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Endocrinology Vol. 143, No. 10 3974-3983
Copyright © 2002 by The Endocrine Society

ARTICLE

Expression of Estrogen Receptor-{alpha} and cFos in Norepinephrine and Epinephrine Neurons of Young and Middle-Aged Rats during the Steroid-Induced Luteinizing Hormone Surge

Sehime Temel, Winston Lin, Shruti Lakhlani and Lothar Jennes

Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky 40536

Address all correspondence and requests for reprints to: Lothar Jennes, Ph.D., Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, 428 Health Science Research Building, Lexington, Kentucky 40536. E-mail: ljenn0{at}pop.uky.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Norepinephrine (NE) and epinephrine are important stimulators of GnRH release during the preovulatory surge in female rats. Previous studies have shown that the catecholaminergic neurons are sensitive to estradiol and that NE release in the hypothalamus is decreased in middle-aged rats at the time when the estrous cycles become irregular and later cease to exist. The aims of the present study were to determine whether the NE and epinephrine neurons continue to express estrogen receptor (ER)-{alpha} in middle-aged rats; temporal expression of ER-{alpha} and cFos changes with age during the steroid-induced surge; and tyrosine hydroxylase (TH), dopamine-ß-hydroxylase (DBH), and phenylethanol-N-methyltransferase mRNA content in catecholaminergic neurons of the brain stem changes during the surge with age. The results show that there was no difference in TH mRNA content; however, DBH mRNA levels in areas A1, A2, and C1 of the middle-aged animals did not rise during the surge as was observed in the young animals. Although the percentage of NE and epinephrine neurons that express ER-{alpha} was unchanged during the surge in both young and middle-aged animals, cFos expression was enhanced in areas A1 and A2 of the middle-aged animals but not in the young animals. Together the results suggest that NE and epinephrine neurons in the middle-aged rat continue to express appropriate basal levels of TH, DBH, and phenylethanol-N-methyltransferase mRNAs as well as ER-{alpha} and cFos; however, the enhancement of DBH expression, as seen in the young animals during the steroid-induced surge, was not detected in middle-aged animals. On the other hand, cFos expression in the middle-aged rat was higher in areas A1 and A2 during the surge. It is concluded that the reduced catecholamine release during the surge in middle-aged rats is caused, in part, by an altered sensitivity of the NE neurons to estradiol, which results in an aberrant cFos expression and probably not by major deficits in the expression of transmitter synthesizing enzymes or steroid receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DECAPEPTIDE GnRH is the key hormone in the brain that controls the reproductive cycle in all mammals. The activity of GnRH-producing neurons in turn is governed by complex positive and negative feedback loops within the brain as well as with hormones of the anterior pituitary and the gonads (1). Of importance for the present studies are the interactions with the ovarian steroids, particularly with estradiol, which inhibits GnRH release during all stages of the cycle, except during proestrus when rising circulating levels of estradiol induce an increase in the pulse frequency and amplitude of GnRH release, which causes a massive release of the gonadotropins from the anterior pituitary. Previous studies have shown that certain GnRH neurons express estrogen receptor (ER)-ß(2); however, afferent input to the GnRH neurons by steroid-receptive neurons is needed for the induction of a preovulatory or steroid-induced GnRH-LH surge (3). Among these afferent neurons are the norepinephrine (NE)- and epinephrine-containing neurons that reside in the medulla and project to the GnRH perikarya in the medial septum-diagonal band-preoptic area as well as to the median eminence, which is the site of GnRH release. Thus, from an anatomical point of view, these catecholaminergic neurons could modify GnRH release at both the GnRH perikaryon and axon terminals.

