Immunocytochemical localization of nuclear estrogen receptors and progestin receptors within the rat dorsal raphe nucleusкод для вставкиСкачать
THE JOURNAL OF COMPARATIVE NEUROLOGY 391:322–334 (1998) Immunocytochemical Localization of Nuclear Estrogen Receptors and Progestin Receptors Within the Rat Dorsal Raphe Nucleus STEPHEN E. ALVES,1* NANCY G. WEILAND,1 SHINJI HAYASHI,2 AND BRUCE S. MCEWEN1 1Laboratory of Neuroendocrinology, The Rockefeller University, New York, New York 10021 2Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183, Japan ABSTRACT Estradiol and progesterone modulate central serotonergic activity; however, the mechanism(s) of action remain unclear. Recently, estradiol-induced progestin receptors (PRs) have been localized within the majority of serotonin (5-HT) neurons in the female macaque dorsal raphe nucleus (DRN; Bethea  Neuroendocrinology 60:50–61). In the present study, we investigated whether estrogen receptors (ERs) and/or PRs exist within 5-HT and/or non-5-HT cells in the female and male rat DRN and whether estradiol treatment alters the expression of these receptors. Young adult female and male Sprague-Dawley rats were gonadectomized, and 1 week later, half of the animals received a subcutaneous Silastic implant of estradiol-17b. Animals were transcardially perfused 2 days later with acrolein and paraformaldehyde, and sequential dual-label immunocytochemistry was performed on adjacent sections by using either a PR antibody or an ERa antibody. This was followed by an antibody to either the 5-HT-synthesizing enzyme, tryptophan hydroxylase (TPH), or to the astrocytic marker, glial fibrillary acidic protein (GFAP). Cells containing immunoreactivity (ir) for nuclear ERs or PRs were identified within the rat DRN in a region-specific distribution in both sexes. No colocalization of nuclear ER-ir or PR-ir with cytoplasmic TPH-ir or GFAP-ir was observed in either sex or treatment, indicating that the steroid target cells are neither 5-HT neurons nor astrocytes. Females were found to have approximately 30% more PR-labeled cells compared with males throughout the DRN (P , 0.05), but no sex difference was detected in the number of neurons demonstrating ER-ir. In both sexes, 2 days of estradiol exposure decreased the number of cells with ER-ir, whereas it greatly increased the number of cells containing PR-ir in several DRN regions (P , 0.005). Collectively, these findings demonstrate the existence of nonserotonergic cells that contain nuclear ERs or PRs within the female and male rat DRN, including estradiol-inducible PRs. These findings point to a species difference in ovarian steroid regulation of 5-HT activity between the macaque and the rat, perhaps transsynaptically via local neurons in the rat brain. J. Comp. Neurol. 391:322–334, 1998. r 1998 Wiley-Liss, Inc. Indexing terms: serotonin; tryptophan hydroxylase; astroglia; macaque; species difference Several lines of convincing evidence point to a modulatory role for the ovarian steroids, estradiol (E) and progesterone (P), on the activity of midbrain serotonin (5-HT) neurons in rodents and primates. In particular, a sex difference exists in the 5-HT system of the rat brain, with females displaying elevated 5-HT synthesis and turnover within the raphe nuclei as well as in forebrain targets compared with males (Rosencrans, 1970; Watts and Stanely, 1984; Dickinson and Curzon, 1986; Kennett et al., 1986; Carlsson and Carlsson, 1988; Haleem et al., 1990). Although the physiological basis for this sex difference in r 1998 WILEY-LISS, INC. 5-HT function is not fully understood, numerous studies have reported that brain 5-HT levels and activity are altered during periods of natural ovarian hormone fluctua- Grant sponsor: National Institutes of Health; Grant numbers: F32 NS10047, NS07080, and NS30105. *Correspondence to: Dr. Stephen E. Alves, Laboratory of Neuroendocrinology, Box 165, The Rockefeller University, 1230 York Avenue, New York, NY 10021. E-mail: firstname.lastname@example.org Received 25 March 1997; Revised 18 August 1997; Accepted 19 September 1997 ESTROGEN AND PROGESTIN RECEPTORS IN THE RAPHE tion, including the estrous cycle (Myer and Quey, 1975; Kueng et al., 1976; Biegon et al., 1980; Uphouse et al., 1986; Desan et al., 1988), pregnancy, and the postpartum period (Greengrass and Tongue, 1974; Desan et al., 1988). Furthermore, E and/or P treatment to ovariectomized (OVX) rats has been shown to enhance 5-HT synthesis, turnover, and receptor binding in numerous brain regions (Crowley et al., 1979; Cone et al., 1981; Biegon et al., 1983; Di Paolo et al., 1983; Chomicka, 1986; Cohen and Wise, 1988; Mendelson et al., 1993; Sumner and Fink, 1995). Ovarian steroid modulation of serotonergic activity has been linked to the regulation of several neuroendocrine processes. For example, a combination of E and P treatment to OVX rats significantly increases 5-HT synthesis and, thus, 5-HT concentrations within the dorsal raphe nucleus (DRN; Cone et al., 1981) and the preoptic areaanterior hypothalamus (POA-AH; King et al., 1986), a circuitry by which 5-HT is believed to stimulate luteinizing hormone (LH) release. Concurrent with these neural changes, this treatment does induce an E-dependent surge in LH, which is advanced and amplified by P (Kawakami et al., 1978; Chen et al., 1981). Walker and Wilson (1983) demonstrated that 5-HT is involved in this potentiating effect of P upon the E-induced LH surge, because the inhibition of 5-HT synthesis blunted the effects of P. More recent findings have identified serotonergic regulation of the