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Endocrinology, doi:10.1210/en.2007-0827
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Endocrinology Vol. 149, No. 2 802-811
Copyright © 2008 by The Endocrine Society

Gonadal Steroid Hormones Maintain the Stress-Induced Acetylcholine Release in the Hippocampus: Simultaneous Measurements of the Extracellular Acetylcholine and Serum Corticosterone Levels in the Same Subjects

Dai Mitsushima, Kenkichi Takase, Toshiya Funabashi and Fukuko Kimura

Department of Physiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan

Address all correspondence and requests for reprints to: Dai Mitsushima, D.V.M., Ph.D., Department of Neuroendocrinology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. E-mail: dm650314{at}med.yokohama-cu.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To examine the role of gonadal steroid hormones in the stress responses of acetylcholine (ACh) levels in the hippocampus and serum corticosterone levels, we observed these parameters simultaneously in intact, gonadectomized, or gonadectomized steroid-primed rats. In both sexes of rats, neither gonadectomy nor the replacement of gonadal steroid hormone affected the baseline levels of ACh. However, gonadectomy severely attenuated the stress response of ACh, whereas the replacement of corresponding gonadal hormone successfully restored the response to intact levels. The gonadal hormones affected the serum corticosterone levels in a different manner; the testosterone replacement in orchidectomized rats suppressed the baseline and the stress response of corticosterone levels, whereas the 17β-estradiol replacement in ovariectomized rats increased the levels. We further found that letrozole or flutamide administration in intact male rats attenuated the stress response of ACh. In addition, flutamide treatment increased the baseline levels of corticosterone, whereas letrozole treatment attenuated the stress response of corticosterone. Moreover, we found a low positive correlation between the ACh levels and corticosterone levels, depending on the presence of gonadal steroid hormone. We conclude that: 1) gonadal steroid hormones maintain the stress response of ACh levels in the hippocampus, 2) the gonadal steroid hormone independently regulates the stress response of ACh in the hippocampus and serum corticosterone, and 3) the sex-specific action of gonadal hormone on the cholinergic stress response may suggest a neonatal sexual differentiation of the septohippocampal cholinergic system in rats.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEPTOHIPPOCAMPAL CHOLINERGIC NEURONS play an important role in the formation of hippocampal-dependent memory (1, 2, 3, 4, 5, 6). In behaving animals, extracellular acetylcholine (ACh) levels in the hippocampus are known to increase during stress (7, 8, 9) and learning (10, 11, 12). The released ACh not only enhances synaptic plasticity via the postsynaptic M1 muscarinic receptors (13) but also is responsible for neurogenesis in the dentate gyrus (14). Moreover, the septohippocampal cholinergic inputs generate theta rhythm (15) that modulates the induction of long-term potentiation in the hippocampal CA1 neurons (16).

Although the role of the released ACh in the hippocampus has been mostly studied in male animals, we found the sex difference in the stress response of ACh in the hippocampus: immobilization stress acutely increased the extracellular ACh levels in the hippocampus, but the stress response of ACh in cycling female rats was significantly smaller than in males (17). The sex-specific cholinergic response could be attributed to the neonatal organizational effect of testosterone and/or the activational effects of gonadal steroid hormone. Although there is no evidence that sex neonatally differentiates the cholinergic system in rats, several findings strongly suggest activational effects of gonadal steroid hormones in the control of the septohippocampal cholinergic neurons: 1) 40–60% of cholinergic neurons in the septum possess estrogen receptor-{alpha} immunoreactivity in both sexes of rats (18, 19); 2) the number of choline acetyltransferase-immunoreactive neurons in the septum decreases after gonadectomy (18, 20) but is restored by testosterone (20) or 17β-estradiol replacement (21); and 3) circulating testosterone in male rats (22, 23) and 17β-estradiol in female rats (24, 25, 26) enhance memory consolidation. In the present study, therefore, to examine the activational effects of gonadal steroid hormones concerning sex difference in the stress response of ACh in the hippocampus, an in vivo microdialysis study was performed in intact, gonadectomized, or gonadectomized steroid-primed rats.

Exposure to stress also increases corticosterone release from adrenal cortex in a sex-specific manner (27, 28, 29). The increased circulating corticosterone not only controls the hippocampal pyramidal neurons (30) but also modulates memory consolidation in rats (31). Moreover, hippocampus partly mediates the negative feedback effect of corticosterone on the hypothalamic-pituitary-adrenal (HPA) axis (32, 33, 34). To further analyze the relation between extracellular ACh levels in the hippocampus and serum corticosterone levels in both sexes of rats, we observed these parameters simultaneously in the same subjects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young male (239.5 ± 2.0 g) and female rats (176.8 ± 2.9 g) of the Wistar-Imamichi strain at 7–8 wk of age were obtained from Animal Reproduction Research Co. (Omiya, Japan). Unisex groups of two to three rats were housed in plastic cages (length 31 cm, width 47 cm, height 20 cm) at a constant temperature of 23 ± 1 C under a constant cycle of light and dark (lights on from 0500 to 1900 h). Experiments were performed in an electromagnetic- and sound-shielded room (length 1.2 m, width 2.2 m, height 2.3 m) (27). Food and water were available ad libitum in all experimental periods. All the animal housing and surgical procedures were in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Animal Research Center, Yokohama City University Graduate School of Medicine.

