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Department of Pharmacology, Boston University School of Medicine, Boston, Massachusetts 02118
Address all correspondence and requests for reprints to: Dr. Mark J. Alexander, Department of Pharmacology, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118. E-mail: mjalex{at}bu.edu
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Introduction |
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Several lines of evidence indicate that the activity of ARC NT neurons is regulated either directly or indirectly by ovarian steroids. NT and the structurally related peptide neuromedin N (N) are synthesized together as part of a precursor protein encoded by NT/N messenger RNA (mRNA) (5). Estrogen induces NT/N mRNA selectively in the ARCdm, and a corresponding increase in NT immunoreactivity in the external zone of the median eminence confirms the neuroendocrine phenotype of these ARCdm cells (6). Significantly, there is a 5-fold increase in NT release at the median eminence of female rats coincident with the onset of estrogen-induced surges of PRL and GnRH (7). Moreover, neutralization experiments with anti-NT serum under several different endocrine conditions have provided evidence that estrogen and progesterone modulate the effects of endogenous NT on PRL secretion (8). Taken together, these results indicate that NT neurosecretory cells may be important hypothalamic targets for the actions of ovarian steroids.
Estrogen and progesterone exert many coordinated actions through
cognate receptors belonging to the superfamily of ligand-activated
transcription factors. The rat hypothalamus contains at least two
subtypes of nuclear estrogen receptor (ER
and ERß) (9, 10, 11) and at
least two isoforms of nuclear progesterone receptor (PR-A and PR-B)
(12). In the female ARC, estrogen slightly up-regulates ERß mRNA
(11), but markedly down-regulates ER mRNA (13, 14), the predominant
form of ER mRNA in this region (10), thereby complicating detection of
ER mRNA-expressing cells. In contrast, estrogen markedly
up-regulates PR mRNA and PR in the ARC (15, 16), consistent with the
observation that estrogen priming is required for many of the central
effects of progesterone. It is generally accepted that
estrogen-inducible synthesis of PR is triggered by an action of
ligand-bound ER within the cell nucleus, and several studies in
vivo (17, 18) support the view that estrogen-inducible PR occurs
only in cells expressing ER and is therefore a conservative cellular
marker for ER in the rodent ARC.
If NT neurons in the ARC express ER and PR, then ovarian steroids could
act directly on these neurons to potentially alter the expression of
transcription factors, enzymes, receptors, or other proteins such as
the NT/N precursor and thereby influence neuronal activity in complex
ways. Although many estrogen-responsive NT neurons in the rat preoptic
area (19, 20) have been shown to contain ER immunoreactivity (21, 22, 23),
there is no information regarding possible colocalization of NT with
either ER or PR in rat ARC neurons. An immunohistochemical study in the
estrogen-treated guinea pig found little evidence for colocalization of
NT and PR in the ARC (24); however, there is currently no evidence that
ovarian steroids regulate the activity of NT neurosecretory cells in
this species. To determine whether NT neurons in the rat ARC are
capable of expressing receptors for ovarian steroids, we used
estrogen-inducible PR mRNA as a marker for PR and for ER as well
because the elevated estrogen levels leading to induction of NT/N mRNA
in the ARCdm cause marked down-regulation of ER mRNA. Using double
label in situ hybridization, we sought to determine whether
and to what extent NT/N mRNA and PR mRNA are colocalized in individual
ARC neurons of the female rat under estrogen-stimulated conditions.
Preliminary results of this study have been published in abstract form
(25).
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Materials and Methods |
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Rats were decapitated between 1600–2000 h on the seventh day after OVX, and thus, the duration of estradiol treatment was either 2.5 or 7 days. Brains were rapidly removed, frozen in chilled isopentane, and stored at -85 C. A cryostat microtome was used to prepare coronal sections (20 µm) through the forebrain, which were thaw mounted on slides pretreated with aminoalkylsilane (26) and stored at -85 C. For quantitative assessment of the extent to which NT/N mRNA and PR mRNA are colocalized, a series of sections (1 in 2) through the rostral half of each ARC were generated from brains of OVX females treated with estradiol for 7 days.