proestrous LH surge to be mediated specifically via a 5-HT2 receptor mechanism (Dow et al., 1994). Interestingly, the 5-HT2A receptor gene is regulated by E. Administration of E to OVX rats greatly increases this receptor’s message in the DRN (Sumner and Fink, 1993) as well as the density of the receptor protein in several forebrain regions (Sumner and Fink, 1995). In addition to reproductive regulation, the serotonergic system is believed to play an important role in the etiology of several common psychopathologies. Included among these are depression and anxiety, both of which occur with higher frequency in women than in men (Weissman and Klerman, 1985). It is not known whether this sex difference in susceptibility is a result of circulating gonadal steroids or of steroid hormone action during the sexual differentiation of the brain. However, E therapy has been shown to significantly reduce symptoms of depression and/or anxiety in women (Klaiber et al., 1979; Oppenheim, 1983; McEwen, 1994) as well as in mice in a rodent model of depression (Bernardi et al., 1989). E treatment has been shown to decrease the number of 5-HT reuptake sites assessed by [3H]paroxitine binding in the hippocampus of GDX female and male rats (Mendelson et al., 1993). Interestingly, GDX females were found to exhibit significantly lower binding within several of the hippocampal regions examined in response to E compared with GDX males. A decrease in 5-HT reuptake would presumably allow more 5-HT to remain in the synapse, thereby prolonging the actions of this transmitter. Because many therapeutic drugs for depression act to block 5-HT reuptake (Meltzer, 1990), this presynaptic action of E on 5-HT nerve terminals may contribute to the antidepressant effects of this steroid. However, with the discovery of a novel antidepressive agent, tianeptine, which appears to exert its effects by actually enhancing 5-HT reuptake (Wilde and Benfield, 1995), it becomes evident that relatively little is known regarding the mechanisms of serotonergic-active drugs in treating depression. Thus, to more clearly understand the apparent E-mediated alteration in 323 serotonergic activity at a terminal field, it is necessary to consider how E may affect serotonergic activity at the cell body, where gene regulation occurs. Cells that concentrate radiolabeled E (Pfaff and Keiner, 1973) or P (MacLusky and McEwen, 1980) have been documented within the rat midbrain, but, to our knowledge, no study has reported on the presence of estrogen receptors (ERs) or progestin receptors (PRs) specifically within the raphe nuclei or in 5-HT neurons in the rat brain. E-concentrating cells, determined by autoradiography, have been reported previously in the raphe nucleus in the male and female lizard Anolis carolinensis, but it was not determined whether the cells were serotonergic (Morrell et al., 1979). An immunocytochemical (icc) study by Turcotte and Blaustein (1993) demonstrated the presence of both ER- and PR-containing cells of undetermined phenotype within midbrain raphe nuclei in female guinea pigs. Recent icc findings have demonstrated the presence of E-induced nuclear PRs within the majority of 5-HT neurons of the DRN in the female macaque brain (Bethea, 1993, 1994). These data from the macaque are the first to demonstrate a means by which the ovarian steroids can exert a direct receptor-mediated effect on serotonergic function. In the present study, we have investigated 1) whether ERs and/or PRs occur within the DRN of the rat brain and, if so, whether they are found in serotonergic and/or nonserotonergic cells; 2) how localization, regional distribution, and/or number of receptor-containing cells compare between females and males; and 3) whether E priming alters any of these parameters. MATERIALS AND METHODS Animal treatment and tissue preparation Animal care, maintenance, and surgery were in accordance with the applicable portions of the Animal Welfare Act and the U.S. Department of Health and Human Services ‘‘Guide for the Care and Use of Laboratory Animals.’’ Young adult female (n 5 12) and male (n 5 11) Sprague-Dawley rats (200–250 g; Charles River, Wilmington, MA) were GDX under Metofane anesthesia. One week later, half of the animals received a 2-cm subcutaneous (s.c.) silastic implant of estradiol-17b (200 µg/ml sesame oil) under Metofane anesthesia, and half were anesthetized but received no implant. This E replacement results in plasma levels well within physiological range (approximately 25 pg/ml; Weiland and Orchinik, 1995). Two days later, animals were injected with a lethal dose of sodium pentobarbital (150 mg/kg) and were transcardially perfused with 120 ml of 3.75% acrolein and 2% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.4. We had previously determined that this fixation protocol allows for the most sensitive detection of ERs and PRs with icc in our rat model. Brains were immediately removed and postfixed in 2% paraformaldehyde in 0.1 M PB at 4°C overnight; transferred to 0.1 M PB, pH 7.4; coronally sectioned at 40 µm on a vibratome; and stored in cryoprotectant (30% sucrose, 30% ethylene glycol in O.1 M PB, pH 7.4) at 220°C. For icc confirmation that the ER antibody recognizes both the ligand-bound and unbound receptor (Okamura et al., 1992), we administered estradiol benzoate (EB; 10 µg in sesame oil s.c.; n 5 4) or vehicle (n 5 4) to OVX rats and killed them 1 hour later, as described above. 