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Modulation of glutamate receptor expression by gonadal steroid hormones in the rat striatum
Brain Research Bulletin, Vol. 59, No. 4, pp. 289–292, 2003 Copyright © 2002 Elsevier Science Inc. All rights reserved. 0361-9230/02/$–see front matter
PII: S0361-9230(02)00885-7
Modulation of glutamate receptor expression by gonadal steroid hormones in the rat striatum Deborah N. D’Souza, 1† Richard E. Harlan1,2 and Meredith M. Garcia2,3∗ 1 Department of Structural and Cellular Biology, Tulane University Medical School, New Orleans, LA, USA; 2 Program in Neuroscience, Tulane University Medical School, New Orleans, LA, USA; and 3 Department of
Otolaryngology, Tulane University Medical School, New Orleans, LA, USA [Received 5 March 2002; Revised 2 July 2002; Accepted 20 July 2002] but completely inhibited morphine-mediated c-Fos expression in females [15]. This sex difference was eliminated by castration and restored by the administration of testosterone in males, but ovariectomy with or without estrogen replacement did not alter the response to MK-801 in females [14]. Gender differences have been previously reported in the responses to both morphine [5,8,10,23,30] and MK-801 [15,22,24]. Other studies have shown that sex hormones may alter glutamatergic transmission in the brain by regulating expression of glutamate receptors. Estrogen has been found to modulate the AMPA subtype of glutamate receptor (subunits GluR1 and GluR2/3) in a sexually dimorphic manner [13]. Although Brann et al. [6] found that in males or females, castration and steroid replacement did not affect the mRNA levels of the NMDA receptor within the hypothalamus, Gazzaley et al. [19] reported that gonadal hormones do modulate the pharmacological and physiological properties of the NMDA receptor. Thus, the role of gonadal steroids on glutamatergic neurotransmission is somewhat unclear. In our previous study [14], we found that testosterone in males, but not estradiol in females, regulated the ability of MK-801 to block morphine-induced c-Fos expression in the CPu. One mechanism by which this could occur is through steroid regulation of glutamate receptor abundance. Therefore, we tested the hypothesis that gonadal hormones could modulate levels of glutamate receptor subtypes in the CPu and medial thalamus, regions of the brain showing gender differences and effects of hormones on morphineand MK-801-mediated IEG expression. We chose to study the NR1 subunit of the NMDA receptor because it is expressed in all functional NMDA receptors, and the GluR2 subunit of the AMPA receptor because it regulates the permeability of the AMPA receptor to Ca2+ and thus is important in modulating neuronal excitability. We used autoradiographic immunocytochemistry [20,26] to determine levels of striatal NR1 and GluR2 subunits in male (intact, castrated, or castrated + testosterone) and female (intact, ovariectomized, or ovariectomized + estrogen) rats. We predicted that testosterone treatment in males, but not estradiol treatment
ABSTRACT: The non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) attenuates morphine-induced immediate-early gene expression in the rat striatum in a sexually dimorphic manner that depends in part on gonadal steroids. To determine if this effect was dependent on modulation of glutamate receptor gene expression, we studied the effect of gonadal hormones on levels of the NR1 subunit of NMDA receptor and the GluR2 subunit of the AMPA-subtype of glutamate receptor in the rat striatum, using autoradiographic immunocytochemistry. We found that ovariectomy decreased GluR2 immunoreactivity in the striatum, but no changes were seen in levels of NR1 following gonadectomy in either sex. Thus, the effects of gonadal steroids on NMDA receptor-mediated responses are not due to regulation of NR1 expression. © 2002 Elsevier Science Inc. All rights reserved. KEY WORDS: Immediate-early genes, Gonadal hormones, Glutamate receptors, Morphine.
