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J Appl Physiol 92: 2457-2466, 2002. First published March 1, 2002; doi:10.1152/japplphysiol.01236.2001
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Vol. 92, Issue 6, 2457-2466, June 2002

Neonatal sex steroids affect ventilatory responses to aspartic acid and NMDA receptor subunit 1 in rats

Yijiang Shi and Evelyn H. Schlenker

Division of Basic Biomedical Sciences, School of Medicine, University of South Dakota, Vermillion, South Dakota 57069


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that administration of estradiol benzoate to males and testosterone propionate to female neonatal rat pups alters sex-specific ventilatory responses to aspartic acid with correspondent changes in N-methyl-D-aspartate receptor subunit 1 (NR1) expression determined by Western blot in specific brain regions. One-day-old rat pups received estradiol benzoate, testosterone propionate, or vehicle and were studied at weanling and adulthood. Different groups had distinct patterns of changes in tidal volume and frequency of breathing after aspartic acid administration. NR1 expression in hypothalamus was altered by age, sex, and treatment. Medullary and pontine NR1 expression correlated with baseline ventilation and magnitude of the ventilatory response to aspartic acid in some groups. Thus 1) tidal volume and breathing frequency patterns in response to aspartic acid are gender, age, and treatment dependent; 2) sex, age, and exogenous steroid hormones affect NR1 expression primarily in the hypothalamus; and 3) there is correlation between NR1 expression in pons and medulla with ventilatory parameters.

estradiol benzoate; testosterone propionate; hypothalamus; pons; medulla; body weight; anogenital distance; gender; N-methyl-D-aspartate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SEXUAL DIMORPHISM IN CONTROL OF BREATHING has been demonstrated by a number of studies (2, 19, 46, 56), including those using aspartic acid, an N-methyl-D-aspartate (NMDA) receptor agonist (48-50). Specifically, 580mg/kg aspartic acid depressed ventilation in male but not in female rats (48). Additionally, dextromethrophan, an NMDA receptor (NR) antagonist, depressed ventilation in female but not in male rats (47). Thus it is plausible that NRs function differently in modulating ventilation in adult female and male rats. Moreover, treatment of neonatal female rat pups with testosterone propionate (TP) resulted in an adult ventilatory response to aspartic acid similar to that of the male adult rats (49) but different from those of intact or ovariectomized female rats. Finally, perinatal estradiol benzoate (EB) treatment of male rat pups resulted in smaller, hypogonadal adult animals whose ventilatory response to hypercapnia was diminished and whose ventilatory responses to aspartic acid were female-like relative to those of control rats (50). All these studies strongly suggest interactions between sex steroid hormones, NRs, growth, and the ventilatory control that may be influenced during critical periods of development.

Native NRs are heterodimers composed of NR subunit 1 (NR1) and NR2A, B, C, and D or NR3A or B subunits (37, 40). The distribution patterns of some of these subunit types differ in male and female rat brains (21) and throughout development (1, 31, 35). Furthermore, there is evidence that sex steroid hormones influence NRs (7, 16, 25, 61, 62). For example, estrogen significantly increases mRNA levels of hypothalamic NR2B in 30-day-old but not in 15-day-old female rats (25). In the anteroventral periventricular nucleus of the hypothalamus, estrogen appears to suppress levels of NR1 mRNA (18). Moreover, systemic estradiol treatment increases the density of NRs (61), NR1 immunofluorescence (16), and postsynaptic sensitivity to NR-mediated synaptic transmission (63) in rat hippocampus.

On the basis of the effects of sex steroids on body growth and development (4, 15, 17, 20, 27), NRs (7, 16, 25, 61, 62) and gender differences in control of ventilation (2, 19, 46, 48-50, 56, 65), we generated several hypotheses. First, neonatal treatment of female rats with TP and male rats with EB will result in a "masculinization" or "feminization," respectively, of growth and development and in control of ventilation in response to aspartic acid in weanling and adult animals. Second, there is a gender, age, and treatment (TP or EB) difference in protein expression levels of NR1, with Western blotting, in the hypothalamus, pons, and medulla areas that affect growth (32, 34) and also control breathing (43, 59). Third, neonatal treatment of rats with TP and EB will result in changes in NR1 protein expression in these brain regions, which correspond to changes in ventilatory responses to aspartic acid. Finally, increased steroid hormone production during adulthood counteracts the effects of neonatal treatments.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Groups and Sex Hormone Treatments

Sprague-Dawley rat pups were bred at the Lee Medical Animal Facility. After birth, rat pups were sexed according to their anogenital distances (AGD). Neonatal rat pups were treated 1 day after birth with 0.1 ml sesame oil vehicle, 100 µg TP/6 g body wt (suspended in 0.1 ml sesame oil) in females, or 100 µg EB/6 g body wt (suspended in 0.1 ml sesame oil) in males. Animals were housed in plastic cages in a temperature-controlled room (~22°C) on a 12:12-h light-dark cycle with their dams. Food and water were available ad libitum. The University of South Dakota Animal Care and Use Committee approved all procedures.

