- Email: [email protected]
Regulation by estradiol of GABAA and GABAB binding sites in the diencephalon of the rat: an autoradiographic study
144
Brain Research, 503 (1989) 144-147 Elsevier
BRES 23793
Regulation by estradiol of GABAA and GABAe binding sites in the diencephalon of the rat: an autoradiographic study Anne-Marie Fran~ois-Bellan l, Louis Segu 2 and Micheline H6ry ~ SLaboratoire de M#decine Exp~rimentale, INSERM U297, Marseille and 2Laboratoire de Neurobiologie, C.N.R.S., Marseille (France) (Accepted 25 July 1989)
Key words: Estradiol; GABA receptor; GABA n binding site; Quantitative autoradiography; Suprachiasmatic nucleus; Rat
Using in vitro quantitative aute~'adiography we studied the in vine effects of estradiol on G A B A A and GABA n receptors in the rat brain. In all the areas studied (suprachiasmatic nucleus, medial preoptic area, striatum, frontal cortex), estradiol failed to significantly affect the GABAA receptor density. Chronic treatment with estradiol led however in the suprachiasmatie nucleus and the striatum to a decrease in the density of GABA B receptors. GABA n receptor regulation by estradiol was found to be area-specific within the hypothalamus since it was not observed in the medial preoptic area. The down regulation of GABA~, receptors in the suprachiasmatie nucleus induced by estradiol treatment might thus explain the inhibitory effect of the steroid on the GABA control of serotonin metabolism we recently reported.
In a previous study, we showed that the stimulation of y-aminobutyric acid (GABA) transmission by systemic administration of either amino-oxyacetic acid (AOAA), a GABA transaminase inhibitor, or RS-baclofen, a GABA e receptor agonist, leads to an increase in serotonin (5-HT) release and synthesis in the suprachiasmatic area (SCA) of the male rat s. More recently, we reported that the serotoninergic system of the SCA of ovariectomized (OVX) rats responds to AOA~. :r.'atment in the same way as that of the males; however, when OVX are chronically treated with a subcutaneous implant of estradiol (E2), A O A A treatment fails to affect 5-HT metabolism in the SCA 9. We likewise observed that systemic administration of RS-baclofen affected neither the 5-HT content nor the release and synthesis of the amine in the SCA of ovariectomized-estradiol-treated (OVX-E2) rats, while the same RS-baclofen treatment increased all of these parameters of 5-HT metabolism in male and OVX SCA's (unpublished data). The latter observation raised the possibility that the steroid might have an effect on the density of GABA B receptors involved in the GABAergic control of 5-HT metabolism in the SCA. Could oestrogenization possibly produce a down-regulation of G A B A . receptors in the SCA, which would explain the inhibitory effect of E 2 on GABA/5-HT interaction? Gonadal steroids have been shown to affect the number and/or affinity of receptor sites for several neurotransmitters in the central nervous system TM. A
chronic E2 treatment has been shown to regulate the binding of neurotensin 25 in the suprachiasmatic nucleus. Numerous conflicting reports indicate that E 2 may influence the GABAA receptor density. The [3H]muscimoi binding to crude membranes from the hippocampus, striatum and cerebral cortex has been shown to either decrease tLt2 or increase 2° after chronic treatment by E 2. Lasaga et al. 16 recently demonstrated that hypothalamic membranes from female rats showed an increase in [3H]muscimol binding in response to chronic E2 administration. Recently, using autoradiography, O'Connor et ai. 26 have shown that in vine E2 decreases muscimol binding in the preoptic suprachiasmatic nucleus. At present there is no information available on the effects of E2 on G A B A n receptors. The aim of the present study was to examine the influence of E 2 on the density of the two types of GABA receptors in the suprachiasmatic nucleus, since we postulated that the inhibitory effect of E 2 on GABAIS-HT interaction, which we recently reported, could be explained in terms of a down-regulation of GABAB receptors induced by the steroid. To test the regional specificity of the effect of E 2 on G A B A B receptors, we also studied other diencephalic regions known to have differential capacitities to specifically a c c u m u l a t e [3H]estradiol2S: the cerebral cortex, the striatum and the medial preoptic area. For this purpose we developed an in vitro quantitative autoradiographic technique based on the principle of the method published by Bowery et al. 2.
Correspondence: M. H~ry, Lab. M~decine Experimentale, INSERM U297, Fac. M6dicine, Boulevard Pierre Dramard, 13326 Marseille eedex 15, France.