INTRODUCTION Previous studies from this and other laboratories have shown that morphine induces the expression of the immediate-early gene (IEG) c-Fos, predominantly in neurons of the dorsomedial caudate-putamen (CPu; [9,18,25]) and that this expression can be attenuated by pretreatment with the non-competitive NMDA receptor antagonist, dizocilpine (MK-801; [4,15,25]). The intralaminar nuclei of the thalamus, which express mu opiate receptors [7], send a glutamatergic projection to the dorsomedial CPu [3]. Studies done by Frankel et al. [16] have demonstrated that the morphine-induced increase in c-Fos expression in the CPu may be subsequent to activation of these thalamostriatal glutamatergic projections. Thus, glutamate and its cognate receptors may play a central role in mediating the effects of morphine in the striatum. It has previously been shown that MK-801 reduces morphineinduced c-Fos expression in the striatum in a sexually dimorphic manner, where MK-801 partially inhibited this effect in males
Abbreviations: Acb, nucleus accumbens; cc, corpus callosum; Cg, cingulate cortex; CM, central medial nucleus of the thalamus; CPu, caudate-putamen; Par, parietal cortex; PV, paraventricular nucleus of the thalamus; Re, reuniens nucleus of the thalamus; Rh, rhomboid nucleus of the thalamus; Tu, olfactory tubercle; VP, ventral pallidum; 3V, third ventricle ∗ Address for correspondence: Dr. Meredith Mason Garcia, Ph.D., Program in Neuroscience, Tulane University Medical School, 1430 Tulane Ave. SL-59, New Orleans, LA 70112-2699, USA. Fax: +1-504-582-7846; E-mail: [email protected] † Present address: Department of Pharmacology, Loyola University Stritch School of Medicine, Chicago, IL 60153, USA.
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in females, would alter levels of NR1, but perhaps not GluR2, in the CPu. MATERIALS AND METHODS Treatment of Animals All treatment of animals was performed in accordance with NIH guidelines and was approved by the Advisory Committee on Animal Resources of the Tulane University Medical School. All efforts were made to minimize animal suffering and to reduce the number of animals used. Adult male or random-cycling female Sprague–Dawley rats (200–225 gm) were housed in group cages (3–4 per cage) in the Vivarium of the Tulane University Medical School, with controlled temperature and light/dark cycles of 12 h/12 h (lights on, 0600 h; lights off, 1800 h). Animals were provided with unlimited access to food (Purina rat chow) and tap water. The animals were assigned to groups as follows: intact males, castrated males, castrated males treated with testosterone, intact females, ovariectomized females, ovariectomized females with estrogen replacement; there were 3–5 animals per group. Sections of these same brains were processed for immunocytochemical localization of c-Fos, and significant sex differences and effects of gonadal hormones were revealed, even with the rather small N’s per group [14]. Male rats were anesthetized with methoxyflurane and were castrated by removal of the testes and testicular fat with a single 2-cm midscrotal incision. Half the castrated rats were given daily injections of 2 mg/kg testosterone propionate dissolved in sesame oil for a total of 8 days, beginning the day of surgery. A previous study has shown that similar injections induce a large increase in circulating testosterone levels, which rapidly fall to physiological levels [29]. Female rats were also anesthetized with methoxyflurane and were ovariectomized via bilateral dorsal incisions and, at the same time, implanted subcutaneously with either empty silastic capsules or capsules containing crystalline estradiol (5 mm releasing length, 0.24 mm wall thickness). These capsules produce constant circulating levels of estradiol within the normal physiological range [27]. After surgery, all animals were allowed to recover for 7–8 days.
Tissue Collection Animals were anesthetized with an overdose of pentobarbital (100 mg/kg, i.p.) and subjected to transcardiac perfusion with phosphate buffered saline (PBS) followed by 3% neutral buffered paraformaldehyde. Brains were removed, blocked, and post-fixed for 2 h in the same fixative at room temperature; they were then cryoprotected in 30% sucrose at 4◦ C until they sank, and rapidly frozen on powdered dry ice. Brains were stored at −70◦ C until immunocytochemical analysis was performed. Autoradiographic Immunocytochemistry To localize NR1 and GluR2 immunoreactivity was performed using a modification of the method of Lu and Wolf [26]. The brains were cut on a freezing microtome (60 µm coronal sections) and the sections were washed five times in PBS to remove the residual fixative. The sections were then incubated for 1 h at room temperature in blocking buffer (normal goat serum, 15 µl/ml in 0.4% Triton (TX)-PBS). The blocking buffer was then aspirated and the primary antibodies were added to each well (rabbit anti-NMDAR1 at a dilution of 1:1000 or rabbit anti-GluR2 at a dilution of 1:2000; Chemicon). The sections were incubated with the primary antibody at 4◦ C for 2 days. The sections were again washed extensively in PBS to remove the residual primary antibody. They were then incubated for 1 h with the secondary antibody (35 S anti-rabbit IgG, Amersham, 1:200 dilution—prepared in 0.4% TX-PBS). The sections were then washed in PBS 5 times, and then mounted on chrom-alum subbed slides from 10 mM sodium acetate containing 0.5% Dreft’s detergent. The sections were then dried using a slide warmer, dehydrated in 100% ethanol for 2 min, air dried and placed in contact with X-ray film (Classic SX) for 3 days. Data Analysis The film was developed using an automatic X-ray film processor and the autoradiographs were analyzed by computer-assisted densitometry using an Aims Technology camera interfaced through a Data Translation frame-grabber, with a Macintosh IIci computer controlled by the NIH Image 1.52 software. The captured images were enhanced using the ‘contrast enhancement’ and
FIG. 1. Schematic diagram showing regions of the brain that were analyzed, as marked with shading, i.e., the midline-intralaminar thalamic nuclei (A) and the dorsomedial CPu (B), corresponding to plates 28 and 14 of Paxinos and Watson [28].
