behavior: a review, Behav. Revs., 1 (1977) 213-224. 4 Chan, L. and O'Malley, B. W., Mechanism of action of sex steroid hormones, New Engl. J. Med., 294 (1976) 1322-1328. 5 Dolphin, A., Adrien J., Hamon M. and Bockaert J., Identity of [aH]dihydroalprenolol binding sites and fl-adrenergic receptors coupled with adenylate cyclase in the central nervous system: pharmacological properties, distribution and adaptive responses, Molec. Pharmacol., 15 (1979) 1-15. 6 Fillion, G., Fillion, M. P., Spirakis, C., Bahers, J.-M. and Jacob, J., 5-Hydroxytryptamine binding to synaptic membranes from rat brain, Li]e Sci., 18 (1976) 65-74. 7 Greengrass, P. M. and Tonge, S. R., Changes in brain monoamine concentrations during the estrous cycle in the mouse: possible pharmacological implications, J. Pharm. Pharmacol. 23 (1971) 897-898. 8 Krall, J. F., Mori, H., Tuck, M. L., Leshon, S. L. and Korenman, S. G., Demonstration of adrenergic catecholamine receptors in rat myometrium and their regulation by sex steroid hormones, Life Sci., 23 (1978) 1073-1082. 9 Kueng, W., Wirz-Justice, A., Menzi, B. and Chappuis-Arndt, E. Regional brain variation of tryptophan monoamines, monoamine oxidase activity, plasma free tryptophan and total tryptophan during the estrous cycle of the rat, Neuroendocr., 21 (1976) 289-296. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurements with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 11 MacLusky, N. J. and McEwen, B. S., Oestrogen modulates progestin receptor concentrations in some rat brain regions but not in others, Nature (Lond.), 274 (1978) 276-278. 12 Meyer, D. C. and Quay, W. B., Hypothalamic and suprachiasmatic uptake of serotonin in vitro: twenty four hour changes in male and proestrous female rats, Endocrinology, 98 (1976) 1160-1165. 13 Meyerson, B. J., Correr, H. and Eliasson, M., 5-Hydroxytryptamine and sexual behavior in the female rat. In E. Costa, G. L. Gessa and M. Sandler, (Eds.), Advances in Biochemical Psychopharmacology, VoL II, Raven Press, N.Y., 1974, pp. 229-242. 14 Morell, J. I. and Pfaff, D. W., A neuroendocrine approach to brain function: localization of sex steroid concentrating cells in vertebrate brains, Amer. Zool., 18 (1978) 447-460. 15 Nelson, D. L., Herbert, A., Bourgoin, S., Glowinski, J. and Hamon, M., Characteristics of central 5-HT receptors and their adaptive changes following intracerebral 5,7,dihydroxytryptamine administration in the rat, Molec. PharmacoL, 14 (1978) 983-995. 16 O'Steen, W. K., Serotonin suppression of luteinization in gonadotropin treated immature rats, Endocrinology, 74 (1964) 885-888. 17 Roberts, J. M., Goldfien, R. D., Insel, P. A. and Tsuchiya, A. M., Uterine a adrenergic receptors increase but platelet a adrenergic receptors decrease with estrogen, Clin. Res., 26 (1978) 494. 18 Tagliamonte, A., Tagliamonte, P., Gessa, G. L. and Brodies, B. B., Compulsive sexual activity induced by p-chlorophenylalaninein normal and pinealectomized male rats, Science, 166 (1969) 1433-1435.
225 19 Tokarz, R. R., Seaver, S. R. and Harrison R. W., Estrogen regulation of testosterone receptor concentration in the chick oviduct, J. Cell BioL, 79 (1978) 193. 20 Whitaker, P. M. and Seeman, P., High affinity ZH-serotonin binding to caudate: inhibition by hallucinogens and serotoninergic drugs, Psychopharmacology, 59 (1978) 1-5.