Rats were weaned 21 days after birth, and the ventilatory response to aspartic acid was measured in half of the rats. Subsequently, rats were weighed, AGDs were measured, and rats were killed and had their brains removed. The remaining rats were raised until adulthood. Procedures described above for weanlings were repeated in the adult rats (~50 days old). There were a total of eight groups of rats with six animals in each group: weanling female TP-treated, weanling female control, weanling male EB-treated, weanling male control, adult female TP-treated, adult female control, adult male EB-treated, and adult male control.

Ventilation Measurements

Ventilation was determined in conscious rats placed in a 20 × 8 cm Plexiglas cylindrical chamber. One side of the chamber contained ports to measure 1) the flow rate of air exiting the chamber by using a Gilmont flowmeter (Barnant, IL) and 2) the chamber temperature by using a thermometer (Cole Parmer, IL). The other side of the chamber contained ports to 1) allow air to enter the chamber, 2) measure the pressure fluctuations associated with ventilation by use of a low-pressure transducer (Validyne, CA) coupled to a Bio-Pac data acquisition system, and 3) calibrate the system by using a 1-ml glass syringe. Ventilatory parameters determined were tidal volume (VT), frequency of breathing (f), and the product of the two: minute ventilation (VE).

For the experimental procedures, rats were weighed and placed into the chamber. Baseline ventilatory parameters were measured 30 min later. Then, rats were injected subcutaneously with 0.15-0.3 ml of phosphate-buffered saline (PBS). After the injection of PBS, rats were placed back into the chamber, and ventilatory parameters were evaluated 30 min later. Finally, 580 mg/kg aspartic acid was injected subcutaneously and followed by respiratory measurements 30 min later. The dose of aspartic acid was chosen from a previous dose-time response study (48). The body weight-corrected VE (BWCVE) at baseline was evaluated to determine whether ventilation was different among the various groups before any kind of treatment (saline or aspartic acid). The magnitude of response to aspartic acid relative to PBS [BWCVE (P-A)/P] was also determined.

Tissue Collection and Homogenization

Rats were killed by inhalation of CO2. To evaluate the effectiveness of the neonatal treatment, AGDs and body weights were measured. To collect the brains, an incision was made in the scalp, and the whole brain was extracted. The hypothalamus, pons, and medulla were then dissected on ice. For 0.1 g of tissue, 300 µl of 2× Laemmli's buffer [62.5 mM Tris · HCl (pH 6.8), 25% glycerol, 2% SDS, 5% beta -mercaptoethanol, and 0.01% bromophenol blue] were added, followed by sonication, using a 550 sonic dismembranator, for 30 s. Disrupted tissues were stored at -70°C.

Protein Assay and Western Blotting

Preparation of samples. Samples of hypothalamus, pons, and medulla from each animal prepared as described above were thawed at 37°C for 5 min and centrifuged at 14,000 g at room temperature for 5 min. Supernatant was removed and diluted 1:3.5 with 6/7× Laemmli buffer to make the final concentration of sample solution equal to 1× Laemmli buffer. Then the solution was mixed, boiled for 5 min, and centrifuged at 14,000 g for 5 s. Finally, the solution was sonicated for 6 s and stored at -70°C.

Normalization of protein amount. To evaluate the relative protein concentration of each sample, samples were thawed at 37°C for 5 min, centrifuged at 14,000 g for 5 min, and 10 µl supernatant was taken for loading. Samples were run at 200 V for 20 min, and gels were stained in Coomassie solution [0.0016% Coomassie brilliant blue (Bio-Rad Laboratories), 9.5% ethanol, 5% acetic acid, (66)]. Densitometry was used to determine the integrated density value (IDV) of each lane by using ChemiImager 400 software. According to their IDV values, volumes of samples were adjusted so that amount of protein from each sample was the same in each lane within a gel (14, 39). This volume for each sample was used for the following two procedures.