324 S.E. ALVES ET AL. Dual-label immunocytochemistry Free-floating 40 µm sections were washed in ice cold 0.1 M phosphate-buffered saline (PBS), pH 7.4, to thoroughly remove cryoprotectant. To deter nonspecific staining, sections were washed in 1% sodium borohydride (NaBH4) in PBS for 30 minutes at 4°C and rinsed eight to ten times with PBS to remove all NaBH4. Then, endogenous peroxidase activity was inhibited by washing tissue with 0.3% H2O2 in 40% methanol in PBS for 15 minutes. Following several washes in PBS, tissue was blocked with 2% normal goat (ER) or horse (PR) serum in PBS with 0.4% Triton X-100 (PBST) for 1 hour. Tissue was then exposed to one primary antibody: either a polyclonal antibody against the isoform ERa made in rabbit (Okamura et al., 1992, 1: 40,000) over 7 days or an anti-PR mouse monoclonal antibody (1:1,600; Affinity Bioreagents, Golden, CO) overnight in blocking buffer diluted to 1% normal serum at 4°C. Both of these antibodies have been well characterized and affinity purified to ensure the specificity of binding and are reported to recognize both the unbound and bound receptors (for the anti-ER antibody, see Okamura et al., 1992; Weiland et al., 1997; for the anti-PR antibody, see Traish and Wotiz, 1990). The anti-ER antibody used, as indicated above, is specific for the a isoform as opposed to the newly identified ER b isoform (see Kuiper et al., 1996). Sections incubated without the primary antibody were also included and served as negative controls. All sections were washed in PBS and exposed to the appropriate biotinylated secondary antibody (anti-rabbit made in goat against ER; rat-adsorbed anti-mouse made in horse against PR; 1:600; Vector Laboratories, Burlingame, CA) in PBST and 1% normal serum for 1 hour. Following several PBS washes, sections were exposed to the avidinbiotin complex (ABC Elite kit; Vector Laboratories) in PBS for 45 minutes, washed, and exposed to the substrate metal-enhanced 3,38-diaminobenzidine tetrachloride (DAB) in a peroxide buffer (Pierce, Rockford, IL, or Sigma, St. Louis, MO). The reaction product appears as a punctate, dark nuclear stain. Following PBS washes, this procedure was repeated, incubating the tissue overnight at room temperature with an affinity-purified rabbit polyclonal antibody to the 5-HT-synthesizing enzyme, tryptophan hydroxylase (TPH; 1:1,000; Protos Biotech, New York, NY). This antiserum was used as a marker for 5-HT neurons for two reasons: 1) It produces excellent labeling of 5-HT cell bodies and processes, and 2) it is completely compatible with tissue previously processed for ER or PR icc. To test whether any steroid receptor-immunopositive cells could be glial, a subset of sections was incubated with an affinity-purified mouse monoclonal antibody to the astrocyte marker, glial fibrillary acidic protein (GFAP; 1:100; Boeringher-Mannheim, Indianapolis, IN). Standard DAB (0.05%; Sigma) with 0.003% H2O2 in PBS was used to visualize the antigen and appears as a light golden-brown cytoplasmic stain. Upon completion of dual labeling, sections were mounted onto gelatin-coated slides and air dried overnight. Tissue was then dehydrated in a series of ethanol concentrations, cleared in histoclear, and coverslipped with DPX mounting medium. Tissue analysis Sequential 40 µm sections approximately 240 µm apart, representing the DRN from rostral to caudal (from approximately Bregma 27.00 mm to 29.30 mm), were immuno- stained as described above. Each section was anatomically categorized according to distance from Bregma and was grouped into one of five approximate levels of the DRN by using the atlas of Paxinos and Watson (1986) and the brain maps of Swanson (1992) as guides. The distribution of cells containing TPH-ir served as a marker for the boundaries of the DRN. Cells containing ER-ir or PR-ir within the DRN of each section were counted at a magnification of 1003 on a Nikon light microscope (Tokyo, Japan). Cell counts/level/brain were derived from the sum of labeled cells counted in two representative 40 µm sections. The cell counts from each DRN level were averaged within groups, and these mean values were compared between groups. Brain mapping Representational maps of the rat brain DRN at five approximate levels (Bregma 27.1, 7.7, 8.3, 8.8, and 9.3) were made on a Power MacIntosh computer (Apple Computers, Cupertino, CA) employing the program Adobe Illustrator (Adobe Systems, Mountain View, CA) and by using a computerized version of the brain maps of Swanson (1992) together with a printed version of the atlas of Paxinos and Watson (1986) as guides. These maps serve to illustrate the regional distribution and relative density of cells containing ER-ir or PR-ir; each black dot drawn equals approximately two receptor-immunopositive cells within a representative 40 µm section. The cell densities illustrated in these maps depict optimal conditions, i.e., the ER map represents an average number of ERcontaining cells per level of a GDX brain, whereas the PR map depicts an average density of PR cells per level of a GDX 1 E brain. An approximate distribution of cells containing ER-ir or PR-ir within the adjacent periaqueductal gray (PAG) was included in these maps, because large concentrations of steroid receptor-immunopositive cells, sometimes continuous with those in the DRN, were observed through this nucleus. Statistical analysis A fully factorial three-way analysis of variance (ANOVA) with two between measures, sex and treatment, and one within measure, level of the DRN, was performed on the data, and significance was determined at P , 0.05. Posthoc comparisons were made between relevant pairs (treatment for five regions) by using an unpaired, two-tailed t test