Probes for NT/N mRNA
Probes for NT/N mRNA were synthesized with a template
plasmid (prNT4, provided by P. Dobner) consisting of a 336-bp
EcoRV/BglII fragment (nucleotides 626–961) of
the rat NT/N gene (5) ligated into
BamHI/SmaI-digested pGEM4 (Promega Corp., Madison, WI). This insert corresponds to part of exon 4
encompassing the NT-coding domain and proximal 3'-untranslated region.
Antisense probes synthesized from this sequence detect both short and
long isoforms of NT/N mRNA, which result from differential utilization
of alternate poly(A) adenylation signals (5). Radiolabeled antisense
probe was transcribed from EcoRI-digested prNT4 with T7 RNA
polymerase (Riboprobe System-T7 kit, Promega Corp.) and
[35S]CTP (1250 Ci/mmol; New England Nuclear Corp.,
Boston, MA) in the absence of unlabeled CTP. Unincorporated nucleotides
were removed by chromatography with Sephadex G-50 (Quick Spin column,
Roche Molecular Biochemicals, Indianapolis, IN).
Probe yield was calculated from the number of [35S]CTP
molecules incorporated in each full-length transcript and the specific
activity of [35S]CTP. Antisense probe labeled with
digoxigenin (dig) was transcribed from EcoRI-digested
prNT4 with T7 RNA polymerase (MEGAscript T7 kit, Ambion, Inc., Austin, TX) and dig-UTP (2 mM;
Roche Molecular Biochemicals) in the presence of unlabeled
UTP (2 mM). Unincorporated nucleotides were removed as
described above. The yield of dig-labeled probe was estimated from the
RNA concentration obtained by spectrophotometric analysis of diluted
probe samples at 260 nm.
Probes for PR mRNA
Probes for PR mRNA were synthesized with a template plasmid
(pPR-1, provided by O.-K. Park-Sarge and K. Mayo) consisting of a
548-bp rat PR complementary DNA (cDNA) (27) ligated into
BamHI/SalI-digested pGEM4. This insert
corresponds to part of the ligand-binding domain that is present in
both A and B isoforms of PR mRNA (12) and displays only partial
nucleotide sequence identity with corresponding regions of androgen
receptor (62%), glucocorticoid receptor (59%), and ER (41%) cDNAs
(27). Radiolabeled antisense probe was transcribed from
EcoRI-digested pPR-1 with T7 RNA polymerase and
[35S]CTP in the absence of unlabeled CTP. Radiolabeled
sense probe was transcribed similarly from HindIII-digested
pPR-1 with SP6 RNA polymerase (Riboprobe System-SP6 kit, Promega Corp.). Unincorporated nucleotides were removed, and probe yield
was calculated as described above. Dig-labeled antisense probe was
transcribed from EcoRI-digested pPR-1 with T7 RNA polymerase
(MEGAscript T7 kit, Ambion, Inc.) and dig-UTP (2
mM; Roche Molecular Biochemicals) in the
presence of unlabeled UTP (2 mM). Unincorporated
nucleotides were removed, and probe yield was estimated as described
above for dig-labeled NT/N mRNA probe.
Single label in situ hybridization histochemistry
Single label in situ hybridization was performed
according to a modified version (28) of a protocol originally described
by Cox et al. (29). In preparation for probe application,
sections were warmed to room temperature and treated for 10 min with
4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH
7.4) at 4 C, rinsed in 0.1 M sodium phosphate containing
0.15 M sodium chloride, rinsed in 0.1 M
triethanolamine (pH 8.0), treated for 10 min with 0.25% acetic
anhydride in 0.1 M triethanolamine, rinsed in 2 x SSC
(0.3 M sodium chloride-0.03 M sodium citrate,
pH 7.0), dehydrated in a graded ethanol series, and defatted in
chloroform. Air-dried sections were stored at room temperature until
probe application.