Experimental design
Experiment I.
To examine the role of gonadal steroid hormones concerning sex difference in the stress response of ACh in the hippocampus, the two sexes were each divided into four groups. Male groups included: 1) gonadally intact male rats, 2) orchidectomized rats (group ORX), 3) orchidectomized 17β-estradiol-primed rats (group ORX+E), and 4) orchidectomized testosterone-primed rats (group ORX+T); female groups included: 1) gonadally intact diestrous female rats, 2) ovariectomized rats (group OVX), 3) ovariectomized 17β-estradiol-primed rats (group OVX+E), and 4) ovariectomized testosterone-primed rats (group OVX+T). Gonadectomy was performed 15.0 ± 1.6 d before the in vivo experiment, and a testosterone or 17β-estradiol capsule was sc implanted on the day of the gonadectomy. To make a 17β-estradiol capsule, we packed a 1:4 mixture of 17β-estradiol (Sigma, St. Louis, MO) and cholesterol crystals in a piece of SILASTIC brand tubing (15 mm length per 250 g body weight, inner diameter 2.0 mm, outer diameter 3.0 mm; Dow Corning, Midland, MI). To make a testosterone capsule, we packed testosterone crystals (Sigma) in a piece of SILASTIC brand tubing (30 mm length per 250 g body weight, inner diameter 2.0 mm, outer diameter 3.0 mm; Dow Corning). The packed capsules were soaked in saline for more than 24 h before use (35).

Four to six rats in each group were used for gonadal steroid hormone assay. The rats were deeply anesthetized with sodium pentobarbital and trunk blood was taken to determine the serum levels of 17β-estradiol and testosterone. Separated serum samples were stored at –70 C until 17β-estradiol and testosterone assay.

Experiment II.
To specify the hormone pathway maintaining the cholinergic stress response, gonadally intact male rats were divided into three groups. The three groups of rats were orally administered an aromatase inhibitor (5 mg/kg·d; Letrozole, Novartis Pharma, East Hanover, NJ), an androgen receptor antagonist (15 mg/kg·d; Flutamide, Sigma), or a vehicle (0.2 ml of 4% carboxymethylcellulose) at 0900 h daily for 7 d, and the in vivo microdialysis study was performed on the seventh day.

Four rats in each group were used for gonadal steroid hormone assay. Trunk blood were taken as described in experiment I, and serum samples were stored at –70 C until the assay.

17β-Estradiol assay
The serum 17β-estradiol concentration was measured by estradiol enzyme immunoassay (EIA) kit (Cayman Chemical Co., Ann Arbor, MI). Before the assay, 500 µl of diethyl ether were mixed with 50 µl of serum sample and the supernatant evaporated. Then EIA buffer was added to reconstitute the sample for assay. Minimum detectability of the assay was 10.8 pg/ml in experiment I and 6.1 pg/ml in experiment II. The intraassay standard variation was 3.8%, and the interassay coefficient of variation was 8.2%.

Testosterone assay
The serum testosterone concentration was measured by testosterone EIA kit (Cayman). Before the assay, 500 µl of diethyl ether were mixed with 50 µl of serum sample and the supernatant evaporated. Then EIA buffer was added to reconstitute the sample for assay. Minimum detectability of the assay was 9.1 pg/ml in experiment I and 16.4 pg/ml in experiment II. The intraassay standard variation was 4.9%, and the interassay coefficient of variation was 8.7%.

In vivo microdialysis
Under anesthesia with sodium pentobarbital (30–50 mg/kg, ip), a stainless-steel guide cannula (outer diameter 0.52 mm) was implanted stereotaxically into the right side of the dorsal hippocampus. Coordinates were 3.2–3.5 mm anterior from the ear bar, 3.0 mm lateral to the midline, and 2.1–2.2 mm below the surface of the brain according to the brain atlas of Albe-Fessard et al. (36). The coordinates were adjusted based on sex and body weight (37). After the cannula implantation, a stylet was inserted into the guide cannula until the microdialysis experiment. The animals were housed individually and allowed to recover in a cylindrical plastic cage (diameter 35 cm, height 45 cm) for 10.2 ± 1.8 d. During this period, vaginal smears were taken to confirm expression of the normal estrous cycle in female rats. Male rats were handled for a short time daily.