GONADAL STEROIDS AND GLUTAMATE RECEPTORS
291 TABLE 1
MEAN (±SEM) PIXEL DENSITIES OF NMDAR1 AND GluR2 IMMUNOREACTIVITIES IN DORSOMEDIAL CPu AND MIDLINE-INTRALAMINAR THALAMIC NUCLEI UNDER DIFFERENT TREATMENT CONDITIONS
Glutamate Receptors
Intact Male
Castrated Male
Cast + Test Male
NMDAR1 (CPu) NMDAR1 (thalamus)
115.493 (0.185) 109.647 (0.142)
115.203 (1.611) 110.807 (1.392)
GluR2 (CPu) GluR2 (thalamus)
120.223 (2.107) 103.523 (0.863)
117.947 (1.636) 106.666 (0.592)
∗
OVX + E2 Female
Intact Female
OVX Female
117.270 (4.010) 111.415 (1.175)
115.493 (1.441) 102.893 (2.005)
114.240 (0.200) 105.630 (0.160)
113.425 (0.495) 105.405 (0.855)
117.970 (3.380) 105.300 (2.000)
117.497 (0.936) 109.633 (1.029)
113.250* (0.070) 108.935 (1.495)
113.850* (0.170) 109.065 (1.405)
p < 0.03 versus intact female.
‘noise reduction’ feature of the NIH image program. A comparison of the optical densities between intact, gonadectomized, and hormone-replacement groups was performed and the data were analyzed statistically with ANOVA followed by post hoc analysis with the Scheffe test. The regions of the brain examined were the midline intralaminar nuclei of the thalamus (Fig. 1A) and the dorsomedial CPu (Fig. 1B). Because we were not looking at sex differences, but rather the effect of gonadal hormones on the receptor levels, we did not put the male and female sections together on the same film. Thus, the ANOVA did not compare across the sexes.
sex differences in the behavioral and neurochemical effects of MK-801 [14] are not likely to be due to a similar hormone-driven regulation of either NMDAR1 or GluR2 in the striatum. However, the results suggest that female gonadal hormones may regulate glutamate-mediated calcium entry in the CPu, via modulation of GluR2 levels in the striatum. These results may provide insight into how gonadal hormones affect behaviors that are dependent on the striatum, such as certain types of reference memory (e.g. [17]) and the rewarding aspects of sexual behavior [2].
RESULTS
These studies were supported in part by LEQSF RD-A-29 to MMG.