Radiolabeled probe was denatured at 90 C for 10 min and diluted to a
final concentration of 0.5–1.0 pmol/ml (
2 x 108
dpm/ml) in hybridization buffer consisting of 50% formamide, 10%
dextran sulfate, 300 mM sodium chloride, 10 mM
Tris-HCl (pH 8.0), 1 mM EDTA, 0.2% BSA, 0.2% Ficoll,
0.2% polyvinylpyrrolidone, yeast transfer RNA at 0.5 mg/ml, and 200
mM dithiothreitol (DTT). Sections were covered with
hybridization mix (50 µl/slide) and a pretreated glass coverslip,
then incubated in a slightly humidified chamber at 45 C (NT/N) or 50 C
(PR) for 18–20 h. Sections were washed three times for 15 min each in
2 x SSC containing 10 mM DTT; incubated at 37 C for
30 min with ribonuclease A (Pharmacia Biotech, Piscataway,
NJ) diluted to 20 µg/ml in buffer composed of 500 mM
sodium chloride, 10 mM Tris-HCl (pH 8.0), 2 mM
DTT, and 1 mM EDTA; incubated at 37 C for an additional 30
min in ribonuclease buffer; washed twice for 15 min each time in 2
x SSC; washed at 58 C (NT/N) or 64 C (PR) for 1 h in 0.1 x
SSC; rinsed briefly in 0.1 x SSC; dehydrated in a graded ethanol
series; and allowed to air-dry.
Double label in situ hybridization histochemistry
Double label in situ hybridization was performed
according to a modified version of a protocol described by Miller
et al. (30). After pretreatment of tissue as outlined above,
dig-labeled probe was added to hybridization mix to yield an estimated
final concentration of 2–3 pmol/ml. Overnight incubation and
subsequent wash steps were performed as described above, except that,
after the final rinse in 0.1 x SSC, sections treated with
dig-labeled probe were stored overnight at 4 C in 2 x SSC
containing 2% normal sheep serum and 0.05% Triton X-100. Sections
were washed twice for 10 min each time in Tris-buffered saline (TBS;
0.1 M Tris-HCl and 0.15 M sodium chloride, pH
7.6), then incubated for 3 h at 28 C in TBS containing 1% normal
sheep serum, 0.3% Triton X-100, and anti-dig-Fab fragments coupled to
alkaline phosphatase (1:1000 dilution of stock solution from
Roche Molecular Biochemicals). Sections were washed for 15
min in TBS and for 15 min in Tris-buffered saline containing magnesium
chloride (TBSM; 0.1 M Tris-HCl, 0.1 M sodium
chloride, and 0.05 M magnesium chloride, pH 9.5). Sections
were then incubated overnight at 4 C in filtered chromagen solution
[TBSM with 0.34 mg/ml 4-nitro blue tetrazolium chloride (Roche Molecular Biochemicals), 0.175 mg/ml
5-bromo-4-chloro-3-indolyl-phosphate (Roche Molecular Biochemicals), and levamisole (1:100 dilution of stock solution
from Vector Laboratories, Inc., Burlingame, CA) to inhibit
endogenous alkaline phosphatase]. Sections were transferred to room
temperature, and chromogenic development was monitored by microscopic
inspection. Chromogenesis was terminated in dilute Tris buffer (0.01
M Tris-HCl and 0.001 M EDTA, pH 8.0), and
sections were rinsed very briefly in 70% ethanol and dried quickly in
a stream of cool air.
Autoradiography
Sections were apposed to Hyperfilm-ßmax (Amersham Pharmacia Biotech, Arlington Heights, IL) for about 2 weeks (PR
probe) or about 5 weeks (NT/N probe), after which films were developed
for 4 min in Kodak D19 (Eastman Kodak Co.,
Rochester, NY), rinsed in water, and fixed for 5 min in
Kodak Rapid Fixer. Sections processed for double label
in situ hybridization were dipped in 3% parlodion (in
isoamyl acetate) and allowed to air-dry overnight. Sections were then
dipped in NTB2 nuclear track emulsion (Kodak) diluted 1:1
with distilled water and stored at 4 C for approximately 2 weeks (PR
probe) or approximately 4 weeks (NT/N probe). Emulsion was developed
for 4 min in D19 (diluted 1:1 with distilled water), rinsed in water,
and fixed for 5 min in Kodak Fixer. Sections processed for
single label in situ hybridization were counterstained
lightly with thionin, dehydrated in an alcohol series, immersed in
xylene, and coverslipped with Permaslip (Alban Scientific, St. Louis,
MO). Sections processed for double label in situ
hybridization were dried in a stream of cool air and coverslipped with
Permaslip.