221
Serotonin receptor concentration during the estrous cycle of the rat
A. BIEGON, H. BERCOVITZand D. SAMUEL Dept. of Isotope Research, Weizmann Institute of Science, Rehovot (Israel)
(Accepted November 29th, 1979) Key words: serotoninreceptor -- estrous cycle
Changes in receptor density are known to modify the responsiveness of the tissue to transmitter agonists and antagonists. An increase in receptor levels causes supersensitivity, while a reduction in receptor levels produces subsensitivity. Thus, changes in receptor levels could be part of the regulation of neurotransmission in the CNS. However, all of the published data on receptor supersensitivity or subsensitivity have been derived from experiments involving surgical or pharmacological intervention, e.g. denervation, transmitter depletion or chronic treatment with receptor agonists or antagonistsz,5,15. So far, there is no report of changes in receptor levels in the brains of intact animals under physiological conditions. We wish to present data demonstrating that serotonin receptor levels undergo regular changes during the estrous cycle of the rat. We have measured the binding of labeled serotonin [aH]5HT, (New England Nuclear, 28.2 Ci/mmol)to several brain regions from intact, freely cycling female rats. Males were included in the study in order to compare our binding data with those previously reported 6,15,20. Adult (12-14week-old) male and female Wistar rats from a local breeding colony were kept under controlled lighting (12 h dark/12 h light) and temperature (24 :~ 1 °C), with food and water available ad lib. Females underwent daily vaginal smears and only those exhibiting at least 3 consecutive regular 4-day cycles were included in the study. Animals were sacrificed by decapitation between 12 and 14 h. The brains were quickly removed and dissected in the cold. Brain regions (basal forebrain, hippocampus, cortex and caudate) from 2-3 animals were pooled and homogenized in 50 mM Tris.HC1 buffer, pH 7.6, using a polytron. The homogenate was spun at 30,000 × g for 10 min, washed and spun again. The pellet was resuspended in 20 ml buffer and incubated for 10 min at 37 °C in a shaker bath; followed by centrifugation at 30,000 × g for 10 min. The pellet was then homogenized in 50 mM Tris.HC1 buffer, pH 7.4, containing 100/~M pargyline and 0.02 ~ ascorbic acid, and incubated at 37 °C for 10 mln. Samples (1 ml) of this homogenate were added to test tubes containing the appropriate concentrations of label in 0.5 ml, and 0.5 ml of buffer with or without cold serotonin to a final concentration of 1 #M. After 10 min at 37 °C, the tubes were transferred to an ice-water bath and the contents filtered through GF/B glass fiber
222
_'5f 0
20
'-'
15
I
°=
o2 I
PE~
J
EI g rn
~:~o
O
BASAL F~EBRAIN HIPPOCAMPUS
/111 CORTEX
CAUDATE
Fig. 1. Specificbinding of [aH]5HT (5 nM) to several brain regions from female rats at different stages of the estrous cycle. D1, diestrous 1 ; D2, diestrous 2 ; PE, proestrous ; E, estrous. Results are means :E S.E.M. of 2-6 determinations. * Significantly lower than estrous and diestrous (Anova: F = 7.92, P < 0.002; Duncan's test: P < 0.05). filters, followed by 2 × 5 ml washes with ice-cold buffer. The filters were then transferred to vials and shaken overnight with 10 ml of toluene-Triton scintillation fluid, and counted in a Packard Tri-Carb scintillation counter, at 30~o efficiency. Protein was determined according to Lowry et al. 1°. In one series of experiments, we measured the specific binding of serotonin (5 nM) to several brain regions from cycling female rats (Fig. 1). The results were subjected to an analysis of variance followed by Duncan's test for multiple comparisons. In the basal forebrain (including the hypothalamus, septum and preoptic area) we found a clear and significant difference between the binding on diestrous-1 and 2 and proestrous-estrous. The binding on proestrous and estrous was about 40 °/o lower than on diestrous. In the hippocampus, a similar direction was observed, which, however, did not reach statistical significance. There was no effect of the cycle on serotonin binding in the cortex and caudate. A reduction in binding could be the result of changes in receptor affinity and/or concentration. In order to investigate these possibilities, we have performed complete saturation experiments on the basal forebrain region. Fig. 2 illustrates the binding curves and Scatchard plots obtained in one experiment for males, and females in diestrous-1 compared to proestrous. Similar comparisons were made for the other stages of the cycle. On the basis of the first series of experiments we have decided to pool the (very similar) Ka and Bmax values from diestrous-I and 2; and f r o m proestrous and estrous females. The results are summarized in Table I. Serotonin is thought to be strongly involved in the regulation of sexual behavior. Many reports have demonstrated an inhibitory role for serotonin on sexual behavior in both males and females in several species a,la,xs. It has also been shown to inhibit ovulation in the female 16. In this context, our results are not surprising: since serotonin is inhibitory for both sexual behavior and ovulation, one would expect it to be down-regulated when sexual behavior and ovulation occur - - namely, on the days
223 [SH]-SHT Bound(cpm xlO-3) 0
I I
2 I
3 I
4 I
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\
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[3HI 5 HT, nM Fig. 2. Binding o f [SH]5HT to basal forebrain from: (a) male, (b) female rats. The top drawings are the Scatchard plots of the binding data. ~', males; PE, proestrous females; DI, diestrous-1 females.
of proestrous and estrous. Interestingly, serotonin levels have been reported to go downY, 9 on proestrous, while serotonin uptake increases1, lz. Thus the reduction in serotonin receptor levels seems to be one of a number of mechanisms activated on proestrous day so as to decrease serotoninergic transmission. The mechanism responsible for the changes in receptor levels reported here is not known. However, the basal forebrain, containing the hypothalamus and septalpreoptic area, is considered to be the main target of sex steroids in the brain14; and steroidal sex hormones are known to exert a large part of their effects through an TABLE I
Binding characteristics o f serotonin to basal forebrain from males and females at different stages o f the estrous cycle
Results are the means + S.E.M. o f 4-8 experiments. Males (n = 5)
Proestrousandestrous
Diestrous I and 2 (n = 4)
(n = 8)
Ka (nM) 2.6 -t- 0.5 Bm==x (pm/rng protein) 0.18 -t- 0.02
1.9 -4- 0.6 0.11 ± 0.01"
2.2 -t- 0.3 0.17 -t- 0.009
* Significantly lower than other groups. (Anova: F = 6.72, P < 0.01 ; D u n c a n ' s test: P < 0.05).