On the day before the microdialysis experiment, the stylet was replaced with a microdialysis probe (outer diameter 0.31 mm, AI-8–1; Eicom Co., Kyoto, Japan), and a cardiac catheter was implanted through the jugular vein under light ether anesthesia. A two-channel fluid swivel device (SSU-20; Eicom) was connected to the inlet and outlet of the probe, and an artificial cerebrospinal fluid solution (147 mM NaCl; 4 mM KCl; 1.2 mM CaCl2; 0.9 mM MgCl2) was infused through the dialysis probe with a 1.0-mm-long semipermeable membrane (molecular mass cutoff 50,000 Da) at a rate of 2.5 µl/min using a microdialysis pump (CMA/102; Carnegie Medicin, Stockholm, Sweden). We administered 7 µM eserine through the dialysis probe to enhance the dialysate ACh levels. The rats were housed individually in their home cage, and the dialysis was performed under unanesthetized, freely moving conditions (27, 38, 39). After the stabilization period (overnight), the dialysates were collected (37.5 µl) in 15-min fractions for 5 h. After the collection of the first four samples, the immobilization stress was applied from 1200 to 1300 h, when the extracellular ACh levels were relatively stable (39, 40). In intact female rats, we collected dialysates on diestrous days (i.e. diestrus 1 or diestrus 2). Details of the immobilization and behavioral responses to the stress were described previously (27). We strapped the animals’ legs onto a wooden board (19 x 30 cm) with soft cotton ties for 60 min.

At the same time, blood samples were taken from the same rats we described previously (35, 41). Blood (150 µl) was drawn twice before immobilization (at 1100 and 1200 h), twice during immobilization (at 1230 and 1300 h), and four times after release from immobilization (at 1330, 1400, 1500, and 1600 h). An equal volume of heparinized saline was replaced after each bleeding. Separated serum samples were stored at –70 C until corticosterone assay. After the sampling experiment, the animals were deeply anesthetized with sodium pentobarbital and perfused with 10% formalin solution. The location of the dialysis probe was microscopically verified in frozen sections of the brain (Fig. 1Go).


Figure 1
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  		FIG. 1. Location of the microdialysis probes within the dorsal hippocampus. Vertical lines represent the 1.0 mm length of dialysis membrane. CC, Corpus callosum; DG, dentate gyrus; LP, lateral posterior nucleus.

 
ACh assay
ACh was quantified by a combination of HPLC, enzyme reaction, and electrochemical detection (ECD-100; Eicom). Details of the ACh assay procedures were described previously (38, 39, 40). A 30-µl aliquot of dialysate was used for subsequent measurements of the ACh concentrations. Thirty microliters of ethylhomocholine solution (100 nM) were mixed with the aliquot for the internal standard solution, and the mixture was injected into the HPLC system. A solution that consisted of 0.1 mM Na2HPO4 (pH 8.5) containing 200 mg/liter sodium 1-decanesulfonate (Aldrich Chemical Co., Inc., Milwaukee, WI) and 65 mg/liter tetramethylammonium chloride (Wako Pure Chemical Industries Co., Osaka, Japan) was delivered to the HPLC as the mobile phase at a rate of 0.6 ml/min. After separation with a styrene polymer column (AC-GEL; Eicom), ACh was converted to hydrogen peroxide by a postcolumn enzyme reactor (AC-ENZYMPAK; Eicom) with immobilized acetylcholinesterase and choline oxidase. The hydrogen peroxide was detected with an electrochemical detector (ECD-100; Eicom). The least amount detected was 30–70 fmol/sample.

To calculate the recovery rate in each dialysis probe, in vitro experiments were also performed. The amount of ACh collected every 15 min was divided by the in vitro recovery rate to estimate extracellular ACh levels. The in vitro recovery rate was determined for individual probes and applied to results from individual rats (mean ± SEM; experiment I, 6.6 ± 0.3%, experiment II, 6.2 ± 0.2%).

Corticosterone assay
The serum corticosterone levels were determined by the protein-binding method (17, 27, 42, 43) in a single assay. Briefly, 1-ml ethanol was mixed with 5 µl of serum sample and the supernatant evaporated. Then 1 ml of corticosterone-binding globulin-isotope solution was added to each tube. To make the corticosterone-binding globulin-isotope solution, 250 ml of 0.2% pooled human serum solution were filtrated and mixed with 5 µCi of 1,2,6,7-3H-corticosterone (PerkinElmer Life Sciences, Inc., Boston, MA). An insoluble absorbing agent (Florisil; Wako Pure Chemical Industries) was used to separate protein-bound and unbound steroids. The minimal detectable amount of corticosterone was 2.3 µg/dl. The intraassay standard variation of the assay was 5.8%, and the intraassay coefficient of variation was 7.1%.

Data analysis
Serum 17β-estradiol and testosterone levels were analyzed by one-way ANOVA followed by post hoc analysis with the Fisher protected least significant difference (PLSD) test, in which the variable was the group. For time course analyses, extracellular ACh levels and serum corticosterone levels were analyzed by one-way ANOVA with repeated measures followed by post hoc analysis with the Fisher PLSD test, in which the variable was time.