ACKNOWLEDGEMENTS
As seen in Table 1, we found that castration or hormonal treatment did not significantly alter NMDAR1 or GluR2 receptor subunit levels in males within the CPu or the thalamus. In females, after ovariectomy, there was a small but significant reduction in the GluR2 receptor subunit levels in the CPu (p < 0.05), which was not restored with estrogen treatment. In the thalamus, we saw no changes in GluR2 receptor subunit levels across the treatment groups in female rats. We saw no effect of gonadal steroids on NMDAR1 receptor subunit levels in females in either CPu or thalamus. DISCUSSION The prediction in this study was that testosterone in males, but not estradiol in females, would regulate abundance of NR1, but perhaps not GluR2, in the CPu. However, the results demonstrated that testosterone had no effect on NR1 or GluR2 levels in either the CPu or the thalamus. On the contrary, ovariectomy, but not estradiol replacement, decreased levels of GluR2, but not NR1, in the CPu. Our results are in agreement with previous studies indicating a lack of effect of ovariectomy on NMDA receptors in the striatum [11,12]. Estrogen has been reported to alter NMDA receptor expression in several other brain regions, including the hypothalamus, hippocampus and cerebral cortex [1,6,11,12,19,21,31]. In agreement with a previous study [6], testosterone also did not have any significant effects on NMDA receptor abundance in the striatum or thalamus. In contrast to the NR1 subunit of NMDA receptors, we found that ovariectomy resulted in a decrease in the GluR2 receptor density that was not restored with estrogen treatment. These results are in contrast to those of Cyr and coworkers [11,12], who found no effect of ovariectomy on AMPA receptor binding in the striatum, but a downregulation in response to estradiol. There are substantial differences in duration of ovariectomy and estradiol treatment that could explain this discrepancy. Because estradiol did not restore GluR2 receptor density, there is a possibility that the absence of progesterone following ovariectomy might be responsible for the alteration in the GluR2 receptor subunit level that we report here. Because we saw a significant effect of castration on GluR2 receptor subunit levels in females but did not see any effect in males, our studies suggest that the hormone-driven
REFERENCES 1. Adams, M. M.; Morrison, J. H.; Gore, A. C. N-Methyl-d-aspartate receptor mRNA levels change during reproductive senescence in the hippocampus of female rats. Exp. Neurol. 170:171–179; 2001. 2. Becker, J. B.; Rudick, C. N.; Jenkins, W. J. The role of dopamine in the nucleus accumbens and striatum during sexual behavior in the female rat. J. Neurosci. 21:3236–3241; 2001. 3. Berendse, H. W.; Groenewegen, H. J. Organization of the thalamostriatal projections in the rat, with special emphasis on the ventral striatum. J. Comp. Neurol. 299:187–228; 1990. 4. Bontempi, B.; Sharp, F. R. Systemic morphine-induced Fos protein in the rat striatum and nucleus accumbens is regulated by mu opioid receptors in the substantia nigra and ventral tegmental area. J. Neurosci. 17:8596–8612; 1997. 5. Boyer, J. S.; Morgan, M. M.; Craft, R. M. Microinjection of morphine into the rostral ventromedial medulla produces greater antinociception in male compared to female rats. Brain Res. 796:315–318; 1998. 6. Brann, D. W.; Zamorano, P. L.; Chorich, L. P.; Mahesh, V. B. Steroid hormone effects on NMDA receptor binding and NMDA receptor mRNA levels in the hypothalamus and cerebral cortex of the adult rat. Neuroendocrinology 58:666–672; 1993. 7. Brodsky, M.; Elliott, K.; Hynansky, A.; Jenab, S.; Inturrisi, C. E. Quantitation of mu-opioid receptor (MOR-1) mRNA in selected regions of the rat CNS. Neuroreport 6:725–729; 1995. 8. Candido, J.; Lutfy, K.; Billings, B.; Sierra, V.; Duttaroy, A.; Inturrisi, C. E.; Yoburn, B. C. Effect of adrenal and sex hormones on opioid analgesia and opioid regulation. Pharmacol. Biochem. Behav. 42:685– 692; 1992. 9. Chang, S. L.; Squinto, S. P.; Harlan, R. E. Morphine activation of c-Fos expression in rat brain. Biochem. Biophys. Res. Commun. 157:698– 704; 1988. 10. Cicero, T. J.; Nock, B.; Meyer, E. R. Gender-related differences in the antinociceptive properties of morphine. J. Pharmacol. Exp. Ther. 279:767–773; 1996. 11. Cyr, M.; Ghribi, O.; Di Paolo, T. Regional and selective effects of oestradiol and progesterone on NMDA and AMPA receptors in the rat brain. J. Neuroendocrinol. 12:445–452; 2000. 12. Cyr, M.; Thibault, C.; Morissette, M.; Landry, M.; Di Paolo, T. Estrogen-like activity of tamoxifen and raloxifene on NMDA receptor binding and expression of its subunits in rat brain. Neuropsychopharmacology 25:242–257; 2001.