Controls for labeling specificity
Specificity of labeling obtained with antisense probe for NT/N
mRNA has been verified previously (28). Labeling obtained with
antisense probe for PR mRNA was validated in the present study by
several methods. In situ hybridization with sense strand
probe yielded no discernible signal on sections through the mediobasal
hypothalamus of estradiol-treated females. Moreover, there was
excellent agreement between the regional distribution of signal
obtained with dig-labeled antisense probe for PR mRNA and that obtained
with its 35S-labeled counterpart. Finally, the regional
distribution of estrogen-inducible signal for PR mRNA was in excellent
agreement with that described previously (16) and with the regional
distribution of estrogen-inducible PR described by others (15).
Image reproduction
Photographs of film autoradiograms were obtained with
Kodak Plus-X Pan film. Photomicrographs of emulsion
autoradiograms were obtained with an Axioskop microscope (Carl Zeiss, Inc., Thornwood, NY) and Kodak Technical Pan
film, and darkfield images were obtained under transverse illumination
(Microvid Darklite Illuminator, Micro Video, Inc., Avon, MS).
For labeling and reproduction, all photomicrographs were converted to
digital images with a high resolution scanner, an Apple Power
Macintosh computer (Apple Computers, Cupertino, CA), and Adobe
Photoshop, then printed with an Epson Photo 700 six-color printer.
Changes were made in the brightness and contrast of the original
photomicrographs, and dust artifacts were removed, but the data content
of these images was not altered in any way.
Quantitative and statistical analysis
For quantitation of double label in situ
hybridization results, NT/N mRNA was detected with
35S-labeled probe, and PR mRNA was detected with
dig-labeled probe, as this combination yielded the best overall
sensitivity in preliminary studies. All sections were processed
together during hybridization and autoradiographic procedures.
NT/N-positive cells were defined as clusters of silver grains 5 times
or more the average density of grains overlying the ventromedial
hypothalamic nucleus (VMH), a density classified as background labeling
on the basis of film autoradiographs. To guard against false
classification of cells as PR positive due to excessive chromagenic
development, autoradiograms previously generated with
35S-labeled PR probe were used as a guide to determine the
extent of chromogenic development for the dig-labeled PR probe.
Analysis was conducted on the rostral half of the ARC, which was divided into three principal levels (A–C) corresponding to those depicted in the Swanson rat brain atlas (31) as plates 26–28, respectively. Level A, the most rostral, denotes that portion of the ARC located rostral to the infundibular recess of the third ventricle. At this level, the ARC is principally a midline structure that extends bilaterally into the ventral part of the hypothalamic periventricular nucleus. Labeled cells in this periventricular portion (ARCd) and the remaining portion (ARCv) were analyzed separately by analogy with the ARCdm and ARCvl divisions present at other levels. Level B denotes the most rostral extent of the ARC as a completely bilateral structure, whereas level C denotes that portion of the ARC coincident with the main A13 DA cell group in the medial zona incerta. At levels B and C, it is possible to distinguish an ARCdm and an ARCvl, and labeled cells in these two divisions were analyzed separately. Based on the observed boundary of estrogen-inducible NT/N signal as well as a demarcation system used by others (32), we differentiated the ARCdm from the ARCvl with a line intersecting the midline at the base of the third ventricle and extending laterally at an angle of 30° from horizontal. No attempt was made to formally distinguish a ventromedial division due to the relatively small number of NT/N cells present in this region. NT/N cells located slightly outside the formal ARC border (particularly at its ventrolateral aspect) but within the distribution of estrogen-inducible PR signal were classified as ARC cells for the purpose of quantitation.