Mean ACh levels or corticosterone levels before the stress exposure were defined as the baseline, and the stress-induced maximum levels during stress was defined as the stress response. In experiment I, to evaluate the sex difference in the baseline levels and the stress-induced levels, we performed three-way ANOVA, in which the variables were stress, sex, and hormone treatment. In experiment II, we performed two-way ANOVA, in which the variables were stress and drug treatment. The data were analyzed by post hoc ANOVAs with Fisher PLSD test to examine specific differences.

To examine the correlation between the ACh levels and serum corticosterone levels, eight points of ACh levels and serum corticosterone concentration (at 1100, 1200, 1230, 1300, 1330, 1400, 1500, and 1600 h) were evaluated by Pearson’s correlation coefficient (38, 39). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum concentration of 17β-estradiol and testosterone (experiment I)
Serum 17β-estradiol levels in experiment I are shown in Fig. 2AGo. The levels in ORX+E and OVX+E rats were equivalent to the peak levels in proestrous female rats (44, 45). In ANOVA followed by post hoc analysis, ORX+E rats showed significantly higher 17β-estradiol levels than male rats (F3,13 = 12.176, P < 0.01). In addition, OVX+E rats showed significantly higher, but OVX rats showed significantly lower, 17β-estradiol levels than diestrous female rats (F3,14 = 18.043, P < 0.01).


Figure 2
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  		FIG. 2. A, Serum 17β-estradiol concentration in intact, gonadectomized, and gonadectomized steroid-primed rats. B, Serum testosterone concentration in intact, gonadectomized, and gonadectomized steroid-primed rats. SILASTIC brand capsule containing 17β-estradiol or testosterone was sc implanted for 2 wk in gonadectomized rats. Each data point represents the mean ± SEM. The number of rats in each group is shown in parentheses. a, P < 0.05 vs. males; b, P < 0.05 vs. females.

 
Serum testosterone levels in experiment I are shown in Fig. 2BGo. The levels in ORX+T and OVX+T rats were equivalent to the levels in intact male rats. Both ORX and ORX+E rats showed significantly lower testosterone levels than intact male rats (F3,13 = 94.824, P < 0.01). Similarly, both OVX and OVX+E rats showed significantly lower testosterone levels than diestrous female rats, and OVX+T rats showed significantly higher testosterone than diestrous female rats (F3,14 = 27.434, P < 0.01).

Time-course analysis in the extracellular ACh levels (experiment I)
In male groups, extracellular ACh levels increased rapidly at the onset of immobilization stress but gradually decreased thereafter, even before the immobilization ended. The ACh levels increased rapidly again after the end of immobilization stress and then dropped gradually when the animals were returned to their home cage (Fig. 3AGo). In gonadally intact male rats, ANOVA followed by post hoc analysis showed that the ACh levels during (at 1215, 1230, and 1245 h) and after (at 1315 and 1330 h) release from immobilization stress were significantly higher than the baseline levels before immobilization stress (F16,80 = 9.953, P < 0.01). Although the response duration was short in ORX rats (F16,64 = 3.374, P < 0.01), that was successfully restored by the replacement of either sex steroid (ORX+T, F16,80 = 5.256, P < 0.01; ORX+E, F16,80 = 5.338, P < 0.01).


Figure 3
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  		FIG. 3. A, Effects of orchidectomy and gonadal steroid hormone replacement on the stress response of extracellular ACh levels in the hippocampus and serum corticosterone levels in male rats. B, Effects of ovariectomy and gonadal steroid hormone replacement on the stress response of extracellular ACh levels in the hippocampus and serum corticosterone levels in female rats. Horizontal black bars indicate time of immobilization stress, and the vertical gray lines represent the onset and end of the immobilization. SILASTIC brand capsule containing 17β-estradiol or testosterone was sc implanted for 2 wk in gonadectomized rats. The number of rats in each group is shown in parentheses. *, P < 0.05; **, P < 0.01 vs. mean levels before immobilization stress. C, Gonadectomy significantly attenuated the stress response of ACh, whereas replacement of testosterone in ORX rats and 17β-estradiol in OVX rats restored the response. D, The baseline levels and stress response of serum corticosterone in the same subjects. a, P < 0.05 vs. males; b, P < 0.05 vs. females. All data are presented as means ± SEM.

 
Female groups showed a similar change in extracellular ACh levels, although the stress response of ACh was relatively low (Fig. 3BGo). In gonadally intact female rats, the ACh levels during (at 1215, 1230, and 1245 h) and after (at 1315 h) immobilization stress were significantly higher than the baseline levels (F16,96 = 7.147, P < 0.01). Although the response duration was short in OVX rats (F16,80 = 5.602, P < 0.01), that was successfully restored by the replacement of either sex steroid (OVX+T, F16,80 = 7.290, P < 0.01; OVX+E, F16,112 = 10.773, P < 0.01).