292 13. Diano, S.; Naftolin, F.; Horvath, T. Gonadal steroids target AMPA glutamate receptor-containing neurons in the rat hypothalamus. Endocrinology 138:778–789; 1997. 14. D’Souza, D. N.; Harlan, R. E.; Garcia, M. M. Sexually dimorphic responses to morphine and MK-801 are mediated by sex steroid-dependent and -independent mechanisms in the rat. J. Appl. Physiol. 92:493–503; 2002. 15. D’Souza, D. N.; Harlan, R. E.; Garcia, M. M. Sexual dimorphism in the response to N-methyl-d-aspartate receptor antagonists and morphine on behavior and c-Fos induction in the rat brain. Neuroscience 93:1539– 1547; 1999. 16. Frankel, P. S.; Garcia, M. M.; Harlan, R. E. Infusion of β-FNA into the thalamus attenuates morphine-induced c-Fos induction in the rat caudate putamen. Brain Res. 838:222–226; 1999. 17. Galea, L. A.; Wide, J. K.; Paine, T. A.; Holmes, M. M.; Ormerod, B. K.; Floresco, S. B. High levels of estradiol disrupt conditioned place preference learning, stumulus response learning and reference memory but have limited effects on working memory. Behav. Brain Res. 126:115–126; 2001. 18. Garcia, M. M.; Brown, H. E.; Harlan, R. E. Alterations in immediate-early gene proteins in the rat forebrain induced by acute morphine injection. Brain Res. 692:23–40; 1995. 19. Gazzaley, A. H.; Weiland, N. G.; McEwen, B. S.; Morrison, J. H. Differential regulation of NMDAR1 mRNA and protein by estradiol in the rat hippocampus. J. Neurosci. 16:6830–6838; 1996. 20. Glazer, E. J.; Ramachandran, J.; Basbaum, A. I. Radioimmunocytochemistry using a tritiated goat anti-rabbit second antibody. J. Histochem. Cytochem. 32:778–782; 1984. 21. Gu, G.; Varoqueaux, F.; Simerly, R. B. Hormonal regulation of glutamate receptor gene expression in the anteroventral periventricular nucleus of the hypothalamus. J. Neurosci. 19:3213–3222; 1999.
D’SOUZA, HARLAN AND GARCIA 22. Honack, D.; Loscher, W. Sex differences in NMDA receptor-mediated responses in rats. Brain Res. 620:167–170; 1993. 23. Krzanowska, E. K.; Bodnar, R. J. Morphine antinociception elicited from the ventrolateral periaqueductal gray is sensitive to sex and gonadectomy differences in rats. Brain Res. 821:224–230; 1999. 24. Lipa, S. M.; Kavaliers, M. Sex differences in the inhibitory effects of the NMDA antagonist, MK-801, on morphine and stress-induced analgesia. Brain Res. Bull. 24:627–630; 1990. 25. Liu, J.; Nickolenko, J.; Sharp, F. R. Morphine induces c-Fos and junB in striatum and nucleus accumbens via D1 and N-methyl-daspartate receptors. Proc. Natl. Acad. Sci. U.S.A. 91:8537–8541; 1994. 26. Lu, W.; Wolf, M. E. Repeated amphetamine administration alters AMPA receptor subunit expression in rat nucleus accumbens and medial prefrontal cortex. Synapse 32:119–131; 1999. 27. Parsons, B.; MacLusky, N. J.; Krieger, M. S.; McEwen, B. S.; Pfaff, D. W. The effects of long-term estrogen exposure on the induction of sexual behavior and measurements of brain estrogen and progestin receptors in the female rat. Horm. Behav. 13:301–313; 1979. 28. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates, 2nd ed. San Diego: Academic Press; 1986. 29. Sodersten, P.; Eneroth, P.; Ekberg, P.-H. Episodic fluctuations in concentrations of androgen in serum of male rats: Possible relationship to sexual behavior. J. Endocrinol. 87:463–471; 1980. 30. Vathy, I.; Rimanoczy, A.; Eaton, R.; Katay, L. Modulation of catecholamine turnover rate in brain regions of rats exposed prenatally to morphine. Brain Res. 662:209–215; 1994. 31. Watanabe, T.; Inoue, S.; Hiroi, H.; Orimo, A.; Maramatsu, M. NMDA receptor type 2D gene as target for estrogen receptor in the brain. Brain Res. Mol. Brain Res. 63:375–379; 1999.