The hypothalamus receives its main NE innervation from the ventrolateral medulla (A1) and the nucleus of the solitarii tract (A2) and, to a lesser extent, from the locus ceruleus (A6) (4, 5). Many catecholaminergic neurons in the areas A1 and A2 express the ER-{alpha} (6, 7, 8, 9) and, to a lesser extent, ER-ß(10). Activation of these receptors leads to an increase in electrical excitability (11), enhanced NE turnover rates in many hypothalamic nuclei (12), and enhanced NE release in the mediobasal hypothalamus (13), which in turn stimulates GnRH secretion. During proestrus, NE release in the mediobasal hypothalamus (14) and the medial preoptic area (15) are enhanced during the afternoon, which is paralleled by an enhanced GnRH release. This stimulatory action of NE is essential for the generation of a preovulatory and steroid-induced GnRH-LH surge because a reduction in NE synthesis (16, 17, 18, 19, 20) or administration of {alpha}1-adrenergic receptor antagonists prevents the surge (21, 22, 23). The finding that GnRH neurons contain {alpha}1-adrenergic receptors in their plasma membrane and that such receptor patches are juxtaposed by norepinephrine axons indicates that the stimulatory effects of norepinephrine could be exerted directly on the GnRH neurons (24).

The epinephrine innervation of the central nervous system originates from neurons of three cell groups that are located in the ventrolateral medulla (C1), the nucleus of the solitarii tract, and dorsal vagal nucleus (C2) and in and next to the medial longitudinal fascicle (C3) (25). Axonal transport studies have shown that the hypothalamus receives its epinephrine input from all three cell groups (26), and we have shown previously that many epinephrine neurons in the medulla express the ER-{alpha} protein, which suggests that these neurons could propagate the steroid signal to the GnRH neurons (27). Similar to NE, epinephrine binds with high affinity to {alpha}1-adrenergic receptors that are present in GnRH neurons. That endogenous epinephrine is important for the control of GnRH neurons is indicated by the findings that inhibition of epinephrine synthesis with drugs that selectively block the synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) prevents the steroid induced GnRH-mediated LH surge without interfering with NE synthesis (28, 29, 30). Similarly central administration of epinephrine stimulates LH release and is more effective than NE (31, 32, 33, 34).

Several studies have shown that in aging rodents there is a decline in the activity of NE system beginning at 12–14 months of age (35). Thus, NE content (36), release (14), and turnover in the hypothalamus (37) are reduced, which correlates with the reduced height of the LH surge in middle-aged animals, but no data are available on epinephrine system in the middle-aged rat. In the present studies, we aimed to determine whether the NE and epinephrine neurons continue to express ER-{alpha} and the temporal expression of ER-{alpha} changes during the steroid-induced LH surge. In addition, we measured whether the sequential administration of estradiol and progesterone activates these neurons and causes a transient induction of cFos synthesis in these neurons, and lastly whether there is a deficiency in the expression of the catecholamine synthesizing enzymes in middle-aged rats, compared with young rats, that could explain a decline in the activity of the catecholaminergic system as well as the reduced and delayed LH surge in middle-aged animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young (60 d old) and middle-aged (8–10 months old) female Sprague Dawley rats were purchased from Zivic-Miller (Zeltenolpe, PA) and housed in the central animal facilities at the University of Kentucky in a temperature- and light-controlled environment (14 h light and 10 h dark; lights on at 0500 h). All procedures involving animals were conducted in accordance with NIH guidelines for the care and use of laboratory animals and were approved by the Animal Care and Use Committee of the University of Kentucky.

Regularly cycling young and constant estrous middle-aged rats were ovariectomized 12–14 d before a sc implantation of a SILASTIC capsule 20 mm long, 1.57-mm inner diameter, 3.18-mm outer diameter (Dow Corning, Inc. Corp., Midland, MI) containing estradiol (180 µg/ml in sesame oil) at 0800 h. Two days later, the animals received one sc injection of progesterone (5 mg/100 g body weight) in sesame oil at 1000 h. This treatment results in a robust LH surge on the day of progesterone administration.

For in situ hybridization experiments, groups of five animals were decapitated on the day of LH surge at 0800, 1200, 1600, and 2000 h and blood was collected for measurements of LH levels;the brains were quickly removed, frozen on dry ice, and stored at -80 C.

For immunohistochemical experiments, groups of five animals were perfusion fixed on the day of LH surge under anesthesia at 0800, 1200, 1600, and 2000 h with PBS (0.1 M, pH 7.4), followed by 4% paraformaldhyde and 7.5% saturated picric acid in PBS. Blood was collected by cardiac puncture for measurements of LH levels, and the brains were removed and postfixed in above fixative overnight at 4 C. Serial 40-µm coronal vibratome sections were collected from the brain stem, infiltrated with cryoprotectant and stored at -20 C.