with Bonferroni criteria. RESULTS Figure 1 demonstrates the robust labeling of the DRN with the TPH antiserum used in this study. Cells containing specific nuclear ER-ir or PR-ir were identified through the DRN in a region-specific distribution in both female and male rat brains. The immunolabeled nuclei of these cells were found adjacent to, but not colocalized with, the cytoplasmic TPH-labeled cells (Fig. 2A–G). Likewise, no colocalization of ER-ir or PR-ir with GFAP-ir was observed in the DRN (Fig. 2H). It can be seen on the photomicrographs in Figure 2 that the labeled nuclei of the steroid target cells were often smaller in size compared with the unstained nuclei of the 5-HT cells but were larger than the nuclei of the astroglial cells. These findings indicate that the steroid target cells identified within the DRN are neither serotonergic neurons nor astrocytes. ESTROGEN AND PROGESTIN RECEPTORS IN THE RAPHE 325 Fig. 1. Tryptophan hydroxylase (TPH) immunoreactivity (-ir) within the dorsal raphe nucleus (DRN) of the rat brain at approximately 27.3 mm (A) and 28.0 mm (B) from Bregma. Note the robust and specific labeling of cell bodies and fibers within the DRN. Photomicrographs were taken under a Nikon light microscope with a 35-mm camera using Kodak (Eastman Kodak, Rochester, NY) ASA 100 Technical Pan film (503). aq, Cerebral aqueduct. Scale bar 5 80 µm. Figure 3 illustrates the regional distribution and relative density of ER-immunolabeled (Fig. 3A) and PRimmunolabeled (Fig. 3B) cells at five representative levels of the DRN. These brain maps show that the distributions of cells containing ER or PR were found to be quite similar, but some differences were identified. Significant rostral-tocaudal gradients in the numbers of both steroid target cells were found (df 4,76; ER: F 5 108.79; P , 0.0001; PR: F 5 59.95; P , 0.0001), with the greatest numbers of receptorcontaining cells observed within the rostral-to-medial levels of the DRN. In addition, several significant differences in the numbers of ER- and/or PR-containing cells were found based on either gender or treatment. No sex differences were found in the regional distribution or pattern of cells containing detectable ER-ir or PR-ir throughout the DRN or in the number of cells containing ER-ir. However, a sex difference in the number of PRimmunolabeled cells was recorded, independent of the level of the DRN or the treatment (df 1,19; F 5 5.007; P , 0.05). Collectively, females were found to have approximately 30% more cells demonstrating PR-ir within the DRN than males (Fig. 4). The mean numbers of steroid receptor-labeled cells identified within representative sections of GDX vs. GDX 1 E brains are illustrated in Figures 5 (ER) and 6 (PR). Independent of gender, E treatment had a significant effect on the number of cells containing detectable ER-ir within the DRN (df 1,19; F 5 29.54; P , 0.0001). In addition, an interaction between E treatment and region of the DRN was found (df 4,76; F 5 5.93; P , 0.0003). In a post-hoc comparison, E treatment was found to significantly decrease (40–50%; P , 0.005) the number of detectable cells demonstrating ER-ir within two DRN levels examined (Fig. 5). E treatment also had a significant effect on the number of cells containing PR-ir within the DRN, again independent of gender (df 1,19; F 5 35.00; P , 0.0001). An interaction between treatment and region was also identified (df 4,76; F 5 7.82; P , 0.0001). E treatment significantly increased (100–260%; P , 0.005) the number of cells containing detectable PR-ir within several levels of the DRN examined (Fig. 6). Qualitative comparison of receptor immunolabeling in GDX vs. GDX 1 E brains demonstrates an overall decrease in the intensity of ER-ir (Fig. 2A,B) but a great increase in nuclear PR-ir following 2 days of E exposure in both sexes. Figure 7 shows that the intensity of ER-ir is not altered in the brains of OVX rats treated acutely with E (for 1 hour) in contrast to 2 days of E priming. This finding provides further evidence that the ER antibody recognizes both the occupied and unoccupied forms of the receptor. 326 Fig. 2. A–H: Nuclear estrogen receptor (ER)-ir (A,B,G) and progestin receptor (PR)-ir (C–F,H, small arrows) within the dorsal raphe nucleus (DRN) are found adjacent to but not colocalized with cells cytoplasmicly immunolabeled for tryptophan hydroxylase (TPH; A–G) or glial fibrillary acidic protein (GFAP; H, large arrows), suggesting that the steroid targets are not serotonin (5-HT) neurons or astroglia. A qualitative comparison between the intensities of ER-ir in gonadectomized (GDX; A) and GDX 1 estradiol (E; B) brains suggests a decrease in nuclear ER concentration following E treatment, as seen S.E. ALVES ET AL. here in the rostral DRN. In contrast, note the absence of nuclear PR-ir in the rostral DRN of a GDX brain (C) compared with a GDX 1 E brain (D), which demonstrates clearly labeled PR-containing cells. However, in the lateral wings of the DRN, cells can be seen with distinct PR-ir in both the GDX (E) and the GDX 1 E (F) brains, suggesting that these cells may contain PRs that are not E-induced. Photomicrographs were taken under a Nikon light microscope with a Kodak 35-mm camera using Ektachrom 160 film (2003 in A–F, 4003 in G,H). Scale bars 5 40 µm. ESTROGEN AND PROGESTIN RECEPTORS IN THE RAPHE 327 Fig. 3 The distribution of cells in gonadectomized (GDX) rats containing nuclear estrogen receptors (ER)-ir (A) or in GDX 1 estradiol (E) rats containing PR-ir (B) within the DRN and the surrounding periaqueductal gray (PAG) in females and