0006-8993/891503.50 C~) 1989 Elsevier
Science P.hli;h~r~ R V (ginmpdirnl I~;vi~;nn~
145 Adult male and female Wistar rats (weighing 200-250 g) were submitted to a 14-h light/10-h dark cycle (light from 05.00 to 19.00 h). They had food and water available ad libitum in a temperature-controlled (22 + 2 *(2) room. Females were ovarieetomized (OVX). Three weeks later, some of them (OVX-E2) were subcutaneously implanted with a silicone elastomer capsule containing estradiol-17fl (Merck)4. Silastic capsule containing 10 nag E2 gave plasma E2 levels of approximately 100 pg/mi. All the females were sacrificed 4 weeks after ovariectomy. All animals were decapitated between 10.00 and 11.00 h to avoid circadian fluctuations. The brains were rapidly extracted from the skull at 4 °C, and quickly freezed by immersion in isopentane refrigerated by liquid nitrogen. They were stored at -20 *C. Cryostat (-20 *C) 20-gin coronal sections were thaw-mounted on subbed slides (1% gelatin, 0.1% chromealun). They were stored at -20 *C. To eliminate endogenous iigands, sections were preineubated in Tris-HC! (50 raM; pH = 7.4) for one hour at 4 *C. Sections were incubated 45 rain at 4 °C in 50 mM Tris-HCi pH 7.4 added to 2.5 mM CaCI26 and 50 nM [3H]GABA (Amersham, Specific activity 60-100 Ci/mmol) to obtain total binding fiB). The presence of 100#M RS-baclofen (Ciba-Geigy) in the incubation solution displaced the radioligand from type B GABA binding sites2; sections incubated under these conditions allowed measurement of [3H]GABA binding to type A binding sites. Conversely, sections incubated with 40 /~M isoguvacine (RBI) made it possible to determine [aH]GABA binding to type B binding sites. Non-specific binding occurred when 100 btM GABA (Sigma) was added to the incubation solution, After the incubation, sections were rinsed for 1 rain 45 s in 3 successive baths containing 50 mM Tris-HCl pH 7.4 with CaCIz (2.5 mM), and warm-air-dried. Radioactive sections and tritium standards (Microscales 1.3-33 nCi/mg tissue equivalent, Amersham) were apposed to a tritiumsensitive film (Ultrofilm, LKB) in a darkproof box. After 3 months of exposure, films were developed for 6 rain with D19 (Kodak), quickly rinsed, fixed 20 min with ALA (Kodak), and rinsed with tapwater and distilled water. Brain areas were identified by observing the sections stained by Cresyl violet. Quantitative autoradiography (QAR) was carried out with a computerized image analysis system (BIOLAB). From each autoradiogram we sampled 8 'punch' measures in each structure of interest. We repeated these measures on 10-20 different sections, obtained from 3 animals per experimental group, Results were expressed as the mean + S.E.M. (fmol/mg of tissue equivalent) of all these measurements. Statistical comparisons were made using a one-way analysis of variance (ANOVA: VAR3 program29) which provides evidence of the main effects and paired corn-
TABLE I
Estradiol regulation of [3H] GA BA binding sites in the diencephalon Brain sections were incubated in 50 nM [3HIGABA added to 100~M RS-baclofen to obtain A type sites, or to 40 #M isoguvacine to observe B type sites. Quantitative analysis of autoradiograms allowed the determination of specific binding. Measurements were carded out on different brain areas for male, ovariectomized (OVX) and ovarieetomized estradiol-implanted (OVX-Ez) rats. Results expressed as fmol/mg tissue equivalent are the mean + S.E.M. of 10-20 sections obtained from 3 animals per experimental group.
Anatomical structure Suprachiasmatic nucleus
Site Male type
OVX
OVX.Ez
A B
5.13_+0.85 6.38+0.92 13.48-+1.10 12.41_+0.97
4.94_+1.12 9.76_+0.69***
A B
4.73-+0.56 10.40+0.89
4.09_+0.69 9.44-+0.76
A B
17.87-+1.05 15.58+0.61 15.00+0.91 10.14+0.91 10.55_+0.73 7.65-+1.17'
Frontal cortex somatosensory area A B
33.14_+3.36 25.32_+2.33 24.62_ + 1.64" 44.28_+4.34 33.66_+1.89'* 40.80_+4.69
Medial preoptic area
Striatum
4.21_+0.50 9.97+0.74
*P < 0.05, **P < 0.025 when compared to male values; +P < 0,05 when compared to OVX values (VAR3 paired comparisons).
parisons between groups. Our results show that the labeling of brain sections is heterogeneously distributed. The anatomical distribution densities of A and B types observed is in agreement with previous results obtained by [3H]muscimol high affinity binding for A sites27, by [3H]baclofen in the case of B sites 1° and with the competition method as presently used3. The densities of [3H]GABA binding obtained by OAR on some brain structures of male, OVX and OVX-E2 rats (Table I) were compared using a one-way analysis of variance. In the suprachiasmatic nucleus, no difference in GABAA receptor density was observed among the 3 experimental groups of animals (/~2.39 = 0.84, n.s.); whereas the GABAn site densities in this structure were significantly lower in the OVX-E2 group than in the male (Ft.26 = 6.84, P < 0.025) and OVX groups (Fi,39 -'- 4.85, P < 0.05), a Group effect was found (F2.~ = 3.66, P < 0.05). In the medial preoptic area, no differences in either GABAA or GABAB receptor density was observed among the 3 animal groups (F < 1 whatever the comparisons). In the striatum, GABAA site densities in females were lower than in males (OVX and OVXE 2 compared to males F1,49 = 5.66, P < 0.025); GABA n site densities were lower in OVX-E2 than in OVX (Ft,43 = 4.99, P < 0,05). In the somatosensory area of frontal cortex (FCSSA) the GABAA site densities were lower in females than in males (OVX and OVX-E2