In situ hybridization
Preparation of cRNA probes.
A plasmid containing 1.1 kb rat tyrosine hydroxylase (TH) cDNA sequence (pSP65-rTH) was generously provided by Dr. J. Voogt (University of Kansas). A 535-bp rat dopamine-ß-hydroxylase (DBH) cDNA fragment was amplified by RT-PCR from rat adrenal gland total RNA with two oligonucleotide primers (5'-CACTGTAACAAGACCTCTGCCG-3' and 5'-GGAATTCAGTGTTGTCATGGCA TACACC-3') and cloned into pCRII-TOPO vector (Invitrogen, Carlsbad, CA). A plasmid containing 1.1 kb rat PNMT cDNA sequence was generously provided by Dr. Marian Evinger (State University of New York–Stony Brook). The 636-bp PstI-KpnI fragment of rat PNMT cDNA was further subcloned into pBluescript II KS vector (Promega Corp., Madison, WI). All cDNA sequences were verified by DNA sequencing. For the generation of antisense probes, plasmids pSP65-rTH, pCRII-TOPO-rDBH, and pBluescript II KS-rPNMT were linearized with SmaI, HindIII, and PstI, respectively, and transcribed in vitro in the presence of 35S-UTP with Sp6, T7, or T3 RNA polymerase, respectively.

Hybridization
Rat brain sections (40 sections/animal) were equilibrated to room temperature for approximately 10 min, fixed for 5 min in 4% paraformaldehyde in 0.1 M PBS (pH 7.4), followed by a 2-min rinse in PBS and 1-min rinse in freshly prepared 100 mM triethanolamine (pH 8.0). Sections were then incubated for 10 min in 100 mM triethanolamine and 0.25% acetic anhydride, rinsed with 2x standard saline citrate (SSC) buffer (1x SSC = 0.25 M sodium chloride and 0.015 M sodium citrate, pH 7.2) for 2 min. Sections were dehydrated in ascending ethanol concentrations (70%, 95%, and 100%), rehydrated in 95% ethanol, and air-dried. Sections were hybridized overnight in 55 C humidified chambers with 2 x 106 cpm 35S-labeled antisense cRNA probe/200 µl per slide, which was diluted in hybridization cocktail [50% formamide, 10% dextran sulfate, 300 mM sodium chloride, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1xDenhardt’s solution, 0.2 M dithiothreitol]. Hybridized sections were washed in 2x SSC with 0.17% dithiothreitol for 15 min at room temperature in RNase buffer [500 mM sodium chloride, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA] for 30 min at 37 C, and treated with 20 µg/ml RNase A for 30 min at 37 C. Sections were then washed once in 1 x SSC at room temperature (15 min), twice in 0.1x SSC at 63 C (30 min each), and once in 0.1x SSC at room temperature (15 min), followed by incubation in 300 mM ammonium acetate in 70% and 95% ethanol before they were air-dried. Slides were exposed to Hyperfilm-ß Max (Amersham Pharmacia Biotech, Piscataway, NJ) for 2–4 wk, and the films were developed with Developer D19 (Eastman Kodak Co., Rochester, NY) and fixed with Rapid Fixer (Eastman Kodak Co.).

Specificity controls included incubation with 35S-labeled sense probe, pretreatment with RNase and coincubation with 100-fold excess unlabeled antisense probe. In addition, preliminary experiments were conducted to determine the concentration of labeled probe that results in saturation of the hybridization signal.