males. Each dot represents approximately two receptor-labeled cells. The brain levels depicted here are measured as distances from Bregma (B) and are modified from Swanson (1992) and Paxinos and Watson (1986). Note the higher density of cells containing ER-ir at the more rostral levels of the DRN but the higher concentration of progestin receptor (PR)-labeled cells specifically within the lateral wings (LW) of the dorsal raphe nucleus (DRN), depicted at level B 28.30. In contrast to most other regions of the DRN examined, this population of neurons maintains rather abundant PR-ir without E priming, as demonstrated in Figure 2C–F. AQ, cerebral aqueduct; CLi, caudal linear raphe nucleus; V4, fourth ventricle. Although we did not attempt to determine colocalization of ER and PR in this study, we assume that many, if not most, of the cells that contain PR also contain ER. This assumption is based on the large increase in the numbers of detectable cells containing PR-ir (Fig. 2C,D) as well as the darker intensity of the PR-ir following E treatment in most DRN regions examined. However, a group of cells containing PR-ir within the lateral wings of the DRN appeared to be an exception. These cells consistently demonstrated dark, well-defined PR-ir with or without E priming in both sexes (Fig. 2E,F). Interestingly, the number of PR-containing cells within this level of the DRN did not differ significantly between treatment groups (Fig. 6). In addition, this was one region of the DRN that seemed to contain more cells demonstrating PR-ir than ER-ir (Fig. 3). 328 Fig. 4. Sex difference in the number of progestin receptor (PR)labeled cells was identified within the dorsal raphe nucleus (DRN). Values shown here represent the mean number of PR-labeled cells 6 S.E.M. collapsed across treatment groups and levels of the DRN in female and male rats. DISCUSSION Localization and profile of ovarian steroid target cells Early radioligand binding studies revealed the presence of target cells of E (Pfaff and Keiner, 1973) and P (MacLusky and McEwen, 1980) within the rat midbrain. The results of the present study demonstrate the existence of cells specifically within the rat DRN that contain ER or PR protein. These steroid target cells are found in a region-specific distribution in both female and male brains. However, no colocalization of nuclear ER-ir or PR-ir with cytoplasmic TPH-ir was observed in either sex or treatment group. This finding indicates that the steroid target cells that we have identified are not serotonergic in phenotype. In fact, the generally smaller size of the immunolabeled nuclei for ER or PR, compared with the unstained nuclei of the large, cytoplasmically labeled TPH cells, further suggests different cell types. Although central glial cells can express ER (Langub and Watson, 1992; Santagati et al., 1994) and E-induced PR (Jung-Testas et al., 1991), in the present study, neither ER-ir nor PR-ir was found to colocalize with GFAP-ir. Furthermore, the labeled nuclei of the ER-and PRcontaining cells, although they were often smaller than the nuclei of 5-HT neurons, consistently appeared to be larger than the unstained GFAP-labeled astroglia. We have not ruled out possible colocalization with oligodendrocytes or microglia; however, based on their size, these ovarian steroid target cells are likely to be neurons located adjacent to the primary 5-HT cells. The chemical phenotype(s) of these steroid target cells remains to be determined. In addition to the principle 5-HT neurons, which comprise nearly all of the large cells with long projecting fibers (Jacobs and Azmitia, 1992), cells of several different neurochemical phenotypes have been described to exist within the raphe nuclei of the rat brain. Cell bodies demonstrating immunoreactivity to g-aminobutyric acid (GABA; Nanopoulos et al., 1982), the enkephalins (Khachaturian et al., 1983), nitric oxide synthase (NOS; Vincent and Kimura, 1992), and vasoactive S.E. ALVES ET AL. intestinal peptide (VIP; Loren et al., 1979) have been documented specifically within the DRN. Preliminary dual-label icc studies with antibodies to these chemicals and to ER and/or PR have failed to demonstrate colocalization (Alves, unpublished results). However, it should be pointed out that incompatibilities between icc protocols for the steroid receptors and GABA have prohibited a true picture of GABA distribution, and we are currently in the process of working out these difficulties. In general, the regional distributions of cells immunolabeled for ER or PR throughout the DRN were found to be very similar. This was not surprising, considering the observation that the expression of one class of PRs requires the binding of E to the ER and subsequent transcriptional activation (MacLusky and McEwen, 1978). Thus, many of the PR-containing cells must also contain ER. It has been previously shown in the guinea pig brain that E-induced PRs are found only in cells demonstrating ER-ir (Blaustein and Turcotte, 1989). However, not all ER1 cells appear to contain PR. Our data demonstrate that cells containing ER-ir were more abundant compared with PR-ir containing cells, particularly throughout the rostral regions of the DRN. The midbrain of the female guinea pig has also been shown to contain more cells immunoreactive for ER compared with PR (Turcotte and Blaustein, 1993). However, in contrast to our findings in the rat and to those in the macaque (Bethea, 1993, 1994), the guinea pig DRN is reported to have very few cells demonstrating PR-ir and only in the most lateral regions (Turcotte and