Western blotting. To evaluate NR1 protein levels, samples were loaded onto the 7.5% polyacrylamide gel and separated by PAGE at 200 V for 30 min. Prestained molecular weight standards (Amersham) were included in each gel to indicate the molecular weight of the protein bands. After SDS-PAGE, proteins were transferred to polyvinylidene difluoride (Immobilon-P, Millipore) membrane in an electroblotting apparatus (Bio-Rad Laboratories) for 1 h at 100 V (transfer buffer: 25 mM Tris base, 192 mM glycine, 20% methanol, 0.5% SDS).

For immunodetection, blots were blocked for 1 h at room temperature in 2.24 mM Tris base, 7.76 mM Tris · HCl, 100 mM NaCl, and 0.1% Tween 20 (TBST) containing 5% blocker (nonfat dry milk). Incubation with anti-NR1 monoclonal mouse antibody (1:1,000, PharMingen) was carried out for 1 h at 37°C in blocking buffer, followed by 40 min of washing blots with TBST. After washing, blots were incubated with horseradish peroxidase-conjugated antimouse IgG (1:1,000, PharMingen) in blocking buffer for 1 h at room temperature, followed by 40 min of washing with TBST. NR1 protein bands were visualized by using the enhanced chemiluminescence development (Pierce) of the polyvinylidene difluoride membrane followed by exposure to X-ray film. Molecular weights of the bands on the film were identified by comparison to the prestained, low-molecular-weight standards that were run in an adjacent well during electrophoresis.

Verification of relative protein amounts used for the Western blot. A separate electrophoresis followed by Coomassie staining for each set of samples that was run on the Western blot was used to further assure relatively equal amounts of protein in each lane (Fig. 1). The coefficient of variance (CV; mean divided by standard deviation × 100) for IDVs from this procedure in each gel ranged from 4 to 5%. To evaluate the amount of protein transferred to each blot, 0.2% Naphthol blue black (Sigma Chemical) in the mixture solution of 100% methanol, 100% acetic acid, and H2O (5:1:5 in volume) was used to stain the blot, followed by washing with the mixture of 100% methanol, 100% acetic acid, and H2O (1:1:8 in volume). CV for IDV values from this procedure in each blot ranged from 4 to 5%.


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  		Fig. 1.   Representative Coomassie stain. Each lane was a sample from one animal in each group. Integrated density value (IDV) for each lane was determined to make sure that equal amounts of protein were loaded from different samples. Coefficient of variance for IDVs of all lanes was 4.2%.

Data Analysis

Two- or three-way analysis of variance (ANOVA) was used to analyze the effects of age, sex, and treatment on body weight, AGD, BWCVE, and the BWCVE (P-A)/P. If ANOVA was significant (P < 0.05), post hoc unpaired t-tests with Bonferroni corrections were used to further analyze the specific effects of age, sex, and treatment. Paired Student's t-tests were used to analyze the effect of aspartic acid compared with PBS on ventilatory parameters.

NR1 protein bands were semi-quantified by using the densitometry method determined by ChemiImager 400 software to evaluate the IDVs. Because IDVs for samples on different blots may not be directly comparable due to variability associated with running and developing blots, the IDVs of weanling vehicle-treated females in each Western blot were set as 1 and the IDVs of samples from other animals were expressed as ratios relative to 1. The ratio of IDVs on the same blot can indicate the ratio of the NR1 protein levels if both bands are not saturated. A three-way ANOVA with post hoc unpaired Student's t-tests (with Bonferroni corrections) was used to analyze the effects of age, sex, and treatment on these ratios for each brain region (hypothalamus, pons, and medulla).

Finally, for each brain region, the ratios between NR1 IDVs in one pair of animals from two groups (their samples having been run in same blot) were calculated, and their ratios of BWCVE or BWCVE (P-A)/P were used to determine whether there was a correlation between the NR1 and ventilatory responses. For example, the ratio of NR1 IDVs from weanling female vehicle and weanling female treated rats (run on the same blot) was compared with their ratio of BWCVE or BWCVE (P-A)/P. Then all the IDVs from the samples of these two groups (weanling female vehicle and weanling female treated rats) for the remaining blots were evaluated relative to their BWCVE or BWCVE (P-A)/P ratios. A significant Pearson correlation indicated that there is a relationship between either control of breathing at baseline or in magnitude of the ventilatory response to aspartic acid and NR1 levels in a brain region. Significance was accepted at P < 0.05. Data are expressed as means ± SD.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Neonatal Treatment with TP or EB on Animal Growth and Development