Group comparison in the extracellular ACh levels (experiment I)
The baseline levels and the stress-induced ACh levels were analyzed using three-way ANOVA (Fig. 3CGo). Main effects of stress (F1,84 = 124.685, P < 0.01) and sex (F1,84 = 13.783, P < 0.01) were significant. Interactions in stress vs. hormone (F3,84 = 4.345, P < 0.01), stress vs. sex (F1,84 = 4.313, P < 0.01), and stress vs. sex vs. hormone (F3,84 = 3.202, P < 0.05) were also statistically significant.

Post hoc ANOVAs with Fisher PLSD test showed that orchidectomy severely attenuated the stress response of ACh, which was successfully restored by testosterone replacement. However, 17β-estradiol replacement did not restore the stress response of ACh in ORX rats. The stress response of ACh in ORX or ORX+E rats was significantly less than in intact male or ORX+T rats (P < 0.01). The baseline levels of ACh were not statistically different.

In female groups, the stress response of ACh was relatively low, and gonadally intact rats showed significant sex difference (P < 0.05). Ovariectomy severely attenuated the stress response of ACh, but 17β-estradiol replacement successfully restored the response in OVX rats. Testosterone replacement may partially restore the response in OVX rats, but the stress response of ACh in OVX+T rats was not different from that in OVX rats. The stress response of ACh in OVX rats was significantly less than in diestrous female or OVX+E rats (P < 0.05). The baseline levels of ACh were not statistically different.

Time-course analysis in the serum corticosterone levels (experiment I)
In male groups, serum corticosterone levels increased clearly upon exposure to immobilization stress (Fig. 3AGo). ANOVA followed by post hoc analysis showed that the corticosterone levels during and after release from immobilization were significantly higher than the baseline levels (males, F6,30 = 40.842, P < 0.01; ORX, F6,24 = 40.936, P < 0.01; ORX+T, F6,30 = 35.807, P < 0.01; ORX+E, F6,30 = 16.616, P < 0.01).

In all female groups, the stress response of serum corticosterone was relatively high (Fig. 3BGo). Although similar stress response of corticosterone was observed in diestrous female (F6,36 = 36.328, P < 0.01), OVX (F6,30 = 12.925, P < 0.01), and OVX+T rats (F6,30 = 14.130, P < 0.01), OVX+E rats showed longer response duration (F6,42 = 31.301, P < 0.01).

Group comparison in the serum corticosterone levels (experiment I)
The baseline levels and stress-induced corticosterone levels were analyzed using three-way ANOVA (Fig. 3DGo). Main effects of stress (F1,84 = 457.546, P < 0.01), sex (F1,84 = 43.712, P < 0.01), and hormone (F3,84 = 5.920, P < 0.01) were statistically significant. Interactions in stress vs. sex (F1,84 = 51.868, P < 0.01), sex vs. hormone (F3,84 = 5.366, P < 0.01), and stress vs. sex vs. hormone (F3,84 = 3.840, P < 0.05) were also statistically significant.

In post hoc ANOVA with Fisher PLSD test, ORX and ORX+E rats showed significantly higher baseline levels of corticosterone than male and ORX+T rats (P < 0.05). Moreover, ORX rats showed significantly higher stress response of corticosterone than male and ORX+T rats (P < 0.05).

OVX+E rats showed significantly higher baseline levels of corticosterone than diestrous female, OVX, and OVX+T rats (P < 0.01). In addition, OVX+E rats showed significantly higher stress response of corticosterone than diestrous female, OVX, and OVX+T rats (P < 0.05). Diestrous female rats showed significantly higher response than intact male rats (P < 0.01).

Correlation between the ACh levels and serum corticosterone levels (experiment I)
The extracellular ACh levels showed a weak positive correlation with the serum corticosterone levels in intact male and female rats (Table 1Go). Although gonadectomy attenuated the correlation in both sexes of rats, treatment with either 17β-estradiol or testosterone restored the weak correlation.


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  		TABLE 1. Correlation between the extracellular ACh levels and serum corticosterone levels in intact, gonadectomized, and gonadectomized steroid-primed rats

 
Serum concentration of 17β-estradiol and testosterone in letrozole- or flutamide-treated male rats (experiment II)
For experiment II, serum 17β-estradiol and testosterone levels are shown in Fig. 4Go, A and B, respectively. Daily treatment with letrozole significantly decreased 17β-estradiol levels in male rats (F2,9 = 6.247, P < 0.05). In addition, treatment with either letrozole or flutamide significantly increased testosterone levels (F2,9 = 152.295, P < 0.01). Flutamide-treated rats showed significantly higher testosterone levels than letrozole-treated rats.


Figure 4
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  		FIG. 4. Serum 17β-estradiol (A) and testosterone (B) concentration in vehicle-, letrozole-, and flutamide-treated male rats. Rats were orally administered vehicle, letrozole (5 mg/kg·d), or flutamide (15 mg/kg·d) for 1 wk. Each data point represents the mean ± SEM. The number of rats in each group is shown in parentheses. a, P < 0.05 vs. vehicle; b, P < 0.05 vs. letrozole.