146 compared to males FI,49 = 5.85, P < 0.025); the lowest GABAn site densities were observed in the OVX group (male and OVX-E, compared to OVX Fi.6o = 5.14, P < 0.025). All other Group effects or particular paired comparisons were not significant. l'hese results provide the first experimental evidence that brain GABAn receptors may be regulated by gonadal steroids. A chronic estradiol teatment leads in the suprachiasmatic nucleus to a decrease in the density of GABA n receptors as compared with both males and OVX. We recently reported that this receptor subtype is involved in the GABAergic control of SCN 5-HT metabolism7"a. We therefore propose that the downregulation of GABAn receptors in the SCN induced by E 2 may explain the lack of 5-HT responsiveuess to GABA transmission stimulation in OVX-E 2 as the result of either A O A A or RS-baclofen tr.o.at.ment9. In the SCN, a decrease in GABAA receptor density not statistically different may be observed in OVX-E 2 as against OVX. This finding agrees with the results obtained by O'Connor et a126 showing a decrease in [3H]muscimol binding sites in the SCN after acute or chronic E 2 treatment. We have to observe that under our conditions, we detected high and low affinity GABA A binding sites, thus obscuring the changes in high affinity binding sites. O'Connor et al. 26 observed that whatever the duration of treatment, E2 failed to affect the GABA A receptor density in the medial preoptic area. This is also consistent with the present report since in this structure GABA A receptor density is not affected by chronic E 2 treatment. In the striatum and FCSSA, GABA A receptor density is lower when comparing the two female groups to the male group. Since GABAA receptor density does not differ between OVX and OVX-E2, it thus emerges that in these structures chronic E2 treatment fails to affect GABA A receptor binding sites. In this respect, our results are in contrast with those by G6etz et al. tl and Hamon et al. t~" showing a decrease in [3H]muscimol binding sites in the striatum and cerebral cortex following E 2 implantation, and the findings by Maggi and Perez2° reporting an increase in GABA A receptor density in these structures after chronic E 2 administration. Our results are in keeping, however, with those by O'Connor et al. 26 who did not observe any change in [aH]muscimol binding in the striatum of E2-treated rats. The difference in GABA A receptor density in the striatum and FCSSA between the two female groups and the male group suggests a sex difference in the density of this receptor subtype. It has been previously shown that in some brain structures, the number of benzodiazepine receptors, is significantly lower in females than in males 3~. In the present study, the. main target for E 2 effects concerns the GABA a receptor subtype. Within the
hypothalamus, the decrease in GABAn receptor density induced by chronic E 2 treatment is area-specific since it occurs in the SCN and not in the ventral part of the medial preoptic area, which is in the vicinity of the SCN. Treatment of OVX rats with E 2 decreases the GABAn receptor density not only in the SCN but also in the striatum. The functional significance of an effect of E2 on GABAB receptors in the striatum, a brain region that has not been clearly involved in neuroendc.cd~c function, remains an open question. The striatal effects of E 2 can be linked up with clinical studies showing a correlation between variations in E2 plasma levels and modifications in the neuronal activity of the basal ganglia21. Biochemical studies have further shown that an implantation of E2 in the striatum induced a decrease in GAD activity in the substantia nigra 19. The E2-induced decrease in the density of GABAn receptors could be interpreted as a response to an acceleration of presynaptic GABA turnover in OVX-E2. This hypothesis can be ruled out, however, since we recently reported that E 2 has no effect on GABA metabolism in the SCN 9. It is therefore probable that the GABAn receptor down-regulation induced by E2 corresponds to an effect of the steroid on cells postsynaptic to GABAergic terminals. The decrease in GABA a receptor density observed in the SCN after chronic E 2 treatment amounts to around 25%. This difference resembles that observed by numerous authors on various membrane receptors after in vivo E 2 treatment ramS. Since in our study this relatively slight change in GABA n receptor density might be responsible for the total inhibition of the GABA/5-HT functional interaction we previously described, only a minority of the GABA B receptors present in the SCN are presumably involved in this t~euronal interaction. Another explanation for the functional correlate of this reduced alteration in GABAB receptor density may be an additional effect of E 2 o n the coupling of GABA B receptors with adenylate cyclase13,23,3°. There are a number of mechanisms through which E 2 might regulate GABAB binding site density within the SCN and the striatum. One possiblility is that E 2 might directly affect the genomic transcription of GABAB receptors within E2-accumulating c e l l s 22. Such an effect is unlikely to be exerted within the SCN and the striatum themselves, since virtually none of the neurons in these structures have been found to contain E 2 receptors5.24.2s. Another mechanism to account for the action of E 2 is that steroid hormones may alterate some of cell membrane properties is. The third possibility is that the receptor changes we have observed after chronic E 2 treatment might reflect a change in prolactin secretion ~2,~5.17. In conclusion, we have shown for the first time, using a quantitative autoradiographic technique, that chronic
147 E 2 t r e a t m e n t decreases the density of G A B A a receptors within the SCN. We assume that this down-regulation of G A B A a receptors may be involved in the non-responsiveness of 5 - H T metabolism to G A B A transmission stimulation in the SCN of O V X - E z 9, but the mechanism through which the ovarian steroid exerts its effect requires further investigations.
The authors wish to thank Dr. J. Lanoir and Dr. O. Bos!er for critically reading the manuscript. The authors are indebted to Pierre Rage for performing the 'Biolab' program, to Patricia Scardigli for doing the analysis of variance and Bernadette Besson for the typing. They thank Ciba Geigy (Basle) for the generous gift of RS-baclofen and Ciba Geigy (Rueil Malmaison) for financial assistance.