Body weight. A two-way ANOVA indicated that, in regard to body weight, there was a significant interaction between gender and treatment in adult groups [F(1,23) = 28.26, P < 0.001]. In males, neonatal treatment of EB significantly decreased the body weight relative to vehicle-treated males, both at weanling and adulthood (Fig. 2). Neonatal treatment of females with TP significantly increased the body weight at adulthood but not at weanling. Also, adult vehicle-treated males had larger body weights than adult vehicle-treated females. No difference was found between weanling vehicle-treated females and males.


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  		Fig. 2.   Effects of age and gender on body weight. WFV, weanling female vehicle; WFT, weanling female treated; WMV, weanling male vehicle; WMT, weanling male treated; AFV, adult female vehicle; AFT, adult female treated; AMV, adult male vehicle; AMT, adult male treated. * Significant differences between treatment and vehicle groups at same age and gender (P < 0.05).

AGDs. A two-way ANOVA indicated in regard to AGD that there was significant interaction between gender and treatment in weanling groups [F(1,23) = 41.52, P < 0.001] and adult groups [F(1,23) = 17.66, P < 0.001]. In males, neonatal treatment with EB significantly decreased AGD relative to that in the vehicle-treated males, both at weanling and adulthood, suggesting a feminization effect of EB (Fig. 3). However, adult female TP-treated rats had a trend for a larger AGD compared with vehicle-treated animals (P = 0.06). Therefore, in males, neonatal treatment with 100 µg of EB showed a greater effect on body weight and AGD than neonatal treatment of females with 100 µg of TP. Vehicle-treated males had larger AGDs than vehicle-treated females, both at weanling and adulthood.


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  		Fig. 3.   Effects of age and gender on anogenital distances. * Significant differences between treatment and vehicle groups at same age and gender (P < 0.05).

Aspartic Acid-Induced Ventilation

Body weight corrected VE at baseline. A three-way ANOVA indicated that there is an interaction between age, sex, and treatment [F(1,47) = 4.596, P = 0.038]. Overall there was a very large age effect [F(1,47) = 44.524, P < 0.001]. Specifically, adult vehicle-treated females had larger BWCVE than adult vehicle-treated males and lower BWCVE in adult vehicle-treated males than adult EB-treated males (Fig. 4). There were no significant differences among weanling groups.


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  		Fig. 4.   Effects of age and gender on body weight-corrected minute ventilation (BWCVE). Weanling groups (a) differed significantly from adult groups (b and c), independent of gender and treatment (a > b > c).

Ventilation in response to PBS and aspartic acid. Comparison of the effects of PBS to aspartic acid on VT is depicted in Fig. 5A. In both adult and weanling vehicle- or EB-treated males after aspartic acid treatment, VT was not different relative to the response to PBS. By contrast, in weanling TP-treated and adult vehicle-treated females, aspartic acid induced a significant decrease in VT compared with the effect of PBS. Weanling vehicle-treated and adult TP-treated females did not exhibit a significant change of VT in response to aspartic acid.


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  		Fig. 5.   Comparison of tidal volume (A), breathing frequency (B), and minute ventilation (C) in each group after phosphate-buffered saline (PBS) or aspartic acid (ASP) administration. * Significant changes between tidal volume induced by PBS injection and that by ASP (P < 0.05).

The effect of PBS compared with aspartic acid on f is depicted in Fig. 5B. In all females, f decreased after aspartic acid administration, relative to PBS. In weanling vehicle-treated and adult EB-treated males, f decreased after aspartic acid relative to PBS. However, weanling EB-treated and adult vehicle-treated males showed no changes of f in response to aspartic acid.

Differences in VE responses to PBS and aspartic acid are depicted in Fig. 5C. All groups of animals showed a significant decrease in VE in response to aspartic acid. But the pattern of VT and f, resulting in the decrease of VE, was different among groups (Table 1).

                              
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  		Table 1.   Summary of changes of VT, f, and VE induced by acute aspartic acid treatment

Body weight corrected VE change in response to aspartic acid and PBS. A three-way ANOVA indicated that there was a significant effect of gender independent of age and treatment on this parameter [F(1,47) = 4.908, P = 0.032]. Adult vehicle-treated females had a larger BWCVE (P-A)/P than adult vehicle-treated males, but adult vehicle-treated males displayed a smaller BWCVE (P-A)/P than did adult EB-treated males (Fig. 6). Thus there appear to be gender-specific effects on the ventilation magnitude of the response to aspartic acid.