Labeled cells were identified at x400 and mapped manually onto computer-generated diagrams derived from the Swanson atlas (31). Double labeled cells and single labeled NT/N cells were counted bilaterally in either one section (level A) or two noncontiguous sections (levels B and C) per rat, and these values were used to compute the mean number of labeled cells per section as well as the mean percentage of NT/N cells that were double labeled for each rat in each of six ARC regions. These values were intended to provide comparable estimates of colocalization in different ARC regions but not absolute cell counts. Differences in the extent of colocalization among these six regions were evaluated by one-way ANOVA followed by Scheffe’s F test, with P < 0.05 considered significant.
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Results |
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). NT/N signal was seldom observed in
the VMH. Under the same conditions, there were moderate levels of PR
signal in the ventrolateral division of the VMH (VMHvl), but only low
levels of PR signal in the ARC (Fig. 1C). Within the ARC, PR signal was
consistently lower in the ARCdm, where signal levels were difficult to
distinguish from background, than in the ARCvl, where labeled cells
could still be identified on light microscopic autoradiograms.
Consistent with previous reports (12, 33, 34), PR signal was also
readily detectable in extrahypothalamic regions, most noticeably in
cerebral isocortex and the CA3 field of the hippocampus (data not
shown).
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). In contrast, NT/N signal in the ARCvl was not
appreciably affected by estradiol. As expected, estradiol caused a
pronounced increase in PR signal in all divisions of the ARC and in the
VMHvl (Fig. 1D). Estradiol also increased PR signal in scattered cells
within regions immediately surrounding the VMHvl, including the area
between it and the ARCvl. Thus, under estrogen-stimulated conditions,
the regional distributions of NT/N mRNA and PR mRNA overlapped in the
ARC (Figs. 1 and 2) but not in the VMH
(Fig. 1).
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).
Similar, but less vivid, results were obtained in OVX rats treated with
estradiol for 2.5 days. Under OVX conditions, double labeled cells were
sometimes discernible in the ARCvl, but never in the ARCdm due to very
low levels of both NT/N signal and PR signal in this region. Under
estrogen-stimulated conditions, cells containing the highest levels of
PR signal were observed more frequently in the ARCdm than in the ARCvl
(Fig. 3, compare A with B), thus suggesting a greater degree of PR mRNA
induction in ARCdm cells than in their ARCvl counterparts. Regardless
of endocrine status, numerous cells containing both NT/N signal and low
PR signal were observed in certain extra-ARC regions, such as the
lateral hypothalamus and CA1 field of the hippocampus, but not in the
VMH (data not shown).
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and 5A). The largest number of double
labeled cells per section (80 ± 18) occurred in the ARCdm at
level B. Statistical analysis revealed that the ARCdm at levels B and C
contained a significantly higher percentage of NT/N cells that were
double labeled (80%) than did other ARC divisions (60–65%),
including the ARCvl at these same levels (Fig. 5B; F = 18.96;
P < 0.0001; followed by Scheffe’s F test,
P < 0.05). No analogous difference between dorsal and
ventral divisions was observed in the ARC at level A (Fig. 5B). The
percentage of PR cells that were double labeled also varied as a
function of ARC division and rostrocaudal level. At level A, double
labeled cells accounted for about 30% of the total PR cells per
section in the ARCd and approximately 20% of such cells in the ARCv.
At level B, double labeled cells accounted for approximately 50% of PR
cells in the ARCdm, and at level C, double labeled cells accounted for
approximately 30% of PR cells in both the ARCdm and ARCvl.
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Several lines of evidence indicate that PR hybridization signal
obtained in the present study corresponds to authentic PR mRNA and
reliably indicates the presence of PR protein. Riboprobe was generated
from a well characterized cDNA corresponding to the hormone-binding
domain of rat PR (27). Rat cDNAs encoding the structurally related
androgen receptor, glucocorticoid receptor, and ER display only
partial nucleotide sequence identity with this PR cDNA (27), and their
corresponding mRNAs would be unlikely to cross-react with PR probe
under the stringent assay conditions used in the present study.