1 Biegon, A., Reches, A., Snyder, L. and McEwen, B.S., Serotonergic and noradrenergic receptors in the rat brain: modulation by chronic exposure to ovarian hormones, Life Sci., 32 (1983) 2015-2021. 2 Bowery, N.G., Hill, D.R. and Hudson, A.L., Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes, Br. J. Pharmacol., 78 (1983) 191-206. 3 Bowery, N.G., Hudson, A.L. and Price, G.W., GABAA and GABAn receptor site distribution in the rat central nervous system, Neuroscience, 20 (1987) 365-383. 4 Chazal, G., Faudon, M., Gogan, E, H~ry, M., Kordon, C. and Laplante, E., Circadian rhythm of luteinizing hormone secretion in the ovariectomized rat implanted with oestradiol, J. EndocrinoL, 75 (1977) 251-260. 5 Cintra, A., Fuxe, K., Harfstrand, A., Agnat, L.E, Miller, L.S., Greene, J.L. and Gustaffson, J.A., On the cellular localization and distribution of estrogen receptors in the rat tel- and diencephalon using monoclonal antibodies to human estrogen receptor, Neurochem. Int., 8 (1986) 587-595. 6 Cords, M.G. and Guidotti, A., Modulation of GABA receptor binding by Caz+, J. Neurochem., 41 (1983) 277-280. 7 Fran~ois-Bellan, A.M., H~ry, M., Barrit, M., Faudon, M. and H6ry, E, The stimulation of GABAB receptors increases serotonin release in the rat suprachiasmatic area, Neurochem. Int., 11 (1987) 55-62. 8 Fran~ois-Bellan, A.M., H6ry, M., Faudon, M. and H6ry, F., Evidence for GABA control of serotonin metabolism in the rat suprachiasmatic area, Neurochem. Int., 13 (1988) 455-462. 9 Fram;ois-Bellan, A.M., H~ry, M., Segu, L., Faudon, M. and Hdry, E, The effects of estrogenization on GABA metabolism in the hypothalamus: consequences on GABA/5-HT metabolic interaction, ENA/EBBS, Zurich, September 1988. 10 Gehlert, D.R., Yamamura, H.I. and Wamsley, LK., y-Aminobutyric acid s receptors in the rat brain: quantitative autoradiographic localization using [SH](-)-baclofen, Neurosci. Lett., 56 (1985) 183-188. 11 Goetz, C., Bourgoin, S., Cesselin, F., Brandi, A., Bression, D., Martinet, M., Peillon, E and Hamon, M., Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats, Neurochem. Int., 5 (1983) 375-383. 12 Hamon, M., Goetz, C., Euvrard, C., Pasqualini, C., Le Dafnier, M., Kerdelhue, B., Cesselin, E and Peillon, E, B;,ochemical and ~unctional alterations of central GABA receptors during chronic estradiol treatment, Brain Research, 279 (1983) 141-152. 13 Hill, D.R., GABA s receptor modulation of adenylate cyclase activity in rat brain slices, Br. J. PharmacoL, 84 (1985) 249-257. 14 Hrsuka, R.E., Elevation of striatal dopamine receptors by estrogen: dose and time studies, J. Neurochem., 47 (!986) 1908-1915. 15 Hruska, R.E. and Pitman, K.T., Distribution and localization of estrogen sensitive dopamine receptors in the rat brain, J. Neurochem., 39 (1982) 1418-~422. 16 Lasaga, M., Duvilanski, B.H., Seilicovich, A., Afione, S. and
Debeljuk, L., Effect of sex steroids on GABA receptors in the rat hypothalamus and anterior pituitary gland, Fur. ,!. Pharmacol., 155 (1988) 163-166. Locatelli, V., Apud, J.A., Gudelsky, G.A, Cocchi, D., Masotto, C., Casanueva, F., Racagni, G. and Mtiller, E.E., Prolactin in cerebrospinal fluid increases the synthesis and release of hypothalamic 7-aminobutyric acid, J. Endocrinol., 106 (1985) 323328. Mac Ewen, B.S., Neural gonadal steroid actions, Science, 211 (1981) 1303-1311. Mac Ginnis, M.Y., Gordon, J.H. and Gorski, R.A., Time course and localization of the effects of estrogen on glutamic acid decarboxylase activity, J. Neurochem., 34 (1980) 785-792. Maggi, A. and Perez, J., Progesterone and estrogens in rat brain: modulation of GABA (7-aminobutyric acid) receptor activity, Fur. J. PharmacoL, 103 (1984) 165-168. Maggi, A. and Perez, J., Role of female gonadal hormones in the CNS: clinical and experimental aspects, Life Sci., 37 (1985) 893-906. Maggi, A. and Perez, J., Estrogen induced up-regulation of gamma-aminobutyric acid receptors in the CNS of rodents, J. Neurochem., 47 (1986) 1793-1797. Maus, M., Cordier, J., Glowinski, J. and Premont, J., In vitro potentiation by 17fl-oestradiol pretreatment of biogenic aminesensitive adenylate cyclases in mouse embryonic striatal neurones in primary culture, Eur. I. Neurosci., in press. Morrell, J., Krieger, M.S. and Pfaff, D.W., Quantitative autoradiographic analysis of estradiol retention by cells in the preoptic area, hypothalamus a'~d amygdala, Exp. Brain Res., 62 (1986) 343-354. Moyse, E., Miller, M.M., Rost6ne, W. and Beaudet, A., Effects of ovariectomy and estradiol replacement on the binding of ~:Sl-neurotensin in rat suprachiasmatic nucleus, Neuroendocrinology, 48 (1988) 53-60. O'Connor, L.H., Nock, B. and McEwen, B.S., Regional specificity of gamma-aminobutyric acid receptor regulation by estradioi, Neuroendocrinology, 47 (1988) 473-481. Palacios, J.M., Wamsley, J.K. and Kuhar, M.J., High affinity GABA receptors autoradiographic localization, Brain Research, 222 (1981) 285-307. Pfaff, D.W. and Conrad, L.C.A., Hypothalamic neuroanatomy: steroid hormone binding and patterns of axonal projections, in G. Bourne (Ed.), International Review of Cytology, Academic, New York, 1978, pp. 245-265. Rouanet, H. and Lepine, D., Comparisons between treatments in a repeated measurement design: ANOVA and multivariate methods, Br. J. Math. Statis. Psychol., 23 (1970) 147-163. Scherer, R.W., Ferkany, J.W. and Enna, S.J., Evidence for pharmacologically distinct subsets of GABAB receptors, Brain Res. Bull., 21 (1988) 439-443. Shepard, R.A., Nielsen, E.B. and Broadhurst, D.L., Sex and strain differences in benzodiazepine receptor, Fur. J. Pharmacol., 77 (1982) 327-330.