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  		Fig. 6.   BWCVE changes in response to PBS and ASP [BWCVE (P-A)/P] in each group. There is no significant difference among weanling groups. In adult groups, a > c > b, ac > b, and ac is not significantly different from either a or c.

Western Blot Analysis of NR1 Subunit in the Hypothalamus, Pons, and Medulla

Samples of hypothalamus, pons, and medulla of all the animals were evaluated for their NR1 protein levels by using Western blot analysis. One representative blot is shown in Fig. 7. Molecular mass of the NR1 protein was 116 kDa, in accordance with other documented findings.


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  		Fig. 7.   One representative Western blot from the hypothalamus of animals in each group.

NR1 protein expression in the hypothalamus. A three-way ANOVA indicated that there were significant interactions between age and gender [F(1,47) = 7.651, P = 0.009] and between age and treatment [F(1,47) = 11.508, P = 0.002]. In particular, weanling female vehicle-treated rats had significantly higher NR1 levels than weanling TP-treated females (P = 0.0031), than adult vehicle-treated female rats (P < 0.0001), and than weanling male vehicle-treated rats (P = 0.0075). (Table 2)

                              
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  		Table 2.   Summary of IDV ratios of NR1 in hypothalamus of different groups

NR1 protein expression in the pons and medulla. A three-way ANOVA indicated no significant interactions or effects of age, sex, and treatment on NR1 expression in pons and medulla. A three-way ANOVA indicated that there was a borderline significant effect of age in medulla [F(1,47) = 3.675, P = 0.062].

Correlation Between NR1 Levels and Ventilatory Parameters

In the medulla of adult vehicle-treated females and males, there was a positive correlation between ratios of BWCVE for these groups and the respective ratios of their NR1 levels (r = 0.826, P < 0.05) (Fig. 8A). By contrast, in the medulla, weanling vehicle- and TP- treated females exhibited a trend toward a negative correlation between ratios of NR1 expression and their ratios of BWCVE (r = -0.804, P = 0.054). When the ratios were plotted, the data fit a hyperbola (Fig. 8B). Further analysis showed the equation of the hyperbola: y =3.34 - 1.77x2 (adjusted R2= 0.67, P < 0.01), where y is ratio of BWCVE and x is NR1 ratio. A trend toward a negative correlation was also observed between ratios of NR1 levels in pons of weanling vehicle- and EB-treated males and their ratios of BWCVE (r = -0.923, P = 0.051).


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  		Fig. 8.   A: correlation between ratios of N-methyl-D-aspartate receptor subunit 1 (NR1) levels in the medulla of AFV and AMV and their respective ratios of BWCVE. B: correlation between ratios of NR1 levels in the medulla of WFV and WFT and their respective ratios of BWCVE. Here, the data fit into an exponential equation of y = 3.34 - 1.77x2 (adjusted R2 = 0.67, P < 0.01), where y is the ratio of BWCVE and x is the NR1 ratio. C: correlation between ratios of NR1 levels in the pons of WMV and AMV and AFV and AMV and their respective ratios of change of BWCVE (P-A)/P.

Ratios of NR1 protein expression in pons of adult vehicle-treated females and males correlated with their ratios of BWCVE (P-A)/P (r = 0.851, P < 0.05). Moreover, ratios of NR1 levels in pons of weanling and adult vehicle-treated males were correlated with ratios of BWCVE (P-A)/P (r = 0.856, P < 0.05) (Fig. 8C).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Summary of Major Findings

The major findings of this study were that 1) neonatal administration of EB in male rats caused a significant decrease of body weight and AGD both at weanling and adulthood compared with vehicle-treated males, whereas neonatal administration of TP in female rats resulted in a significant increase of body weight at adulthood. 2) There were age- and gender- specific effects on BWCVE and magnitude of the change of BWCVE in response to aspartic acid. 3) There were distinct sex-specific patterns of ventilatory responses (VT and f) to aspartic acid. 4) NR1 protein expression varied in different brain regions among the eight groups. In the hypothalamus, weanling vehicle-treated females had significantly higher NR1 levels than weanling TP-treated females, weanling vehicle-treated males, and adult vehicle-treated females. These differences were not noted in either the pons or the medulla. 5) There was a correlation between ratios of NR1 levels of animals with their ratios of BWCVE and the BWCVE (P-A)/P ratios. Ratios of the NR1 levels in medulla of adult vehicle-treated females and males were positively correlated with their ratios of BWCVE. Also, ratios of NR1 levels in pons between adult vehicle-treated females and males and between weanling and adult vehicle-treated males had a positive correlation with the BWCVE (P-A)/P ratios. The following sections will address each of these issues in detail.