 
Time-course analysis in the extracellular ACh levels in letrozole- or flutamide-treated male rats (experiment II)
Extracellular ACh levels increased rapidly with the onset of immobilization stress (Fig. 5AGo). Vehicle-treated rats showed a clear change as observed in experiment I (F16,80 = 6.604, P < 0.01). Letrozole-treated rats showed small response and short-response duration (F16,80 = 4.694, P < 0.01). Flutamide-treated rats showed small ACh response to stress (F16,96 = 7.271, P < 0.01).


Figure 5
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  		FIG. 5. A, Effects of letrozole or flutamide on the stress response of extracellular ACh levels in the hippocampus and serum corticosterone levels in gonadally intact male rats. Horizontal black bars indicate timing of immobilization stress, and the vertical gray lines represent the onset and end of the immobilization. Rats were orally administered vehicle, letrozole (5 mg/kg·d), or flutamide (15 mg/kg·d) for 1 wk. The number of rats in each group is shown in parentheses. *, P < 0.05; **, P < 0.01 vs. mean levels before immobilization stress. B, Both letrozole and flutamide treatment significantly attenuated the stress response of ACh. C, The baseline levels and stress response of serum corticosterone in the same subjects. a, P < 0.05 vs. vehicle. All data are presented as means ± SEM.

 
Group comparison in the extracellular ACh levels (experiment II)
The baseline levels and the stress-induced ACh levels were analyzed using two-way ANOVA (Fig. 5BGo). Main effects of stress (F1,32 = 71.351, P < 0.01) and treatment (F2,32 = 5.899, P < 0.01) were significant. Interaction in stress vs. treatment (F2,32 = 4.345, P < 0.01) was also statistically significant. In post hoc ANOVA with Fisher PLSD test, treatment with either letrozole or flutamide significantly attenuated the stress response of ACh (P < 0.05).

Time-course analysis in serum corticosterone levels in letrozole- or flutamide-treated male rats (experiment II)
In all groups, the serum corticosterone levels increased clearly upon exposure to immobilization stress (Fig. 5AGo). Although the stress response of corticosterone in vehicle- or flutamide-treated rats was similar to the response in intact male rats (vehicle, F6,30 = 21.676, P < 0.01; flutamide, F6,36 = 43.206, P < 0.01), the stress response was small in letrozole-treated rats (F6,30 = 23.209, P < 0.01).

Group comparison in the serum corticosterone levels (experiment II)
The baseline levels and stress-induced corticosterone levels were analyzed using two-way ANOVA (Fig. 5CGo). Main effect of stress (F1,32 = 316.359, P < 0.01) and treatment (F2,32 = 10.252, P < 0.01) was significant. Interaction in stress vs. treatment (F2,32 = 6.442, P < 0.01) was also statistically significant. In post hoc ANOVA with Fisher PLSD test, flutamide treatment significantly increased the baseline levels of corticosterone (P < 0.01). In contrast, letrozole treatment significantly attenuated the stress response of corticosterone (P < 0.01).

Correlation between the ACh levels and serum corticosterone levels (experiment II)
The extracellular ACh levels consistently showed a weak positive correlation with serum corticosterone levels in experiment II (Table 2Go). Neither letrozole nor flutamide treatment affected the weak correlation.


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  		TABLE 2. Correlation between the extracellular ACh levels and serum corticosterone levels in letrozole- or flutamide-treated male rats

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In experiment I, we found severe impairment of the stress response of ACh in the hippocampus after gonadectomy. The replacement of testosterone in ORX rats and the replacement of 17β-estradiol in OVX rats successfully restored the sex-specific ACh response and the duration of the response. The sex-specific action of gonadal hormone on the cholinergic stress response may suggest a neonatal sexual differentiation of the septohippocampal cholinergic system in rats.

Although stress-induced ACh increase has not been hitherto studied in gonadectomized steroid-primed rats, our results are consistent with previous neuroanatomical and neurochemical findings. Neuroanatomically, orchidectomy decreases the density of cholinergic fibers in the hippocampus, whereas testosterone replacement in ORX rats maintains fiber density in rats (20). Moreover, cholinergic neurons mediate the estradiol-induced increase in N-methyl-D-aspartate receptor binding in the hippocampus and CA1 spine density in female rats (46, 47). Neurochemically, 17β-estradiol increases the induction of choline acetyltransferase in the basal forebrain in OVX rats (48, 49). An in vitro study also demonstrated that treatment with 17β-estradiol increases both high-affinity choline uptake and ACh synthesis in basal forebrain neurons (50). Despite all this evidence suggesting the activational effect of sex hormones on ACh release in the hippocampus, the conclusive evidence such as the physiological release response has been lacking in behaving animals. Our results provide the first evidence that the stress response of ACh is dependent on the presence of gonadal steroid hormones in rats.