PII: S0361-9230(02)00885-7
Modulation of glutamate receptor expression by gonadal steroid hormones in the rat striatum Deborah N. D’Souza, 1† Richard E. Harlan1,2 and Meredith M. Garcia2,3∗ 1 Department of Structural and Cellular Biology, Tulane University Medical School, New Orleans, LA, USA; 2 Program in Neuroscience, Tulane University Medical School, New Orleans, LA, USA; and 3 Department of
Otolaryngology, Tulane University Medical School, New Orleans, LA, USA [Received 5 March 2002; Revised 2 July 2002; Accepted 20 July 2002] but completely inhibited morphine-mediated c-Fos expression in females [15]. This sex difference was eliminated by castration and restored by the administration of testosterone in males, but ovariectomy with or without estrogen replacement did not alter the response to MK-801 in females [14]. Gender differences have been previously reported in the responses to both morphine [5,8,10,23,30] and MK-801 [15,22,24]. Other studies have shown that sex hormones may alter glutamatergic transmission in the brain by regulating expression of glutamate receptors. Estrogen has been found to modulate the AMPA subtype of glutamate receptor (subunits GluR1 and GluR2/3) in a sexually dimorphic manner [13]. Although Brann et al. [6] found that in males or females, castration and steroid replacement did not affect the mRNA levels of the NMDA receptor within the hypothalamus, Gazzaley et al. [19] reported that gonadal hormones do modulate the pharmacological and physiological properties of the NMDA receptor. Thus, the role of gonadal steroids on glutamatergic neurotransmission is somewhat unclear. In our previous study [14], we found that testosterone in males, but not estradiol in females, regulated the ability of MK-801 to block morphine-induced c-Fos expression in the CPu. One mechanism by which this could occur is through steroid regulation of glutamate receptor abundance. Therefore, we tested the hypothesis that gonadal hormones could modulate levels of glutamate receptor subtypes in the CPu and medial thalamus, regions of the brain showing gender differences and effects of hormones on morphineand MK-801-mediated IEG expression. We chose to study the NR1 subunit of the NMDA receptor because it is expressed in all functional NMDA receptors, and the GluR2 subunit of the AMPA receptor because it regulates the permeability of the AMPA receptor to Ca2+ and thus is important in modulating neuronal excitability. We used autoradiographic immunocytochemistry [20,26] to determine levels of striatal NR1 and GluR2 subunits in male (intact, castrated, or castrated + testosterone) and female (intact, ovariectomized, or ovariectomized + estrogen) rats. We predicted that testosterone treatment in males, but not estradiol treatment
ABSTRACT: The non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) attenuates morphine-induced immediate-early gene expression in the rat striatum in a sexually dimorphic manner that depends in part on gonadal steroids. To determine if this effect was dependent on modulation of glutamate receptor gene expression, we studied the effect of gonadal hormones on levels of the NR1 subunit of NMDA receptor and the GluR2 subunit of the AMPA-subtype of glutamate receptor in the rat striatum, using autoradiographic immunocytochemistry. We found that ovariectomy decreased GluR2 immunoreactivity in the striatum, but no changes were seen in levels of NR1 following gonadectomy in either sex. Thus, the effects of gonadal steroids on NMDA receptor-mediated responses are not due to regulation of NR1 expression. © 2002 Elsevier Science Inc. All rights reserved. KEY WORDS: Immediate-early genes, Gonadal hormones, Glutamate receptors, Morphine.
INTRODUCTION Previous studies from this and other laboratories have shown that morphine induces the expression of the immediate-early gene (IEG) c-Fos, predominantly in neurons of the dorsomedial caudate-putamen (CPu; [9,18,25]) and that this expression can be attenuated by pretreatment with the non-competitive NMDA receptor antagonist, dizocilpine (MK-801; [4,15,25]). The intralaminar nuclei of the thalamus, which express mu opiate receptors [7], send a glutamatergic projection to the dorsomedial CPu [3]. Studies done by Frankel et al. [16] have demonstrated that the morphine-induced increase in c-Fos expression in the CPu may be subsequent to activation of these thalamostriatal glutamatergic projections. Thus, glutamate and its cognate receptors may play a central role in mediating the effects of morphine in the striatum. It has previously been shown that MK-801 reduces morphineinduced c-Fos expression in the striatum in a sexually dimorphic manner, where MK-801 partially inhibited this effect in males
Abbreviations: Acb, nucleus accumbens; cc, corpus callosum; Cg, cingulate cortex; CM, central medial nucleus of the thalamus; CPu, caudate-putamen; Par, parietal cortex; PV, paraventricular nucleus of the thalamus; Re, reuniens nucleus of the thalamus; Rh, rhomboid nucleus of the thalamus; Tu, olfactory tubercle; VP, ventral pallidum; 3V, third ventricle ∗ Address for correspondence: Dr. Meredith Mason Garcia, Ph.D., Program in Neuroscience, Tulane University Medical School, 1430 Tulane Ave. SL-59, New Orleans, LA 70112-2699, USA. Fax: +1-504-582-7846; E-mail: [email protected] † Present address: Department of Pharmacology, Loyola University Stritch School of Medicine, Chicago, IL 60153, USA.