Immunocytochemistry
Vibratome sections were equilibrated to room temperature, washed in Tris-HCl buffer (0.05 M, pH 7.6), and treated with the blocking buffer (10% normal horse serum, 0.2% Triton X-100, 0.1% Na-azide in Tris-HCl) for 1 h at room temperature (RT). Sections were then incubated overnight at RT in affinity purified rabbit anti-ER-{alpha} (1:12,000, MC-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or cFos (1:25,000, Oncogene, Cambridge, MA). After several washes with Tris-HCl buffer, sections were incubated 1 h in affinity-purified and cross-absorpt biotinylated donkey antirabbit IgG (1:400, The Jackson Laboratory, West Grove, PA), washed again in Tris-HCl buffer, and exposed for 1 h to avidin-biotin peroxidase complex (Elite, Vector Laboratories, Burlingame, CA). After two 10-min washes in Tris-HCl buffer, sections were washed in Na-acetate buffer (0.1 M, pH 6.4) to equilibrate sections for nickel-diaminobenzidine staining. Sections were then stained with 25 mg diaminobenzidine, 1.5 g nickel ammonium sulfate, and 2 µl H2O2 in 100 ml Na-acetate buffer.

Sections were then washed for 10 min in Na-acetate buffer, followed by two 10-min rinses in Tris-HCl buffer. Sections were exposed for 1 h to blocking buffer and incubated 72 h at 4 C with rabbit anti-PNMT (1:8000; Protos Biotechnology Corp., New York, NY). Sections were washed in Tris-HCl buffer; incubated 1 h with biotinylated donkey antirabbit IgG, followed by avidin-biotin complex;washed several times in Tris-HCl buffer; and stained with diaminobenzidine (50 mg) and 5 µl H2O2 in 100 ml Tris-HCl buffer.

To identify NE neurons, the double-stained sections were washed three times, 10 min each, in Tris-HCl buffer, followed by 1 h in blocking buffer. Sections were then incubated in guinea pig anti-DBH (1:1000, Dr. R. Grzanna, DuPont) overnight at RT, washed in Tris-HCl, and exposed to affinity-purified and cross-absorbed fluorescein isothiocyanate-conjugated donkey anti-guinea pig IgG (1:100, The Jackson Laboratory) for 1 h. After several washes in Tris-HCl, sections were mounted onto glass slides, dried, and coverslipped with mounting media (Vectashield, Vector Laboratories).

Control experiments included omission of primary antibody as well as extensive tests of cross-reactivity of the second antibodies. All control experiments resulted in the absence of staining.

Image analysis
In situ hybridization.
The hybridization signal of the film autoradiograms was assessed with a BH2 light microscope (Olympus Corp., Melville, NY) linked to a semiquantitative analysis system using computer-assisted microdensitometry (NIH image).

Immunohistochemistry.
Each DBH- and PNMT-immunoreactive neuron in areas A1, A2, C1, C2, and C3 was examined for the presence of ER-{alpha} or cFos protein. The anatomical identification of these regions was based on the brain maps of Moore and Card (5) and Hökfelt et al. (25).

RIA for LH
Plasma LH levels were measured by a standard double-antibody RIA using CSU 120 rabbit antirat LH antibody (Dr. Terry Nett, Colorado State University, Fort Collins, CO) at a working dilution of 1:10,000 and purified iodinated rat LH (Covance Laboratories, Inc., Vienna, VA) at 12,000 cpm/100 µl buffer. Plasma LH levels are expressed as nanograms RP-3 per milliliter.

Data analysis
The effects of aging and time of day were assessed by two-way ANOVA. When the F values indicated significant changes, post hoc comparisons were made between groups with the Newman-Keuls test. Significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LH levels
The sequential estradiol-progesterone treatment of ovariectomized young and middle-aged female rats resulted in a transient surge in circulating LH levels during the afternoon. In the young animal, plasma LH concentrations were basal at 0800 h, they began to rise at 1200 h, and they reached their peak level at 1600 h before returning to basal levels in late evening. In the middle-aged animal, plasma LH levels were basal at 0800 and 1200 h, they reached their peak levels at 1600 h, and they returned to basal levels by 2000 h. Peak LH levels in young animals reached about 125 ng/ml, but in middle-aged animals, levels of about 45 ng/ml were reached (Fig. 1Go). Statistical analyses showed that LH levels in young animals were significantly higher, compared with middle-aged animals at 1200 and 1600 h.



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  		Figure 1. Serum LH levels in young and middle-aged rats during the day of the steroid-induced LH surge show that in middle-aged animals, the surge is time delayed and smaller in peak height. **, Statistically significant differences, compared with all other time points during the day of the surge (1600 h, young animals). *, Statistically significant difference, compared with 0800 h of the young animals (1200 h). ##, Statistically significant differences, compared with all other time points during the day of the surge (1600 h, middle-aged animals). #, Statistical differences (2000 h), compared with 0800 and 1200 h of the middle-aged animals (n =10 animals per group; P < 0.05).