Nevertheless, 17β-estradiol replacement was unable to restore the stress response of ACh in ORX rats. Consistently, estradiol treatment failed to increase the amount of N-methyl-D-aspartate receptor binding (51) and spine density in the CA1 area in ORX rats (52). In female rats, testosterone replacement in OVX rats failed to raise the stress response of ACh up to the levels seen in ORX+T rats. Moreover, in our preliminary study, the replacement of androgen receptor agonist (5{alpha}-dihydrotestosterone) in OVX rats failed to enhance the cholinergic response (6.6 ± 0.3 pmol per 15 min, n = 7). These results, together with the present study suggest that the corresponding gonadal steroid hormone maintains the stress response of ACh and that the sex-specific action of gonadal steroid hormone is due to the neonatal sexual differentiation rather than the activational effect of gonadal steroid hormone in adult rats.

What is the physiological relevance of the stress response of ACh in the hippocampus? As mentioned in the introductory text, behavioral studies demonstrated that the extracellular ACh level in the hippocampus increases during the learning period (10, 11, 12) and is positively correlated with memory functions (53, 54). At the cellular level, both pyramidal and nonpyramidal neurons in the hippocampal CA1 area receive direct cholinergic afferents mediated by the muscarinic receptors (55, 56, 57). Moreover, the released ACh in the hippocampus not only enhances synaptic plasticity via the postsynaptic M1 muscarinic receptors (13) but is also responsible for neurogenesis in the dentate gyrus (14, 58). Considering the acute stress response of ACh, enhancement of memory consolidation might be useful to anticipate similar crises in the future. In support of this, the endogenous ACh in the hippocampus is known to play an important role in the encoding of contextual episodes in fear conditioning tests: bilateral injections of scopolamine or selective M1 receptor antagonist into the hippocampus impair the contextual freezing response in male rats (6, 59), and genetic deficiency of M1 receptor reduces the freezing response in male mice (60). Although it is unclear whether this notion is applicable to humans, many people remember where they were and what they were doing when stressful events occur (61).

Male rats show a greater stress response of ACh than female rats (17), which seems to be consistent with the greater memory retention of male rats in contextual fear conditioning tests (27, 62). Moreover, in male rats, orchidectomy impairs memory consolidation for contextual fear, whereas testosterone injection restores the freezing response (63). In female mice, orchidectomy also impairs the contextual freezing response, whereas treatment with estradiol benzoate increases the response (64). Although there is some debate about the role of estrogen during stressful times (65), septohippocampal cholinergic neurons may be responsible for the mnemonic effect of testosterone (22, 23) and 17β-estradiol (24, 25, 26).

Cytochrome P450 aromatase, which converts testosterone to 17β-estradiol, is abundantly expressed in the basal forebrain (66, 67). Because testosterone replacement successfully restored the stress response of ACh in ORX rats, we hypothesized that the androgen and/or the estrogen receptors contribute to maintain the stress response of ACh in male rats. It was suggested that subcutaneous treatment of letrozole or flutamide crosses the blood-brain barrier, changing the CA1 spine synapse density in rats (68, 69). In addition, behavioral analysis suggests that peripherally administered flutamide influences central structures through interactions with androgen receptors (70), and high radioactivity was found in the cerebrum after a single oral administration of 14C-letrozole in rats (Novartis AG, unpublished data). In experiment II, we found that letrozole attenuated the stress response of ACh, suggesting that inhibition of the 17β-estradiol production attenuated the cholinergic response in male rats. Because letrozole treatment significantly increased serum testosterone levels but decreased serum 17β-estradiol levels in male rats, activation of androgen receptor alone may not be sufficient to maintain the cholinergic response. Moreover, our preliminary results showed that the replacement of the nonaromatizable form of testosterone (5{alpha}-dihydrotestosterone) in ORX rats did not restore the cholinergic response; the stress-induced ACh increase in 5{alpha}-dihydrotestosterone-primed ORX rats was 7.6 ± 0.5 pmol (per 15 min, n = 6), which was identical with the response in ORX or ORX+E rats.

These results seem consistent with the observation that testosterone but not 5{alpha}-dihydrotestosterone improves working memory in aged male rats (71). It is also possible that elevated serum testosterone by letrozole down-regulates the expression of androgen receptor mRNA as observed in many target tissues (72). Although the effect is unknown in septohippocampal cholinergic neurons, exogenous testosterone may not down-regulate the expression in cholinergic motoneurons in male hamsters (73). In contrast, activation of estrogen receptors alone may not be sufficient to maintain the cholinergic response because flutamide treatment also attenuated the stress response of ACh. This notion is also supported by the results of ORX+E rats in experiment I. Taken together, these findings led us to the hypothesis that a combination of both androgen and estrogen receptors mediates the action of testosterone in maintaining the stress response in male rats. To prove the hypothesis, further study is necessary to examine the cholinergic stress response in ORX rats after the combined replacement of 5{alpha}-dihydrotestosterone and low dose of estrogen.