Effects of Neonatal Treatment of EB in Males and TP in Females on Growth and Development

Body weight, an important physiological parameter that shows sexual dimorphism (males being heavier than females), depends on energy intake, expenditure, and storage. Regulation of these processes requires a complex network involving diverse neurochemical and neuroendocrine signals from different organs in the body and integration within several regions in the brain (32, 34). In particular, the hypothalamus plays an important integrative function in this process by acting through a variety of systems that involve an interaction between nutrients, amines (noradrenaline, dopamine, serotonin), neuropeptides (neuropeptide Y, peptide YY, galanin, opioids, growth hormone-releasing factor), and hormones (thyroid hormones, estrogen, testosterone, growth hormone) (32).

Sex steroid hormones may regulate body weight through their interactions with these hormones, neurotransmitters, and neuromodulators, affecting food intake and energy utilization. Perinatal administration of sex steroid hormones resulted in an enduring effect on body weight. For example, Bell and Zucker (3) reported that female rat pups treated with TP (1 mg) or EB (50 µg) on day 5 weighed more than oil-treated controls at adulthood. One-day-old male rats treated with EB (100 µg) weighed less and were shorter as adult rats than controls (50).

In our study, neonatal EB treatment resulted in a decreased body weight both at weanling and adulthood, which reflected the weight-limiting effect of estrogen. As for neonatal treatment of TP in females, the weight-promoting effect of testosterone was not observed until adulthood. Tarttelin et al. (55) studied the effects of different doses of TP and showed that when 90 µg was given on postnatal day 3, the female TP-treated rats began to show greater weight gain than controls from week 5 until the end of experiment (week 18). In another study (10), neonatal testosterone (100 µg) treatment of female rats led to an increase in body weight over a period from day 38 to 76 after treatment. As in the present study, animals were weaned at week 3 in both studies. Therefore, the finding that neonatal treatment of females with TP increased body weight in adulthood (~50 days of age) but not in weanling (~21 days of age) is consistent with previous reports.

Another developmental parameter, AGD, has been demonstrated to correlate with size of sexually dimorphic nucleus-preoptic area (SDN-POA) and sexual behavior. For example, administration of testosterone or estrogen during the critical period (late fetal and early neonatal life) increased the size of SDN-POA and AGD in females (17, 54). One day-old female rats with longer AGDs (>1.4 mm) had significantly larger SDN-POA volumes than 1-day-old female rats with short AGD (<= 1.4 mm) as adults (13). Finally androgenized females had smaller SDN-POA than their control females (38).

In this study, AGD was smaller in EB-treated male rats compared with controls, both at weanling and adulthood, and reflected the femininization effect of estrogen. In contrast, TP-treated females did not exhibit an AGD that was significantly different from vehicle controls at either age, but adult treated female rats showed a borderline larger AGD (P = 0.06). This lack of change in AGD could be related to the dose of TP or a small sample size. In Juarez et al.'s (23) study, pregnant rats were injected with 2 mg TP, and female offspring had larger AGDs at 10, 30, 45, 60, and 75 days of age. Therefore, in our study, TP at a dose of 100 µg appeared not to exert a masculine effect on AGD, although it did show such effect on body weight.

Effects of Neonatal Treatment on Ventilation, Including Aspartic Acid-Induced Ventilation

Differences in BWCVE among various groups may reflect effects of neonatal treatment, age, and sex on ventilation. Effects of age were apparent in both female and male rats, with weanlings exhibiting a higher BWCVE than adults. This may be related to the higher metabolism rate of weanlings compared with adults (Inamdar and Schlenker, unpublished results). Moreover, there was a sex difference in this parameter in adults but not in weanlings. Gender differences also existed in BWCVE (P-A)/P in adult vehicle-treated rats but not in weanlings. Thus puberty could play a role in modulating ventilatory responses to aspartic acid (22).