 
In situ hybridization
In situ hybridization with TH, DBH, and PNMT ribroprobes resulted in specific labeling of the locus ceruleus and the regions A1, A2, and C1-3 (Fig. 2Go, A–H). Densitometric analyses of autoradiograms showed that TH mRNA levels in the locus ceruleus and in the areas A1, A2, and C1 did not change during the day of the LH surge in either young or middle-aged animals and that the relative amounts of the mRNA were similar in both age groups (data not shown). DBH mRNA levels also did not change in the locus ceruleus of young and middle-aged animals; however, they were significantly elevated at 1200 h in areas A2 and C1 and at 1600 and 2000 h in area A1 of the young animals, compared with basal mRNA content at 0800 h. No change was measured in the above regions of the middle-aged animal (Fig. 3Go). PNMT mRNA levels did not change during the day of the steroid-induced LH surge in areas C1, C2, and C3 of the young animal; however, a small but statistically significant increase in the mRNA levels was measured at 1600 h in area C1 of the middle-aged animal, compared with 0800 and 1200 h (Fig. 4Go).



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  		Figure 2. A–H, Examples of autoradiograms generated after in situ hybridization with TH cRNA (A–C), DBH cRNA (D–F), and PNMT cRNA probes (G and Hh) showing strong hybridization signals in the locus ceruleus (A and D), areas A1 and A2 (B and E), and areas C1-C3 (F–H) of young (A–C) and middle-aged animals (D–H).

 


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  		Figure 3. DBH mRNA levels in the locus ceruleus did not change during the day of the LH surge; however, they were elevated at 1200 h in areas A2 and C2 and at 1600 and 2000 h in area A1 of the young animals. One asterisk indicates a significant difference, compared with two asterisks at P < 0.05 (n = 5 animals per group).

 


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  		Figure 4. PNMT mRNA levels did not change in young animals during the day of the steroid-induced LH surge; however, they were elevated in the middle-aged animals at 1600 h, compared with 0800 and 1200 h. One asterisk indicates a significant difference, compared with two asterisks at P < 0.05 (n = 5 animals per group).

 
Immunocytochemistry
DBH, PNMT, and ER-{alpha}.
The immunohistochemical procedures revealed that many NE neurons, identified by immunofluorescence for DBH, and epinephrine neurons, identified by a brown diaminobenzidine precipitate over the cytoplasm of epinephrine neurons, contained nuclear ER-{alpha} protein (Figs. 5Go, A–D, and 6Go, A–C). About 50% of the NE neurons in areas A1 and A2 of both young and middle-aged animals expressed ER-{alpha} protein, and this percentage did not change during the day of the steroid-induced LH surge. Similarly, about 30% of the epinephrine, PNMT-positive neurons in areas C1, C2, and C3 of both young and middle-aged animals expressed ER-{alpha} protein, and this percentage also did not change during the day of the LH surge (Fig. 7Go).



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  		Figure 5. A–H, Examples of immunohistochemical colocalization of DBH in areas A1 and A2 (A, C, E, G) and ER-{alpha} (B, D) and cFos (F, H) of young (A, B, E, F) and middle-aged animals. Bar, 20 µm.

 


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  		Figure 6. Examples of immunohistochemical colocalizations of PNMT (brown) and ER-{alpha} (black) in areas C1-3 (A–C) and PNMT (brown) and cFos (black) (D–F) of young (A, E, F) and middle-aged animals (B–D). Bar, 30 µm.

 


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  		Figure 7. Mean percentages ± SEM of DBH neurons in areas A1 and A2 and PNMT neurons in areas C1-3 containing immunoreactive ER-{alpha} protein at different time points during the steroid-induced LH surge in young and middle-aged animals. Note that there is no change during the day of the LH surge in either young or middle-aged animals (n = 5 animals per group).