Blaustein, 1993). In further contrast with the rat, PR-ir was not observed in the guinea pig midbrain without E priming (Turcotte and Blaustein, 1993). The rostral-to-caudal pattern of distribution of the steroid target cells through the DRN was found to be similar between sex and treatment groups. However, differences in the number of immunolabeled cells were found between groups based on either gender or treatment. Gender and ER/PR target cells Gender differences in the density of ER and/or PR target cells within discrete brain regions, at least in part, may dictate differences in responsiveness to these hormones between the sexes. In the present study, the number of cells containing ER-ir was found to be similar in female and male brains. However, a sex difference in the number of cells labeled with PR-ir was identified across the DRN, regardless of treatment. The female brain sections sampled in this study were found to have, on average, approximately 30% more cells containing PR-ir than those of males. Whether a higher concentration of PR target cells within the female DRN is related to the documented sex difference in 5-HT function cannot be determined at this point. It may explain at least partially the greater sensitivity of the female system to P modulation. Because the number of ER-containing cells within the DRN did not differ between females and males regardless of treatment, both sexes may be capable of similar regulation through an E-ER action. Physiologically, perhaps the limiting factor is the level of E in the male brain, which is largely determined by local aromatase concentrations and/or activity. Sex differences have been reported in the density of ERand/or PR-containing cells within numerous brain regions, and simultaneous differences in the two receptor types do not necessarily occur within the same nuclei. For example, Rainbow et al. (1982), by using binding assays, demon- S.E. ALVES ET AL. 329 Fig. 5. Effect of estradiol (E) on the number of estrogen receptors (ER)-labeled cells counted in representative sections of the dorsal raphe nucleus (DRN). Values depicted in the main graph are the mean number of ER-labeled cells 6 S.E.M. in the DRN of female (n 5 12) and male (n 5 11) rats that were either gonadectomized (GDX) or GDX and replaced with estradiol-17b. Inset separates mean values by gender and clearly demonstrates that treatment effects were not dependent upon sex. Cell counts/DRN level/brain were derived from the sum of labeled cells counted in two 40 µm sections representative of the Bregma level indicated (single asterisk, P , 0.005; double asterisks, P , 0.0001). strated that GDX female rats have a much higher concentration of ER within the medial preoptic area (MPOA) than GDX males; however, no sex difference in PR was found in this nucleus. Within the ventromedial hypothalamic nucleus (VMN), a sex difference specifically in PR levels was detected, with no difference in ER binding. The much higher PR concentration within the female VMN appears to explain the ability of E-primed females to respond to P, which, in turn, activates proceptive behavior and enhanced receptivity to a stimulus male (McEwen et al. 1983). In contrast, such behaviors are not seen in similarly treated male rats. 1996), and sex differences have been identified in such regulation (MacLusky and McEwen, 1978; Lauber et al., 1991). In the DRN, E treatment significantly altered the number of steroid receptor-positive cells in both females and males in a region-specific manner. Figures 5 and 6 indicate that E specifically decreased the number of detectable ER1 cells and greatly increased the number of PR1 cells within several DRN levels. Qualitative comparisons of ER-ir and PR-ir between control and E-treated groups demonstrate overall changes in labeling intensities in the same directions. These findings suggest an E-induced increase in the concentration of PR but a decrease in ER content within these E target cells. E has been widely documented to up-regulate PR gene expression in the brains of many species (Thorton et al., 1986; Brown et al., 1987; Bayliss et al., 1991; Bethea et al., 1992, 1996). Physiologically, such regulation is necessary, because many actions of P are dependent upon E priming. E has also been reported to down-regulate the expression of its own receptor within the brain (Simerly and Young, E treatment and ER/PR-containing cells Although the mechanisms that regulate brain steroid receptor levels are not completely understood, the expression of ER and PR, like other steroid receptors, can be modulated by endogenous ligands, the estrogens and progestins (MacLusky and McEwen, 1978; Blaustein and Brown, 1984; Simerly and Young, 1991; Simerly et al., 330 S.E. ALVES ET AL. Fig. 6. Effect of estradiol (E) on the number of progestin receptors (PR)-labeled cells counted in representative sections of the dorsal raphe nucleus (DRN). Values depicted in the main graph are the mean number of PR-labeled cells 6 S.E.M. in the DRN of female (n 5 12) and male (n 5 11) rats that were either gonadectomized (GDX) or GDX and replaced with estradiol-17b. Inset separates mean values by gender. Although a significant sex difference in the number of PR-labeled cells was found (Fig. 4), this graph clearly demonstrates that treatment effects were not dependent upon sex. Cell counts/DRN level/brain were derived from the sum of labeled cells counted in two 40 µm sections representative of the Bregma level indicated (single asterisk, P , 0.005; double asterisks, P , 0.0001). 