17
18 19 20 21 22 23
24
25
26 27 28
29 30 31
Brain Research, 503 (1989) 144-147 Elsevier
BRES 23793
Regulation by estradiol of GABAA and GABAe binding sites in the diencephalon of the rat: an autoradiographic study Anne-Marie Fran~ois-Bellan l, Louis Segu 2 and Micheline H6ry ~ SLaboratoire de M#decine Exp~rimentale, INSERM U297, Marseille and 2Laboratoire de Neurobiologie, C.N.R.S., Marseille (France) (Accepted 25 July 1989)
Key words: Estradiol; GABA receptor; GABA n binding site; Quantitative autoradiography; Suprachiasmatic nucleus; Rat
Using in vitro quantitative aute~'adiography we studied the in vine effects of estradiol on G A B A A and GABA n receptors in the rat brain. In all the areas studied (suprachiasmatic nucleus, medial preoptic area, striatum, frontal cortex), estradiol failed to significantly affect the GABAA receptor density. Chronic treatment with estradiol led however in the suprachiasmatie nucleus and the striatum to a decrease in the density of GABA B receptors. GABA n receptor regulation by estradiol was found to be area-specific within the hypothalamus since it was not observed in the medial preoptic area. The down regulation of GABA~, receptors in the suprachiasmatie nucleus induced by estradiol treatment might thus explain the inhibitory effect of the steroid on the GABA control of serotonin metabolism we recently reported.
In a previous study, we showed that the stimulation of y-aminobutyric acid (GABA) transmission by systemic administration of either amino-oxyacetic acid (AOAA), a GABA transaminase inhibitor, or RS-baclofen, a GABA e receptor agonist, leads to an increase in serotonin (5-HT) release and synthesis in the suprachiasmatic area (SCA) of the male rat s. More recently, we reported that the serotoninergic system of the SCA of ovariectomized (OVX) rats responds to AOA~. :r.'atment in the same way as that of the males; however, when OVX are chronically treated with a subcutaneous implant of estradiol (E2), A O A A treatment fails to affect 5-HT metabolism in the SCA 9. We likewise observed that systemic administration of RS-baclofen affected neither the 5-HT content nor the release and synthesis of the amine in the SCA of ovariectomized-estradiol-treated (OVX-E2) rats, while the same RS-baclofen treatment increased all of these parameters of 5-HT metabolism in male and OVX SCA's (unpublished data). The latter observation raised the possibility that the steroid might have an effect on the density of GABA B receptors involved in the GABAergic control of 5-HT metabolism in the SCA. Could oestrogenization possibly produce a down-regulation of G A B A . receptors in the SCA, which would explain the inhibitory effect of E 2 on GABA/5-HT interaction? Gonadal steroids have been shown to affect the number and/or affinity of receptor sites for several neurotransmitters in the central nervous system TM. A
chronic E2 treatment has been shown to regulate the binding of neurotensin 25 in the suprachiasmatic nucleus. Numerous conflicting reports indicate that E 2 may influence the GABAA receptor density. The [3H]muscimoi binding to crude membranes from the hippocampus, striatum and cerebral cortex has been shown to either decrease tLt2 or increase 2° after chronic treatment by E 2. Lasaga et al. 16 recently demonstrated that hypothalamic membranes from female rats showed an increase in [3H]muscimol binding in response to chronic E2 administration. Recently, using autoradiography, O'Connor et ai. 26 have shown that in vine E2 decreases muscimol binding in the preoptic suprachiasmatic nucleus. At present there is no information available on the effects of E2 on G A B A n receptors. The aim of the present study was to examine the influence of E 2 on the density of the two types of GABA receptors in the suprachiasmatic nucleus, since we postulated that the inhibitory effect of E 2 on GABAIS-HT interaction, which we recently reported, could be explained in terms of a down-regulation of GABAB receptors induced by the steroid. To test the regional specificity of the effect of E 2 on G A B A B receptors, we also studied other diencephalic regions known to have differential capacitities to specifically a c c u m u l a t e [3H]estradiol2S: the cerebral cortex, the striatum and the medial preoptic area. For this purpose we developed an in vitro quantitative autoradiographic technique based on the principle of the method published by Bowery et al. 2.
Correspondence: M. H~ry, Lab. M~decine Experimentale, INSERM U297, Fac. M6dicine, Boulevard Pierre Dramard, 13326 Marseille eedex 15, France.