 
DBH, PNMT, and cFos
Immunohistochemistry for DBH, PNMT, and cFos showed that many catecholaminergic neurons in the areas A1, A2, and C1-3 express cFos in their nuclei (Figs. 5Go, E–H, and 6Go, D–F). Analyses of immunohistochemical stainings to identify cFos-expression in NE and epinephrine neurons during the steroid-induced LH surge reveals that at 1200 h significantly more NE neurons in areas A1 and A2 of the middle-aged animal contain cFos in their nuclei, compared with 0800, 1600, and 2000 h. Such an increase was not measured in young animals. Similarly, the percentage of epinephrine neurons that express cFos did not change during the day of the LH surge in either young or middle-aged animals (Fig. 8Go).



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  		Figure 8. Mean percentages ± SEM of DBH neurons in areas A1 and A2 and PNMT neurons in areas C1-3 containing immunoreactive cFos protein at different time points during the steroid-induced LH surge in young and middle-aged animals. Note that although there is no change in young animals, fos expression is enhanced in middle-aged animals in noradrenergic neurons of areas A1 and A2 at 1200 h, compared with the other times analyzed. One asterisk indicates a significant difference, compared with two asterisks at P <0.05 (n = 5 animals per group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The importance of NE and epinephrine in the generation of basal and surge secretion patterns of GnRH-LH has been well documented (3, 38), and it is known that NE release in the medial preoptic area (14) and turnover rates in the mediobasal hypothalamus are reduced in the middle-aged animals during the steroid-induced LH surge, compared with young animals (35). This decrease in NE release is paralleled by a decrease in GnRH-LH secretion in the middle-aged rats (35), and it is thought that a causal relationship exists between the reduced NE release and decreased GnRH-LH release in that insufficient stimulation of the GnRH neurons by NE leads to a reduced height of the LH surge. Thus, the aim of this study was to identify some of the underlying mechanisms that cause the decrease in secretory activity of the brain stem catecholaminergic neurons.

One possible mechanism is that the catecholaminergic neurons in the middle-aged animals cease to synthesize ERs and therefore would not respond to the steroid hormone signal. However, based on the immunohistochemical data of this study, this possibility apparently does not apply because no differences in the percentage of ER-expressing NE and epinephrine neurons could be measured in young and middle-aged animals. Although these data suggest that estrogen could bind to the appropriate receptors in the brain stem catecholaminergic neurons, it is not clear whether these receptors are functioning or whether the steroid-induced signal transduction cascade is impaired in the middle-aged animals. Based on previous studies in rat and sheep that showed an induction of the synthesis of the transcription factor cFos by estradiol in the NE neurons of the brain stem (39, 40), we determined whether activation of the ER by exogenous steroid as determined by cFos expression continues in the middle-aged rats. The results of this study show that cFos synthesis after administration of estradiol is not impaired in the NE and epinephrine brain stem neurons of the middle-aged animal, compared with the young animal and that, in fact, it is enhanced at 1200 h in the NE neurons of areas A1 and A2 of the middle aged animal. The reasons for such an enhancement in cFos synthesis in middle-aged animals are not clear; however, it is possible that a lack of tight control in the signal transductions or timing mechanisms are indicated. Together the above results suggest that in the middle-aged rat, deficiencies in ER-{alpha} expression or the steroid-induced synthesis of cFos are probably not responsible for a reduced activation of the GnRH-LH axis by estradiol.

The low NE content in the medial preoptic area and arcuate nucleus of middle-aged rats could indicate that the catecholamine is not synthesized in appropriate amounts in the neurons of areas A1, A2, or C1-3. This hypothesis is supported by the findings of Miller and Zhu (41), who reported a significant reduction in the number of immunoreactive NE fibers that contact GnRH neurons in the forebrain of old mice, compared with young animals. These data suggested that either neuronal degeneration of NE or epinephrine axons had occurred or that the levels of the marker enzyme DBH had declined below the detection limits of immunohistochemistry. To determine whether NE or epinephrine synthesis is impaired in the middle-aged rat, mRNA levels for tyrosine hydroxylase, DBH, and PNMT were measured in the locus ceruleus and areas A1, A2, and C1-3 in young and middle-aged rats.