1991; Shughrue et al., 1992; Borras et al., 1994; Zhou et al., 1995; Brown et al., 1996; Simerly et al., 1996), acting as a feed-back mechanism to limit the duration of hormone action. Our finding that acute E exposure (1 hour) does not result in a decrease in ER-ir (Fig. 7) is consistent with a previous report that this antibody recognizes both the ligand-bound and the unbound forms of the receptor (Okamura et al., 1992). Thus, the E-induced changes in ER-ir observed following 2 days of E exposure are likely to be an indication of a decrease in ER gene expression within those cells. The expression of one class of PRs, as mentioned above, requires E induction, whereas a second class of PRs is unaffected by E (MacLusky and McEwen, 1978, 1980). Many, if not most, PR-containing cells in the DRN appear to require E for PR induction, as seen by the scarcity of PR1 cells and the faint PR-ir in the majority of sections from GDX brains. A population of cells within the lateral wings of the DRN may be an exception, however. Cells within this region consistently demonstrated distinct PR-ir with or without E priming. In addition, because fewer ER1 cells occur within this region of the DRN, it seems likely that the receptors within these cells may belong to the class of PRs that do not require E induction (MacLusky and McEwen, 1980). It is tempting to speculate that the presence of non-E-inducible PRs within adjacent neurons may help to explain P modulation of central 5-HT activity without previous exposure to E (Ladisich, 1974). Potential modes of E/P regulation of 5-HT function in the rat brain It is well known that E and P influence neural function at the genomic level by acting at intracellular ER and PR, respectively. When they are bound, the ligand-receptor complexes act as transcription factors and regulate the ESTROGEN AND PROGESTIN RECEPTORS IN THE RAPHE 331 Fig. 7. Estrogen receptors (ER)-labeled cells within the periaqueductal grey (PAG) of an ovariectomized (OVX) 1 acute E brain (estradiol benzoate, 10 µg; A) or an OVX 1 oil brain (B). Rats were killed 1 hour following subcutaneous injection. Note that the intensities of the immunolabeling do not differ between treatments, suggest- ing that the anti-ER antibody recognizes both the unbound and the ligand-bound receptors. Photomicrographs were taken under a Nikon light microscope with a 35-mm camera using Kodak 100 ASA Technical Pan film (4003). Scale bar 5 40 µm. expression of responsive genes. Our findings in the rat are in contrast to the data gathered in the macaque brain, from which it was determined that the majority of 5-HT neurons within the DRN contain nuclear E-induced PR (Bethea, 1993, 1994). Although the existence of ER in macaque 5-HT neurons was not determined in either study, the responsiveness of these neurons to E treatment demonstrated by the dramatic increase in PR expression (Bethea, 1994) and, more recently, in TPH mRNA (PecinsThompson et al., 1996) suggest the presence of ER within 5-HT neurons in this primate species. Collectively, these findings suggest a species difference in the presence of nuclear ovarian steroid receptors within this population of cells. If we compare icc protocols of the primate studies with the present rodent study, differences in tissue-fixation procedures are evident. Although the quality of immunolabeling is very often antibody- and fixative-dependent, we do not believe that methodological differences were the cause of different receptor distributions between these species. Prior to this study, we had performed an extensive comparison of standard fixation procedures to determine the optimal method for ER and PR detection with icc in the rat brain. The paraformaldehyde/acrolein combination consistently produced the cleanest and darkest label for both of the receptor antibodies that we use. In fact, we found that, in tissue fixed with 4% paraformaldehyde alone (which was used in the macaque experiments), the distribution of receptor-labeled cells was similar, but the labeling intensity was considerably lower compared with tissue fixed with paraformaldehyde/acrolein. Thus, in our model, we chose the fixation protocol that produces the best specific signal-to-background labeling and that seems to be the most sensitive for detecting ER and PR. Therefore, in the rat, one can consider that 5-HT cells of the DRN either have so few ERs/PRs that they fall below the level of detection with icc or that these cells lack these specific receptors all together. In fact, both of these conclusions are in sharp contrast to what was determined in the macaque, i.e., that the majority of 5-HT cells in the DRN demonstrate dark, clearly labeled, E-induced PR-ir (Bethea, 1993, 1994). Thus, it seems that a fundamental difference in ovarian steroid regulation of 5-HT activity may exist between the macaque and the rat. A direct steroid hormoneintracellular receptor interaction leading to transcriptional regulation of specific genes appears to be at least one mode of 5-HT regulation that occurs in the macaque brain. In contrast, the apparent absence of detectable intracellular ER or PR within 5-HT neurons of the rat DRN and the presence of these receptors in adjacent cells suggest a possible transsynaptic interaction of the ovarian steroids and 5-HT neurons in this rodent species. It should be noted that, in the macaque, in addition to the large numbers of 5-HT neurons demonstrating PR-ir, adjacent non-5-HT cells were also found to be PR1 (Bethea, 1993). Thus, transsynaptic action could also be a part of ovarian steroid regulation of 5-HT activity within the macaque