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Immunocytochemical localization of nuclear estrogen receptors and progestin receptors within the rat dorsal raphe nucleus

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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 [1994] 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: alvess@rockvax.rockefeller.edu
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.
ACKNOWLEDGMENTS
The authors thank Efrain Azmitia, Nick Hastings, and
Louis Lucas for their insightful discussions and advice and
Maryse Aubourg and Patima Tanapat for their technical
assistance. This work was supported by National Institutes of Health grants F32 NS10047 to S.E.A., NS07080 to
B.S.M., and NS30105 to N.G.W.
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estrogen, progestin, nuclear, dorsal, localization, within, receptors, rat, nucleus, raphe, immunocytochemical
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