In previous studies, aspartic acid has been shown to affect ventilation differently in male and female Sprague-Dawley rats and was influenced by neonatal administration of exogenous sex steroid hormones (48). A dose of 580 mg/kg aspartic acid depressed ventilation in male rats for 45 min by decreasing VT, inspiratory flow rate, and f. In contrast, female rats exhibited a transient decrease of ventilation only at 15 min (48). Adult female rats androgenized by the administration of 1 mg TP 1 day after birth responded to aspartic acid administration with a marked depression of ventilation similar to that noted above in males (49). Moreover, aspartic acid administration depressed ventilation in intact adult males but had no effect on ventilation in adult male rats that had received 100 µg of EB 5 days after birth (50).

Subcutaneous injection of aspartate increases concentrations of aspartate within 15 min in the circumventricular organ regions of the brain, including the arcuate nucleus of the hypothalamus, organum vasculosum of the lamina terminalis, and subfornical (44, 45). In contrast, no appreciable increases were detected in other brain regions, such as ventromedial hypothalamic nuclei and medial preoptic nucleus (44, 45). Thus systemic administration of aspartic acid can act on specific areas of the hypothalamus. Moreover, systemically administered aspartic acid may affect the release of prolactin, beta -endorphins, catacholamines, somatostatin, and luteinizing and growth hormone-releasing hormones (5, 30, 57). In particular, subcutaneous administration of aspartic acid can affect ventilation by releasing endogenous opioids and somatostatin (51). Thus aspartic acid may act directly (on NRs) and indirectly (by affecting the release of neuromodulators) to affect breathing.

In the present study, all eight groups decreased VE after aspartic acid administration relative to PBS. However, each group showed a distinct pattern of VT and f response, suggesting sexual dimorphism of control of ventilation in response to aspartic acid (Table 1). In response to aspartic acid, all males showed no change in VT, whereas all females showed a significant change in f. These two ventilatory characteristics may be considered as genetically determined by sex but not affected by neonatal treatment. We call them "male-pattern" and "female-pattern," respectively. Female and male weanling vehicle-treated rats exhibited similar changes in VT, f, and VE in response to aspartic acid, suggesting that during the weanling period this pattern of response to aspartic acid may result from low levels of circulating sex steroid hormones.

In response to aspartic acid, adult vehicle-treated males showed no change in f, a pattern that was also found in weanling EB-treated males, suggesting that neonatal treatment with EB induces a response similar to that noted when male animals are mature. This may indicate that, in weanling males, treatment with EB has a "masculine" effect. In contrast, when comparing adult vehicle and adult EB-treated males, the latter showed a decrease in f, which is the female-pattern change, suggesting that, after puberty, EB treatment in males will induce a feminization effect.

When comparing weanling and adult vehicle-treated females, the latter showed a significant decrease in VT, which was also found in weanling TP-treated females, suggesting that neonatal treatment of TP in females induces a change that normally happens when animals are mature. Comparing adult vehicle and adult TP-treated females, the latter showed no change in VT in response to aspartic acid, which is the male-pattern change. This may suggest that, after puberty, TP treatment in females will induce a masculine effect.

The discrepancy in VE response to aspartic acid between this study and earlier reports (48-50) could potentially relate to several factors, such as different doses of EB or TP used in the studies, times when EB and TP were given, individual genetic variability within each group, and/or different commercial sources of Sprague-Dawley rats. In the studies by Schlenker and colleagues (48-50), rats were obtained from Sasco (Omaha, NE), whereas this study used rats obtained from Harlan (Madison, WI). Moreover, rats obtained from Hilltop (Scottdale, PA) also showed a different sex-specific ventilatory pattern of response to aspartic acid (22). In that study, female vehicle-treated rats decreased VE after aspartic acid administration both at weanling and adulthood, whereas weanling but not adult male vehicle-treated rats decreased VE after aspartic acid administration. Furthermore, female rats treated with 50 µg TP neonatally showed no change in VE after aspartic acid administration both at weanling and adulthood (22). That different rat strains demonstrate variability in sexual differentiation in behavior and brain structure (33) suggests the importance of genetic factors and sexual differentiation. Thus the ventilatory response of rats to aspartic acid appears to be modulated by genetic factors beyond sex influence.