brain. Another monoaminergic system, the tuberoinfundibular dopamine (TIDA) neurons of the hypothalamus, differs in PR distribution between rodents and primates. Although the vast majority (approximately 90%) of neurons containing tyrosine hydroxylase-ir in the arcuate nucleus of the 332 rat contain nuclear PR-ir (Fox et al., 1990), no TIDA neurons in the monkey arcuate have been found to contain PR (Kohama et al., 1992). This species difference was confirmed by the finding that all PR-ir-containing cells detected in the monkey mediobasal hypothalamus appear to be GABAergic, as demonstrated by colocalization with the GABA synthesizing enzyme, glutamic acid decarboxylase-ir (Leranth et al., 1992). Because we are reporting specifically on the distribution of the ‘‘original,’’ or a, isoform of ER, the newly identified b isoform in the rat (Kuiper et al., 1996) must now be considered as a possible means of direct E action. We have recently obtained a commercially available antibody to ERb, and we are currently investigating the distribution of this receptor. Although our preliminary findings agree with a recent report of ERb mRNA distribution in the rat hypothalamus (Shughrue et al., 1996), no seemingly specific signal has been identified in the DRN (Alves, unpublished results). The existence of yet-to-be-identified plasma membrane binding sites for E and/or P must also be considered as a means of direct ovarian steroid modulation of neural activity, particularly for rapid responses. For example, in the rat nucleus accumbens, E has been shown to directly potentiate K1-stimulated dopamine release (Thompson and Moss, 1994). Very recent evidence has demonstrated that E can rapidly inhibit Ca21 currents in rat neostriatal neurons (Mermelstein et al., 1996), a population of cells that apparently lacks intracellular ER (Pfaff and Keiner, 1973; Simerly et al., 1990). By conjugating E to bovine serum albumin, which produced effects similar to those of E alone, this group demonstrated that the E-mediated action on Ca21 currents is likely to occur at the plasma membrane (Mermelstein et al., 1996). Findings from another recent study by Gu and Moss (1996) indicate that E potentiates kainate-induced currents in dissociated hippocampal CA1 neurons through a G protein-coupled, cAMP-dependent event. In addition to providing another example of a rapid, seemingly nongenomic neural action of E, the findings from this study also bring up another point to consider. That is, if E is capable of increasing intracellular cAMP levels within a cell population that does not appear to contain ‘‘classical’’ ERs, such as the CA1 pyramidal neurons (Weiland et al., 1997), then another means by which E may alter neuronal activity at the level of the genome may be at the c-AMP response element (CRE; Aronica et al., 1994). Recent studies have shown that one s.c. injection of E into OVX rats rapidly (within 15–30 minutes) increases the icc distribution of the phosphorylated form of the CRE binding protein (p-CREB; Gu et al., 1996; Zhou et al., 1996), the phosphorylation of which is dependent upon an increase in intracellular cAMP. In turn, p-CREB acts as a transcription factor at the CRE site. Thus, E may alter the expression of specific genes that lack E-response elements but that contain the CRE. Considering these findings, a preliminary experiment in the DRN showed that, although nuclear p-CREB-ir was distributed throughout the region, very few 5-HT neurons appeared to contain p-CREB, even after E treatment (Alves, unpublished results). Thus, it seems that the modulatory actions of E on 5-HT neurons do not involve a direct transcriptional activation via p-CREB. S.E. ALVES ET AL. CONCLUSIONS It has been known for sometime that the ovarian steroids, E and P, have a modulatory effect on central 5-HT activity in many species, including mice, rats, guinea pigs, and primates. However, it was not until quite recently that an actual means by which such regulation could occur was reported. Bethea (1993, 1994) demonstrated that the majority of 5-HT neurons within the female macaque DRN contain nuclear E-induced PR, thus indicating that these cells are likely targets of the ovarian steroids. Findings from the present icc study demonstrate that, whereas 5-HT neurons in the rat DRN do not appear to have detectable intracellular receptors for E or P, adjacent cells of yet-to-be-determined phenotype(s) do contain nuclear ER, E-induced PR, and perhaps non-E-regulated PR in the brains of both females and males. The only sex difference detected was an overall 30% greater number of PRcontaining cells in the brains of females, regardless of E treatment or region of the DRN. Furthermore, our data indicate that 2 days of E exposure induces some downregulation of the ER but stimulates a great up-regulation in the expression of the E-inducible PR gene in E-sensitive cells. The contrasting findings in receptor localization between the macaque and the rat suggest a species difference in ovarian steroid modulation of 5-HT activity, at least through the so-called ‘‘classical’’ mechanism of these hormones. Although a direct steroid-intracellular receptor interaction is likely to occur in the macaque 5-HT system, a transynaptic interaction involving local cells of another (or other) chemical phenotype(s) would appear to be a possible means of E/P regulation of 5-HT neuronal activity in the rat brain. We are currently continuing the search to identify the phenotype(s) of the ovarian steroid target cells within the rat DRN. 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