0006-8993/891503.50 C~) 1989 Elsevier
Science P.hli;h~r~ R V (ginmpdirnl I~;vi~;nn~
145 Adult male and female Wistar rats (weighing 200-250 g) were submitted to a 14-h light/10-h dark cycle (light from 05.00 to 19.00 h). They had food and water available ad libitum in a temperature-controlled (22 + 2 *(2) room. Females were ovarieetomized (OVX). Three weeks later, some of them (OVX-E2) were subcutaneously implanted with a silicone elastomer capsule containing estradiol-17fl (Merck)4. Silastic capsule containing 10 nag E2 gave plasma E2 levels of approximately 100 pg/mi. All the females were sacrificed 4 weeks after ovariectomy. All animals were decapitated between 10.00 and 11.00 h to avoid circadian fluctuations. The brains were rapidly extracted from the skull at 4 °C, and quickly freezed by immersion in isopentane refrigerated by liquid nitrogen. They were stored at -20 *C. Cryostat (-20 *C) 20-gin coronal sections were thaw-mounted on subbed slides (1% gelatin, 0.1% chromealun). They were stored at -20 *C. To eliminate endogenous iigands, sections were preineubated in Tris-HC! (50 raM; pH = 7.4) for one hour at 4 *C. Sections were incubated 45 rain at 4 °C in 50 mM Tris-HCi pH 7.4 added to 2.5 mM CaCI26 and 50 nM [3H]GABA (Amersham, Specific activity 60-100 Ci/mmol) to obtain total binding fiB). The presence of 100#M RS-baclofen (Ciba-Geigy) in the incubation solution displaced the radioligand from type B GABA binding sites2; sections incubated under these conditions allowed measurement of [3H]GABA binding to type A binding sites. Conversely, sections incubated with 40 /~M isoguvacine (RBI) made it possible to determine [aH]GABA binding to type B binding sites. Non-specific binding occurred when 100 btM GABA (Sigma) was added to the incubation solution, After the incubation, sections were rinsed for 1 rain 45 s in 3 successive baths containing 50 mM Tris-HCl pH 7.4 with CaCIz (2.5 mM), and warm-air-dried. Radioactive sections and tritium standards (Microscales 1.3-33 nCi/mg tissue equivalent, Amersham) were apposed to a tritiumsensitive film (Ultrofilm, LKB) in a darkproof box. After 3 months of exposure, films were developed for 6 rain with D19 (Kodak), quickly rinsed, fixed 20 min with ALA (Kodak), and rinsed with tapwater and distilled water. Brain areas were identified by observing the sections stained by Cresyl violet. Quantitative autoradiography (QAR) was carried out with a computerized image analysis system (BIOLAB). From each autoradiogram we sampled 8 'punch' measures in each structure of interest. We repeated these measures on 10-20 different sections, obtained from 3 animals per experimental group, Results were expressed as the mean + S.E.M. (fmol/mg of tissue equivalent) of all these measurements. Statistical comparisons were made using a one-way analysis of variance (ANOVA: VAR3 program29) which provides evidence of the main effects and paired corn-
TABLE I
Estradiol regulation of [3H] GA BA binding sites in the diencephalon Brain sections were incubated in 50 nM [3HIGABA added to 100~M RS-baclofen to obtain A type sites, or to 40 #M isoguvacine to observe B type sites. Quantitative analysis of autoradiograms allowed the determination of specific binding. Measurements were carded out on different brain areas for male, ovariectomized (OVX) and ovarieetomized estradiol-implanted (OVX-Ez) rats. Results expressed as fmol/mg tissue equivalent are the mean + S.E.M. of 10-20 sections obtained from 3 animals per experimental group.
Anatomical structure Suprachiasmatic nucleus
Site Male type
OVX
OVX.Ez
A B
5.13_+0.85 6.38+0.92 13.48-+1.10 12.41_+0.97
4.94_+1.12 9.76_+0.69***
A B
4.73-+0.56 10.40+0.89
4.09_+0.69 9.44-+0.76
A B
17.87-+1.05 15.58+0.61 15.00+0.91 10.14+0.91 10.55_+0.73 7.65-+1.17'
Frontal cortex somatosensory area A B
33.14_+3.36 25.32_+2.33 24.62_ + 1.64" 44.28_+4.34 33.66_+1.89'* 40.80_+4.69
Medial preoptic area
Striatum
4.21_+0.50 9.97+0.74
*P < 0.05, **P < 0.025 when compared to male values; +P < 0,05 when compared to OVX values (VAR3 paired comparisons).
parisons between groups. Our results show that the labeling of brain sections is heterogeneously distributed. The anatomical distribution densities of A and B types observed is in agreement with previous results obtained by [3H]muscimol high affinity binding for A sites27, by [3H]baclofen in the case of B sites 1° and with the competition method as presently used3. The densities of [3H]GABA binding obtained by OAR on some brain structures of male, OVX and OVX-E2 rats (Table I) were compared using a one-way analysis of variance. In the suprachiasmatic nucleus, no difference in GABAA receptor density was observed among the 3 experimental groups of animals (/~2.39 = 0.84, n.s.); whereas the GABAn site densities in this structure were significantly lower in the OVX-E2 group than in the male (Ft.26 = 6.84, P < 0.025) and OVX groups (Fi,39 -'- 4.85, P < 0.05), a Group effect was found (F2.~ = 3.66, P < 0.05). In the medial preoptic area, no differences in either GABAA or GABAB receptor density was observed among the 3 animal groups (F < 1 whatever the comparisons). In the striatum, GABAA site densities in females were lower than in males (OVX and OVXE 2 compared to males F1,49 = 5.66, P < 0.025); GABA n site densities were lower in OVX-E2 than in OVX (Ft,43 = 4.99, P < 0,05). In the somatosensory area of frontal cortex (FCSSA) the GABAA site densities were lower in females than in males (OVX and OVX-E2