Moreover, the distribution of signal obtained with this PR probe in rat
forebrain agrees well with the distribution of PR mRNA determined
previously (12, 33, 34). Most importantly, signal obtained with this
probe was up-regulated by estrogen in the same hypothalamic regions
where estrogen-inducible PR mRNA (16) and PR (15) have been observed by
others. This concordance strongly suggests that PR signal in the
current study reliably indicates the presence of PR as well as PR mRNA
in a given ARC neuron. We did not determine whether these cells contain
one or both PR isoforms detected previously in rat hypothalamus (12).
Evidence from peripheral tissues indicates that the cellular ratio of
PR-A/PR-B has important developmental consequences (35) and can be
regulated by estrogen (36).
There is compelling evidence that estrogen-inducible PR mRNA occurs
only in cells that also express nuclear ER. Although the mechanism is
not yet understood in detail (37, 38), it is generally accepted that
estrogen-induced synthesis of PR is triggered by an action of
ligand-bound ER within the cell nucleus. In female mice, disruption of
the ER gene reduces the number of ARC cells displaying
estrogen-inducible PR immunoreactivity (PR-ir) to only 10% of that in
wild-type controls (39), thus implicating the ER subtype in the
process of PR induction. Strong support for the critical role of
nuclear ER in estrogen-inducible expression of PR comes from two
independent studies in guinea pig forebrain that used double label
immunohistochemistry to investigate cellular colocalization of ER and
PR under estrogen-treated conditions (17, 18). In both studies, PR-ir
in the ARC and certain other hypothalamic regions occurred only in
cells that also contained ER-ir. Thus, it is reasonable to infer that
functional ER is present in all ARC NT neurons expressing PR mRNA in an
estrogen-inducible manner. This approach does not distinguish between
the two known subtypes of ER; however, it does distinguish functional
variants of ER from nonfunctional variants that might be detected by
probe for ER mRNA. Our approach could potentially underestimate the
number of ER-containing NT neurons in the ARC, as there is evidence for
PR-negative, ER-containing neurons in certain brain regions (17, 18).
On the other hand, a small fraction of PR mRNA-containing cells located
specifically in the ARCvl of estrogen-treated rats could have been
falsely classified as containing estrogen-inducible PR mRNA, as some
lightly labeled PR cells were detectable in this region even under OVX
conditions.
Our results are at variance with those of a double label immunohistochemical study in the colchicine-treated guinea pig that found little evidence for colocalization of NT-ir and PR-ir in ARC neurons under estrogen-stimulated conditions (24). As there is currently no evidence that ovarian steroids alter the activity of NT neurons in this species, this discordance may be attributable to physiological differences between rat and guinea pig, and other findings are consistent with this view. For example, data indicate that PR is present in many, if not most, tyrosine hydroxylase (TH)-containing ARC neurons in the rat (40, 41), but is present in comparatively few such neurons in the guinea pig (42). It is noteworthy that a substantial population of NT-ir neurons in the hypothalamic ventrolateral nucleus, a guinea pig structure considered analogous to the VMHvl of the rat, do contain PR-ir under estrogen-stimulated conditions (24). This is clearly not the case in the rat, as NT/N mRNA-containing cells are seldom observed in the VMHvl.
Use of in situ hybridization methodology in the present study allowed us to avoid potential difficulties associated with intracerebroventricular administration of the alkaloid colchicine, which is generally required for visualization of NT-ir cell bodies in immunohistochemical studies. In the aforementioned study by Warembourg and Jolivet (24), opposing effects of colchicine on PR-ir and NT-ir necessitated the use of a colchicine dose that was probably suboptimal for detection of either PR-ir or NT-ir alone. Further complicating interpretation of such studies is the ability of colchicine to increase NT/N mRNA levels dramatically in the ARC nucleus and other brain regions (20, 43). Although it has been investigated only in the rat to date, this phenomenon may occur in other species as well. We have observed that levels of NT/N mRNA in the ARCdm of OVX rats treated intra-cerebroventricularly with a standard dose of colchicine (100 µg) are comparable to those in OVX rats treated systemically with high doses of estradiol (Alexander, M., unpublished observation). Hence, colchicine-associated effects on NT/N mRNA levels could readily obscure physiological changes in expression of this gene.