Previous experiments have shown that TH mRNA levels are, at least in part, regulated by gonadal steroids (42, 43, 44, 45). Thus, after castration, an initial decrease in TH mRNA levels was reversed by 12 h and followed by a significant increase by 24 h, but administration of estradiol prevented the initial decline in TH mRNA levels and caused a significant increase by 24 h (44). In female rats, TH mRNA levels in the A1 area were unchanged during the morning of proestrus, they rose during the LH surge in the afternoon, and they remained high into the evening, but the mRNA levels did not change in the locus ceruleus. Because such changes were not observed during the day of diestrus or in androgenized female animals, it appears that the increased estradiol levels and not diurnal rhythms were responsible for the changes in TH mRNA expression in area A1 (45). These results differ from the data of the present study in that no changes in the mRNA levels were measured in area A1 in the afternoon or evening of the day of the LH surge. One explanation for this discrepancy is that in a regularly cycling animal, the estradiol levels are elevated for only 24–28 h during diestrus 2 and early proestrus before the LH surge (46), but they were high for 54–58 h in the animal model used for the present study. The prolonged exposure of the catecholaminergic neurons to elevated levels of estradiol may have obscured possible short-term effects of estradiol on TH transcription during the first day of the steroid treatment after which the TH mRNA levels returned to basal. This view is supported by the findings of Liaw et al. (44), who reported an initial increase in TH mRNA levels in area A1 12 h after estradiol administration to castrated animals followed by a return to basal mRNA levels after 24 h. Future experiments are needed to determine whether this interpretation is indeed correct.

The finding of the present study, that TH mRNA levels in areas A1, A2, and C1-3 are similar in young and middle-aged animals, indicates that a deficiency in TH gene expression is probably not responsible for a reduced release of NE and/or epinephrine in the hypothalamus during the LH surge.

The physiological significance of the lack of increase in DBH mRNA levels at 1200 h in areas A2 and C1 as well as at 1600 and 2000 h in area A1 of the middle-aged rat is not clear at present. Although TH is generally regarded as the rate-limiting enzyme in the catecholamine synthesis under most baseline conditions, DBH levels can limit NE synthesis, especially under stimulated conditions. It is possible that in the middle-aged animals, reduced levels of DBH mRNA could be insufficient to generate amounts of NE adequate to stimulate preovulatory GnRH release; however, it appears more likely that the precise control of catecholamine release rather than synthesis is compromised. This view is based on the data of Mohankumar et al. (36), who showed that average extracellular NE levels are actually enhanced in the medial preoptic area of the middle-aged rat, compared with the young rats, and peak NE levels were attenuated. These data suggest that sufficient NE is present in the relevant nerve terminals and the mechanisms controlling the release activity appear to be inappropriate. Although no data are available on epinephrine release patterns in young and middle-aged rats, the present results suggest that PNMT mRNA levels are not reduced in middle-aged animals and, in fact, are elevated in area C1 during the surge. These findings indicate that similar to NE, deficiencies in epinephrine release and not synthesis are responsible for a decreased secretion of this neurotransmitter.

In summary, the main findings of the present study are that NE and epinephrine neurons in the middle-aged animals continue to express appropriate basal levels of TH, DBH, and PNMT mRNAs as well as ER-{alpha}and cFos; however, the enhancement of DBH expression, as seen in the young animals during the steroid-induced surge, were not detected in middle-aged animals. It is suggested that the reduced catecholamine turnover during the surge in middle-aged animals is caused, in part, by a decreased sensitivity of the NE neurons to estradiol and probably not by major deficits in the expression of transmitter-synthesizing enzymes or steroid receptors. Other factors, such as changes in catecholamine release control mechanisms or alterations in signal transduction mechanisms in catecholaminergic neurons in response to estradiol may contribute to the decreased transmitter release.


   Footnotes
 
This work was supported by NIH Grants MH-59890, AG-17164, and RR-15592.

Abbreviations: DBH, Dopamine-ß-hydroxylase; ER, estrogen receptor; NE, norepinephrine; PNMT, phenylethanolamine-N-methyltransferase; RT, room temperature; SSC, standard saline citrate; TH, tyrosine hydroxylase.

Received April 22, 2002.

Accepted for publication June 7, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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