146 compared to males FI,49 = 5.85, P < 0.025); the lowest GABAn site densities were observed in the OVX group (male and OVX-E, compared to OVX Fi.6o = 5.14, P < 0.025). All other Group effects or particular paired comparisons were not significant. l'hese results provide the first experimental evidence that brain GABAn receptors may be regulated by gonadal steroids. A chronic estradiol teatment leads in the suprachiasmatic nucleus to a decrease in the density of GABA n receptors as compared with both males and OVX. We recently reported that this receptor subtype is involved in the GABAergic control of SCN 5-HT metabolism7"a. We therefore propose that the downregulation of GABAn receptors in the SCN induced by E 2 may explain the lack of 5-HT responsiveuess to GABA transmission stimulation in OVX-E 2 as the result of either A O A A or RS-baclofen tr.o.at.ment9. In the SCN, a decrease in GABAA receptor density not statistically different may be observed in OVX-E 2 as against OVX. This finding agrees with the results obtained by O'Connor et a126 showing a decrease in [3H]muscimol binding sites in the SCN after acute or chronic E 2 treatment. We have to observe that under our conditions, we detected high and low affinity GABA A binding sites, thus obscuring the changes in high affinity binding sites. O'Connor et al. 26 observed that whatever the duration of treatment, E2 failed to affect the GABA A receptor density in the medial preoptic area. This is also consistent with the present report since in this structure GABA A receptor density is not affected by chronic E 2 treatment. In the striatum and FCSSA, GABA A receptor density is lower when comparing the two female groups to the male group. Since GABAA receptor density does not differ between OVX and OVX-E2, it thus emerges that in these structures chronic E2 treatment fails to affect GABA A receptor binding sites. In this respect, our results are in contrast with those by G6etz et al. tl and Hamon et al. t~" showing a decrease in [3H]muscimol binding sites in the striatum and cerebral cortex following E 2 implantation, and the findings by Maggi and Perez2° reporting an increase in GABA A receptor density in these structures after chronic E 2 administration. Our results are in keeping, however, with those by O'Connor et al. 26 who did not observe any change in [aH]muscimol binding in the striatum of E2-treated rats. The difference in GABA A receptor density in the striatum and FCSSA between the two female groups and the male group suggests a sex difference in the density of this receptor subtype. It has been previously shown that in some brain structures, the number of benzodiazepine receptors, is significantly lower in females than in males 3~. In the present study, the. main target for E 2 effects concerns the GABA a receptor subtype. Within the
hypothalamus, the decrease in GABAn receptor density induced by chronic E 2 treatment is area-specific since it occurs in the SCN and not in the ventral part of the medial preoptic area, which is in the vicinity of the SCN. Treatment of OVX rats with E 2 decreases the GABAn receptor density not only in the SCN but also in the striatum. The functional significance of an effect of E2 on GABAB receptors in the striatum, a brain region that has not been clearly involved in neuroendc.cd~c function, remains an open question. The striatal effects of E 2 can be linked up with clinical studies showing a correlation between variations in E2 plasma levels and modifications in the neuronal activity of the basal ganglia21. Biochemical studies have further shown that an implantation of E2 in the striatum induced a decrease in GAD activity in the substantia nigra 19. The E2-induced decrease in the density of GABAn receptors could be interpreted as a response to an acceleration of presynaptic GABA turnover in OVX-E2. This hypothesis can be ruled out, however, since we recently reported that E 2 has no effect on GABA metabolism in the SCN 9. It is therefore probable that the GABAn receptor down-regulation induced by E2 corresponds to an effect of the steroid on cells postsynaptic to GABAergic terminals. The decrease in GABA a receptor density observed in the SCN after chronic E 2 treatment amounts to around 25%. This difference resembles that observed by numerous authors on various membrane receptors after in vivo E 2 treatment ramS. Since in our study this relatively slight change in GABA n receptor density might be responsible for the total inhibition of the GABA/5-HT functional interaction we previously described, only a minority of the GABA B receptors present in the SCN are presumably involved in this t~euronal interaction. Another explanation for the functional correlate of this reduced alteration in GABAB receptor density may be an additional effect of E 2 o n the coupling of GABA B receptors with adenylate cyclase13,23,3°. There are a number of mechanisms through which E 2 might regulate GABAB binding site density within the SCN and the striatum. One possiblility is that E 2 might directly affect the genomic transcription of GABAB receptors within E2-accumulating c e l l s 22. Such an effect is unlikely to be exerted within the SCN and the striatum themselves, since virtually none of the neurons in these structures have been found to contain E 2 receptors5.24.2s. Another mechanism to account for the action of E 2 is that steroid hormones may alterate some of cell membrane properties is. The third possibility is that the receptor changes we have observed after chronic E 2 treatment might reflect a change in prolactin secretion ~2,~5.17. In conclusion, we have shown for the first time, using a quantitative autoradiographic technique, that chronic
147 E 2 t r e a t m e n t decreases the density of G A B A a receptors within the SCN. We assume that this down-regulation of G A B A a receptors may be involved in the non-responsiveness of 5 - H T metabolism to G A B A transmission stimulation in the SCN of O V X - E z 9, but the mechanism through which the ovarian steroid exerts its effect requires further investigations.
The authors wish to thank Dr. J. Lanoir and Dr. O. Bos!er for critically reading the manuscript. The authors are indebted to Pierre Rage for performing the 'Biolab' program, to Patricia Scardigli for doing the analysis of variance and Bernadette Besson for the typing. They thank Ciba Geigy (Basle) for the generous gift of RS-baclofen and Ciba Geigy (Rueil Malmaison) for financial assistance.