Neuroanatomical studies demonstrated that approximately 60% of septal cholinergic neurons express the estrogen receptor-{alpha} in male rats (19), whereas fewer cholinergic neurons express the androgen receptors in the septum and diagonal band of Broca (20). Therefore, it is possible that aromatized testosterone (i.e. 17β-estradiol) may directly activate the septohippocampal cholinergic neurons, whereas nonaromatized testosterone may transsynaptically activate the cholinergic neurons in male rats. Furthermore, gonadectomized rats showed small but significant stress response of ACh, which appeared to be sex hormone independent. Although the mechanism is presently unknown, significant localization of the cytochromes P45017{alpha} and P450 aromatase was demonstrated in the hippocampus and hypothalamus in adult male rats (74). Using Western immunoblot analysis, they reported that the concentration of P45017{alpha} and P450 aromatase in the hippocampus was approximately 1/100th to 1/200th the levels in the testis (P45017{alpha}) and ovary (P450 aromatase). It is possible, therefore, that the brain-derived sex hormones from endogenous cholesterol are associated with the sex hormone-independent component in gonadectomized rats.

In the serum corticosterone levels, increased stress response in ORX rats was attenuated by the testosterone replacement, which is consistent with previous reports (28, 75, 76). Furthermore, activation of androgen receptor alone by letrozole treatment attenuated the stress response of corticosterone in male rats. These results may suggest that androgen receptor contributes to the suppression of the HPA axis in male rats. In contrast, 17β-estradiol replacement in OVX rats increased the baseline and the stress response of serum corticosterone as reported previously (77, 78, 79). However, in the present study, 17β-estradiol treatment in ORX rats did not enhance the corticosterone response up to the levels seen in female rats. Moreover, testosterone treatment did not attenuate the response in OVX rats. Although the reason is presently unknown, the difference in the stressor and/or the strain of rats may be associated with the issue.

Thus, the effects of gonadectomy and the replacement of gonadal steroid hormone on corticosterone response to stress were different from those on ACh response, suggesting the mechanism of gonadal steroid hormone action on the HPA axis is different from that on the septohippocampal cholinergic neurons. Although the neural mechanism of how gonadal steroid hormone affect the HPA axis has been unclear, androgen inhibits the expression of c-fos and CRH mRNA in the paraventricular nucleus of the hypothalamus in ORX rats (80). In OVX rats, a recent report suggested that 17β-estradiol does not enhance the expression of c-fos and CRH mRNA but increases adrenal gland sensitivity to ACTH (81).

Based on pharmacological and behavioral studies, the corticosterone response to various stressors affects memory consolidation (82, 83, 84, 85). For example, systemic injections of moderate doses of corticosterone enhance memory function (86, 87, 88), whereas long-term exposure to high doses of corticosterone impairs it (89). Considering the inverted-U-shaped relationship between stress and learning (90), it is possible that moderate corticosterone response in male rats has a mnemonic effect, whereas greater corticosterone response in female rats has an amnesic effect. However, this notion may be difficult to explain the testosterone- or the 17β-estradiol-induced enhancement of memory consolidation in gonadectomized rats (22, 23, 24, 25, 26).

In the present study, ACh levels in the hippocampus and serum corticosterone levels were simultaneously monitored in the same subjects because an inhibitory role of the hippocampus on the HPA axis has been suggested in gonadally intact animals (32, 33, 34). Moreover, both ACh and corticosterone directly regulate the CA1 pyramidal neurons (30, 55), in which corticosterone affects the carbachol-evoked depolarization rather than its effect on the synaptic potentials and after hyperpolarization (91). Although we hypothesized that cholinergic activation of the hippocampus may inhibit the stress response of the HPA axis in intact rats (17), no inverse relation between the ACh levels in the hippocampus and serum corticosterone levels was observed in gonadectomized steroid-primed rats. We found only a low positive correlation between the ACh levels and corticosterone levels, depending on the presence of gonadal steroid hormone (Tables 1Go and 2Go). The present results seem to be consistent with the observation that a selective immunotoxic lesion of septohippocampal cholinergic neurons does not affect circulating corticosterone levels in male rats (92). In addition, circulating corticosterone may not affect the ACh levels because the administration of exogenous corticosterone did not change the extracellular ACh levels in the hippocampus (7). These findings, together with the present study, suggest that the activation of septohippocampal cholinergic neurons may have little effect on the corticosterone response to stress, whereas increase in serum corticosterone may not affect the cholinergic response to stress. Although the activation of adrenocorticosteroid receptors in the hippocampal pyramidal neurons (90) may participate in the negative feedback regulation of the HPA axis (32, 93), cholinergic activation of the hippocampus seems to play a different role during stress.


   Acknowledgments
 
The authors thank Kai Yamanashi for critical comments on this manuscript and Novartis Pharma AG for the generous gift of letrozole.


   Footnotes
 
This work was supported by Grant-in-Aid 18590219 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to D.M.).

Disclosure Statement: The authors have nothing to declare.

First Published Online October 25, 2007

Abbreviations: ACh, Acetylcholine; EIA, estradiol enzyme immunoassay; HPA, hypothalamic-pituitary-adrenal; PLSD, protected least significant difference.

Received June 20, 2007.

Accepted for publication October 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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