Under estrogen-stimulated conditions, the percentage of NT neurons containing PR mRNA was significantly higher in the ARCdm than in the ARCvl. This observation is consistent with other evidence for functional differences between these two ARC divisions, including the differential effect of estrogen on NT/N mRNA levels. Immunohistochemical studies conducted in the colchicine-treated male rat indicate that NT-ir can occur in TH-containing neurosecretory cells located in both the ARCdm and ARCvl (reviewed in Refs. 1, 4). Only TH neurons in the former division seem to be capable of appreciable dopamine (DA) synthesis (44, 45) and are thus considered TIDA neurons responsible for tonic inhibition of PRL secretion from the anterior pituitary. In female rats, there is extensive colocalization of NT/N and TH mRNAs in ARCdm neurons under estrogen-stimulated, but not OVX, conditions (Alexander, M., unpublished observation). Thus, the extent to which NT colocalizes with DA, steroid receptors, and other molecules in ARCdm neurons can vary markedly with the endocrine state (46). Consistent with evidence for TH/NT colocalization in the female ARC, our estimates regarding the extent of NT/PR colocalization (60–80% of NT neurons) are within the range of previous estimates regarding the extent of TH/PR colocalization (40–90% of TH neurons) in this region (40, 41). It is unclear to what extent PR mRNA-containing NT neurons located in the ARCvl (present study) overlap with GHRH/galanin/NT neurosecretory cells identified previously (4), although there is evidence that many galanin neurons in this region contain ER (47).
Remaining to be determined is whether nuclear ER or PR mediate changes
in NT/N gene expression in ARC neurons. Our results establish a
molecular substrate for direct effects of ovarian steroids on these
neurons, but do not elucidate the mechanism by which circulating
estrogen alters NT synthesis in this hypothalamic region. The promoter
region of the rat NT/N gene contains recognition sequences for cAMP
response element-binding protein (48) but no readily identifiable
estrogen-response elements, and studies conducted in neuroblastoma
cells indicate that estrogen can stimulate transcription of a NT/N
promoter-containing construct by activating the protein kinase A
signaling cascade in a manner that does not seem to require either
ER or ERß (49). Significantly, mice bearing a targeted disruption
of the protein kinase A gene do not display estrogen-inducible
expression of NT/N mRNA in the medial preoptic nucleus (49), unlike
wild-type mice or rats (19). Whether a similar mechanism might be
operative in the ARCdm is still unclear. There also remains the
possibility that some other hormone, most notably PRL, mediates the
stimulatory effects of estrogen on NT synthesis in the ARCdm.
To our knowledge, there are no reports that progesterone alters the expression of the NT/N gene in the ARC or other brain regions. Regardless of the actual role of progesterone in regulating NT synthesis, nuclear PR is likely to play a fundamental role in integrating hormonal and neuronal inputs to ARC neurosecretory cells, including those that release NT, DA/NT, or GHRH/NT. There is little doubt that PR mediates many neuroendocrine effects of progesterone, such as its modulation of rhythmic TIDA activity (50). Surprisingly, PR may also mediate certain effects of neurotransmitters on gene expression. There is compelling evidence that DA acting through the D1 DA receptor can trigger activation of nuclear PR in a progesterone-independent manner, leading to altered expression of genes containing PR recognition sites (51). Significantly, estrogen could gate certain hormonal and neuronal inputs to ARC cells by determining availability of PR isoforms for both the progesterone-dependent and progesterone-independent pathways. DA-mediated, progesterone-independent activation of PR has been best documented for behavioral regulation involving the VMH, but DA-mediated activation of PR could also occur in the ARC (52). If ARC NT neurons receive DA input (53) and also express D1 receptor (54), then estrogen-inducible PR may participate in DA regulation of gene expression in these NT neurosecretory cells.
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Acknowledgments |
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Footnotes |
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Received May 12, 1999.
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References |
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and -ß mRNA in the
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and -ß mRNA within the female rat brain. Mol Brain Res 54:175–180[Medline]
gene-disrupted mice. J Neurosci 18:9556–9563This article has been cited by other articles:
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