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in females, would alter levels of NR1, but perhaps not GluR2, in the CPu. MATERIALS AND METHODS Treatment of Animals All treatment of animals was performed in accordance with NIH guidelines and was approved by the Advisory Committee on Animal Resources of the Tulane University Medical School. All efforts were made to minimize animal suffering and to reduce the number of animals used. Adult male or random-cycling female Sprague–Dawley rats (200–225 gm) were housed in group cages (3–4 per cage) in the Vivarium of the Tulane University Medical School, with controlled temperature and light/dark cycles of 12 h/12 h (lights on, 0600 h; lights off, 1800 h). Animals were provided with unlimited access to food (Purina rat chow) and tap water. The animals were assigned to groups as follows: intact males, castrated males, castrated males treated with testosterone, intact females, ovariectomized females, ovariectomized females with estrogen replacement; there were 3–5 animals per group. Sections of these same brains were processed for immunocytochemical localization of c-Fos, and significant sex differences and effects of gonadal hormones were revealed, even with the rather small N’s per group [14]. Male rats were anesthetized with methoxyflurane and were castrated by removal of the testes and testicular fat with a single 2-cm midscrotal incision. Half the castrated rats were given daily injections of 2 mg/kg testosterone propionate dissolved in sesame oil for a total of 8 days, beginning the day of surgery. A previous study has shown that similar injections induce a large increase in circulating testosterone levels, which rapidly fall to physiological levels [29]. Female rats were also anesthetized with methoxyflurane and were ovariectomized via bilateral dorsal incisions and, at the same time, implanted subcutaneously with either empty silastic capsules or capsules containing crystalline estradiol (5 mm releasing length, 0.24 mm wall thickness). These capsules produce constant circulating levels of estradiol within the normal physiological range [27]. After surgery, all animals were allowed to recover for 7–8 days.
Tissue Collection Animals were anesthetized with an overdose of pentobarbital (100 mg/kg, i.p.) and subjected to transcardiac perfusion with phosphate buffered saline (PBS) followed by 3% neutral buffered paraformaldehyde. Brains were removed, blocked, and post-fixed for 2 h in the same fixative at room temperature; they were then cryoprotected in 30% sucrose at 4◦ C until they sank, and rapidly frozen on powdered dry ice. Brains were stored at −70◦ C until immunocytochemical analysis was performed. Autoradiographic Immunocytochemistry To localize NR1 and GluR2 immunoreactivity was performed using a modification of the method of Lu and Wolf [26]. The brains were cut on a freezing microtome (60 µm coronal sections) and the sections were washed five times in PBS to remove the residual fixative. The sections were then incubated for 1 h at room temperature in blocking buffer (normal goat serum, 15 µl/ml in 0.4% Triton (TX)-PBS). The blocking buffer was then aspirated and the primary antibodies were added to each well (rabbit anti-NMDAR1 at a dilution of 1:1000 or rabbit anti-GluR2 at a dilution of 1:2000; Chemicon). The sections were incubated with the primary antibody at 4◦ C for 2 days. The sections were again washed extensively in PBS to remove the residual primary antibody. They were then incubated for 1 h with the secondary antibody (35 S anti-rabbit IgG, Amersham, 1:200 dilution—prepared in 0.4% TX-PBS). The sections were then washed in PBS 5 times, and then mounted on chrom-alum subbed slides from 10 mM sodium acetate containing 0.5% Dreft’s detergent. The sections were then dried using a slide warmer, dehydrated in 100% ethanol for 2 min, air dried and placed in contact with X-ray film (Classic SX) for 3 days. Data Analysis The film was developed using an automatic X-ray film processor and the autoradiographs were analyzed by computer-assisted densitometry using an Aims Technology camera interfaced through a Data Translation frame-grabber, with a Macintosh IIci computer controlled by the NIH Image 1.52 software. The captured images were enhanced using the ‘contrast enhancement’ and
FIG. 1. Schematic diagram showing regions of the brain that were analyzed, as marked with shading, i.e., the midline-intralaminar thalamic nuclei (A) and the dorsomedial CPu (B), corresponding to plates 28 and 14 of Paxinos and Watson [28].