1 Biegon, A., Reches, A., Snyder, L. and McEwen, B.S., Serotonergic and noradrenergic receptors in the rat brain: modulation by chronic exposure to ovarian hormones, Life Sci., 32 (1983) 2015-2021. 2 Bowery, N.G., Hill, D.R. and Hudson, A.L., Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes, Br. J. Pharmacol., 78 (1983) 191-206. 3 Bowery, N.G., Hudson, A.L. and Price, G.W., GABAA and GABAn receptor site distribution in the rat central nervous system, Neuroscience, 20 (1987) 365-383. 4 Chazal, G., Faudon, M., Gogan, E, H~ry, M., Kordon, C. and Laplante, E., Circadian rhythm of luteinizing hormone secretion in the ovariectomized rat implanted with oestradiol, J. EndocrinoL, 75 (1977) 251-260. 5 Cintra, A., Fuxe, K., Harfstrand, A., Agnat, L.E, Miller, L.S., Greene, J.L. and Gustaffson, J.A., On the cellular localization and distribution of estrogen receptors in the rat tel- and diencephalon using monoclonal antibodies to human estrogen receptor, Neurochem. Int., 8 (1986) 587-595. 6 Cords, M.G. and Guidotti, A., Modulation of GABA receptor binding by Caz+, J. Neurochem., 41 (1983) 277-280. 7 Fran~ois-Bellan, A.M., H~ry, M., Barrit, M., Faudon, M. and H6ry, E, The stimulation of GABAB receptors increases serotonin release in the rat suprachiasmatic area, Neurochem. Int., 11 (1987) 55-62. 8 Fran~ois-Bellan, A.M., H6ry, M., Faudon, M. and H6ry, F., Evidence for GABA control of serotonin metabolism in the rat suprachiasmatic area, Neurochem. Int., 13 (1988) 455-462. 9 Fram;ois-Bellan, A.M., H~ry, M., Segu, L., Faudon, M. and Hdry, E, The effects of estrogenization on GABA metabolism in the hypothalamus: consequences on GABA/5-HT metabolic interaction, ENA/EBBS, Zurich, September 1988. 10 Gehlert, D.R., Yamamura, H.I. and Wamsley, LK., y-Aminobutyric acid s receptors in the rat brain: quantitative autoradiographic localization using [SH](-)-baclofen, Neurosci. Lett., 56 (1985) 183-188. 11 Goetz, C., Bourgoin, S., Cesselin, F., Brandi, A., Bression, D., Martinet, M., Peillon, E and Hamon, M., Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats, Neurochem. Int., 5 (1983) 375-383. 12 Hamon, M., Goetz, C., Euvrard, C., Pasqualini, C., Le Dafnier, M., Kerdelhue, B., Cesselin, E and Peillon, E, B;,ochemical and ~unctional alterations of central GABA receptors during chronic estradiol treatment, Brain Research, 279 (1983) 141-152. 13 Hill, D.R., GABA s receptor modulation of adenylate cyclase activity in rat brain slices, Br. J. PharmacoL, 84 (1985) 249-257. 14 Hrsuka, R.E., Elevation of striatal dopamine receptors by estrogen: dose and time studies, J. Neurochem., 47 (!986) 1908-1915. 15 Hruska, R.E. and Pitman, K.T., Distribution and localization of estrogen sensitive dopamine receptors in the rat brain, J. Neurochem., 39 (1982) 1418-~422. 16 Lasaga, M., Duvilanski, B.H., Seilicovich, A., Afione, S. and
Debeljuk, L., Effect of sex steroids on GABA receptors in the rat hypothalamus and anterior pituitary gland, Fur. ,!. Pharmacol., 155 (1988) 163-166. Locatelli, V., Apud, J.A., Gudelsky, G.A, Cocchi, D., Masotto, C., Casanueva, F., Racagni, G. and Mtiller, E.E., Prolactin in cerebrospinal fluid increases the synthesis and release of hypothalamic 7-aminobutyric acid, J. Endocrinol., 106 (1985) 323328. Mac Ewen, B.S., Neural gonadal steroid actions, Science, 211 (1981) 1303-1311. Mac Ginnis, M.Y., Gordon, J.H. and Gorski, R.A., Time course and localization of the effects of estrogen on glutamic acid decarboxylase activity, J. Neurochem., 34 (1980) 785-792. Maggi, A. and Perez, J., Progesterone and estrogens in rat brain: modulation of GABA (7-aminobutyric acid) receptor activity, Fur. J. PharmacoL, 103 (1984) 165-168. Maggi, A. and Perez, J., Role of female gonadal hormones in the CNS: clinical and experimental aspects, Life Sci., 37 (1985) 893-906. Maggi, A. and Perez, J., Estrogen induced up-regulation of gamma-aminobutyric acid receptors in the CNS of rodents, J. Neurochem., 47 (1986) 1793-1797. Maus, M., Cordier, J., Glowinski, J. and Premont, J., In vitro potentiation by 17fl-oestradiol pretreatment of biogenic aminesensitive adenylate cyclases in mouse embryonic striatal neurones in primary culture, Eur. I. Neurosci., in press. Morrell, J., Krieger, M.S. and Pfaff, D.W., Quantitative autoradiographic analysis of estradiol retention by cells in the preoptic area, hypothalamus a'~d amygdala, Exp. Brain Res., 62 (1986) 343-354. Moyse, E., Miller, M.M., Rost6ne, W. and Beaudet, A., Effects of ovariectomy and estradiol replacement on the binding of ~:Sl-neurotensin in rat suprachiasmatic nucleus, Neuroendocrinology, 48 (1988) 53-60. O'Connor, L.H., Nock, B. and McEwen, B.S., Regional specificity of gamma-aminobutyric acid receptor regulation by estradioi, Neuroendocrinology, 47 (1988) 473-481. Palacios, J.M., Wamsley, J.K. and Kuhar, M.J., High affinity GABA receptors autoradiographic localization, Brain Research, 222 (1981) 285-307. Pfaff, D.W. and Conrad, L.C.A., Hypothalamic neuroanatomy: steroid hormone binding and patterns of axonal projections, in G. Bourne (Ed.), International Review of Cytology, Academic, New York, 1978, pp. 245-265. Rouanet, H. and Lepine, D., Comparisons between treatments in a repeated measurement design: ANOVA and multivariate methods, Br. J. Math. Statis. Psychol., 23 (1970) 147-163. Scherer, R.W., Ferkany, J.W. and Enna, S.J., Evidence for pharmacologically distinct subsets of GABAB receptors, Brain Res. Bull., 21 (1988) 439-443. Shepard, R.A., Nielsen, E.B. and Broadhurst, D.L., Sex and strain differences in benzodiazepine receptor, Fur. J. Pharmacol., 77 (1982) 327-330.
17
18 19 20 21 22 23
24
25
26 27 28
29 30 31