224 interaction with gene expression a n d regulation of protein synthesis 4. Since m o n o a m i n e receptors are believed to be m e m b r a n a l proteins, it is possible that sex h o r m o n e s are involved in their synthesis or degradation. Estradiol in particular has been shown to influence the levels of progesterone receptors in the brain 11 testosterone receptors in the chick oviduct 19 a n d n o r e p i n e p h r i n e receptors Iboth a and /¢) in the uterusS, ~v. F u r t h e r experiments with gonadectomized animals and sex steroid replacement are needed in order to determine the possible role of this h o r m o n e in the regulation of m o n o a m i n e receptor levels in the brain.
l Biegon, A. and Samuel, D., Effect of sex and endocrine state on neurotransmitter uptake, submitted for publication. 2 Burt, D. R., Creese, I. and Snyder, S. H., Dopamine receptor binding enhancement accompanies lesion induced behavioral supersensitivity, Science, 197 (1977) 596--598. 3 Carter, S. C. and Davis, J. M., Biogenic amines, reproductive hormones and female sexual behavior: a review, Behav. Revs., 1 (1977) 213-224. 4 Chan, L. and O'Malley, B. W., Mechanism of action of sex steroid hormones, New Engl. J. Med., 294 (1976) 1322-1328. 5 Dolphin, A., Adrien J., Hamon M. and Bockaert J., Identity of [aH]dihydroalprenolol binding sites and fl-adrenergic receptors coupled with adenylate cyclase in the central nervous system: pharmacological properties, distribution and adaptive responses, Molec. Pharmacol., 15 (1979) 1-15. 6 Fillion, G., Fillion, M. P., Spirakis, C., Bahers, J.-M. and Jacob, J., 5-Hydroxytryptamine binding to synaptic membranes from rat brain, Li]e Sci., 18 (1976) 65-74. 7 Greengrass, P. M. and Tonge, S. R., Changes in brain monoamine concentrations during the estrous cycle in the mouse: possible pharmacological implications, J. Pharm. Pharmacol. 23 (1971) 897-898. 8 Krall, J. F., Mori, H., Tuck, M. L., Leshon, S. L. and Korenman, S. G., Demonstration of adrenergic catecholamine receptors in rat myometrium and their regulation by sex steroid hormones, Life Sci., 23 (1978) 1073-1082. 9 Kueng, W., Wirz-Justice, A., Menzi, B. and Chappuis-Arndt, E. Regional brain variation of tryptophan monoamines, monoamine oxidase activity, plasma free tryptophan and total tryptophan during the estrous cycle of the rat, Neuroendocr., 21 (1976) 289-296. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurements with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 11 MacLusky, N. J. and McEwen, B. S., Oestrogen modulates progestin receptor concentrations in some rat brain regions but not in others, Nature (Lond.), 274 (1978) 276-278. 12 Meyer, D. C. and Quay, W. B., Hypothalamic and suprachiasmatic uptake of serotonin in vitro: twenty four hour changes in male and proestrous female rats, Endocrinology, 98 (1976) 1160-1165. 13 Meyerson, B. J., Correr, H. and Eliasson, M., 5-Hydroxytryptamine and sexual behavior in the female rat. In E. Costa, G. L. Gessa and M. Sandler, (Eds.), Advances in Biochemical Psychopharmacology, VoL II, Raven Press, N.Y., 1974, pp. 229-242. 14 Morell, J. I. and Pfaff, D. W., A neuroendocrine approach to brain function: localization of sex steroid concentrating cells in vertebrate brains, Amer. Zool., 18 (1978) 447-460. 15 Nelson, D. L., Herbert, A., Bourgoin, S., Glowinski, J. and Hamon, M., Characteristics of central 5-HT receptors and their adaptive changes following intracerebral 5,7,dihydroxytryptamine administration in the rat, Molec. PharmacoL, 14 (1978) 983-995. 16 O'Steen, W. K., Serotonin suppression of luteinization in gonadotropin treated immature rats, Endocrinology, 74 (1964) 885-888. 17 Roberts, J. M., Goldfien, R. D., Insel, P. A. and Tsuchiya, A. M., Uterine a adrenergic receptors increase but platelet a adrenergic receptors decrease with estrogen, Clin. Res., 26 (1978) 494. 18 Tagliamonte, A., Tagliamonte, P., Gessa, G. L. and Brodies, B. B., Compulsive sexual activity induced by p-chlorophenylalaninein normal and pinealectomized male rats, Science, 166 (1969) 1433-1435.
225 19 Tokarz, R. R., Seaver, S. R. and Harrison R. W., Estrogen regulation of testosterone receptor concentration in the chick oviduct, J. Cell BioL, 79 (1978) 193. 20 Whitaker, P. M. and Seeman, P., High affinity ZH-serotonin binding to caudate: inhibition by hallucinogens and serotoninergic drugs, Psychopharmacology, 59 (1978) 1-5.