GONADAL STEROIDS AND GLUTAMATE RECEPTORS
291 TABLE 1
MEAN (±SEM) PIXEL DENSITIES OF NMDAR1 AND GluR2 IMMUNOREACTIVITIES IN DORSOMEDIAL CPu AND MIDLINE-INTRALAMINAR THALAMIC NUCLEI UNDER DIFFERENT TREATMENT CONDITIONS
Glutamate Receptors
Intact Male
Castrated Male
Cast + Test Male
NMDAR1 (CPu) NMDAR1 (thalamus)
115.493 (0.185) 109.647 (0.142)
115.203 (1.611) 110.807 (1.392)
GluR2 (CPu) GluR2 (thalamus)
120.223 (2.107) 103.523 (0.863)
117.947 (1.636) 106.666 (0.592)
∗
OVX + E2 Female
Intact Female
OVX Female
117.270 (4.010) 111.415 (1.175)
115.493 (1.441) 102.893 (2.005)
114.240 (0.200) 105.630 (0.160)
113.425 (0.495) 105.405 (0.855)
117.970 (3.380) 105.300 (2.000)
117.497 (0.936) 109.633 (1.029)
113.250* (0.070) 108.935 (1.495)
113.850* (0.170) 109.065 (1.405)
p < 0.03 versus intact female.
‘noise reduction’ feature of the NIH image program. A comparison of the optical densities between intact, gonadectomized, and hormone-replacement groups was performed and the data were analyzed statistically with ANOVA followed by post hoc analysis with the Scheffe test. The regions of the brain examined were the midline intralaminar nuclei of the thalamus (Fig. 1A) and the dorsomedial CPu (Fig. 1B). Because we were not looking at sex differences, but rather the effect of gonadal hormones on the receptor levels, we did not put the male and female sections together on the same film. Thus, the ANOVA did not compare across the sexes.
sex differences in the behavioral and neurochemical effects of MK-801 [14] are not likely to be due to a similar hormone-driven regulation of either NMDAR1 or GluR2 in the striatum. However, the results suggest that female gonadal hormones may regulate glutamate-mediated calcium entry in the CPu, via modulation of GluR2 levels in the striatum. These results may provide insight into how gonadal hormones affect behaviors that are dependent on the striatum, such as certain types of reference memory (e.g. [17]) and the rewarding aspects of sexual behavior [2].
RESULTS
These studies were supported in part by LEQSF RD-A-29 to MMG.
ACKNOWLEDGEMENTS
As seen in Table 1, we found that castration or hormonal treatment did not significantly alter NMDAR1 or GluR2 receptor subunit levels in males within the CPu or the thalamus. In females, after ovariectomy, there was a small but significant reduction in the GluR2 receptor subunit levels in the CPu (p < 0.05), which was not restored with estrogen treatment. In the thalamus, we saw no changes in GluR2 receptor subunit levels across the treatment groups in female rats. We saw no effect of gonadal steroids on NMDAR1 receptor subunit levels in females in either CPu or thalamus. DISCUSSION The prediction in this study was that testosterone in males, but not estradiol in females, would regulate abundance of NR1, but perhaps not GluR2, in the CPu. However, the results demonstrated that testosterone had no effect on NR1 or GluR2 levels in either the CPu or the thalamus. On the contrary, ovariectomy, but not estradiol replacement, decreased levels of GluR2, but not NR1, in the CPu. Our results are in agreement with previous studies indicating a lack of effect of ovariectomy on NMDA receptors in the striatum [11,12]. Estrogen has been reported to alter NMDA receptor expression in several other brain regions, including the hypothalamus, hippocampus and cerebral cortex [1,6,11,12,19,21,31]. In agreement with a previous study [6], testosterone also did not have any significant effects on NMDA receptor abundance in the striatum or thalamus. In contrast to the NR1 subunit of NMDA receptors, we found that ovariectomy resulted in a decrease in the GluR2 receptor density that was not restored with estrogen treatment. These results are in contrast to those of Cyr and coworkers [11,12], who found no effect of ovariectomy on AMPA receptor binding in the striatum, but a downregulation in response to estradiol. There are substantial differences in duration of ovariectomy and estradiol treatment that could explain this discrepancy. Because estradiol did not restore GluR2 receptor density, there is a possibility that the absence of progesterone following ovariectomy might be responsible for the alteration in the GluR2 receptor subunit level that we report here. Because we saw a significant effect of castration on GluR2 receptor subunit levels in females but did not see any effect in males, our studies suggest that the hormone-driven
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