NR1 Protein Levels in Hypothalamus, Pons, and Medulla

We examined the relative NR1 protein expression in three brain regions of each group to see whether changes in NR1 levels were associated with ventilatory responses to aspartic acid in male and female rats of two age groups who had received different neonatal treatments. The areas of hypothalamus, pons, and medulla were selected because of their important roles in the control of ventilation (43, 59) and also the sexual dimorphism noted in hypothalamic nuclei (36, 52, 58). The role of pons and medulla has been well described in pattern generation (43), and the hypothalamus can modulate ventilatory patterns (59). Moreover, hypothalamic nuclei such as the paraventricular nucleus, the arcuate nucleus, and caudal nuclei send projections to brain areas associated with ventilatory control, such as nucleus of the tractus solitarius, nucleus ambiguus, phrenic motoneurons, and parabrachial nuclei (28, 64, 67). Moreover these brain regions contain NR (12, 41).

NR subunits also exhibit temporal and developmental patterns of distribution in the central nervous system (1, 31, 35). For example, in five male rat brain regions (olfactory bulb, cortex, hippocampus, midbrain, and cerebellum), levels of NR1 protein were low at birth and increased in similar patterns of 2- to 4.5-fold from P2 to P42 (35). Moreover, relationships between NR subunits and sex steroid hormones exist (16, 25, 61, 63). In the present study, there was an effect of age on NR1 levels in hypothalamus. In particular, weanlings had higher NR1 levels within the hypothalamus than adults, dependent on sex and treatment. A similar trend was noted in the medulla.

Most studies investigating developmental effects on NR1 levels have not done so in the hypothalamus, pons, or medulla in weanlings and adults. In contrast, Ohtake et al. (42) showed that adult rats had higher NR1 expression than neonates within the nucleus of the tractus solitarius, with the situation being reversed in the hypoglossal nucleus. In the present study, we noted a decrease of NR1 expression in the hypothalamus and a similar trend in the medulla after sexual maturity.

The hypothalamus is likely a target site for sex steroid hormone effects in the central nervous system. This includes the SDN-POA, arcuate nucleus, anteroventral periventricular nucleus, and ventromedial hypothalamic nucleus (52). Estrogen receptors (8, 9, 24) and NR1 mRNA are contained in many nuclei in the hypothalamus (12). Moreover, the ventromedial nucleus of the hypothalamus shows a high percentage of colocalization of estrogen receptor alpha  and NR2D mRNA (26). Thus there could be interaction between estrogen, estrogen receptors, and NR subunits in specific hypothalamic nuclei. The present study is the first study to compare NR1 protein levels in the hypothalamus according to age, sex, and neonatal treatment. In the present study, weanling vehicle-treated females had higher NR1 levels than weanling TP-treated females. Although we did not measure estrogen receptor levels, it is probable that weanling female vehicle-treated rats had higher estrogen receptor levels than weanling female TP-treated rats because treatment of female rats with TP significantly decreases the E2-receptor content in the pituitary and hypothalamus (29, 53). Thus there could be a correlation between estrogen receptor levels and NR1 levels in the hypothalamus that are affected by neonatal perturbations.

Because we did not examine NR1 levels in specific hypothalamic nuclei, our results represent the overall protein expression of NR1 within the entire hypothalamus. If sex steroid hormones in different hypothalamic nuclei regulate NR1 levels differently, the Western blotting technique as applied in this study is not sensitive enough to investigate regional effects of sex steroid hormones. To overcome this shortcoming, immunohistochemical studies could be used to investigate NR1 expression in specific hypothalamic neurons in rats of different ages, sex, and neonatal treatments.

Correlation Between BWCVE, BWCVE (P-A)/P, and NR1 Levels in Pons and Medulla

The ratio of NR1 levels of adult vehicle-treated females to males in the pons and medulla, areas associated with control of breathing, correlated positively with BWCVE (P-A)/P and BWCVE, respectively. Moreover, there was a positive correlation between the ratio of NR1 levels in pons of weanling and adult vehicle-treated males and the ratio of their BWCVE (P-A)/P. However, ratios of NR1 levels in medulla of weanling vehicle- and TP-treated females and ratios of their BWCVE exhibited a hyperbolic relationship (y = 3.34 - 1.77x2, adjusted R2 = 0.67, P < 0.01). Thus the role of NRs in the pons and medulla on control of ventilation may be influenced by sex, treatment, and age.

In some groups, however, we found no correlation between NR1 levels and the ventilatory response to aspartic acid. This could result from several factors. First, distinct patterns of ventilatory response to aspartic acid could result from effects of sex steroids on subunit expression, channel function, such as altering phosphorylation of NR (7, 11, 18, 25, 62), rather than only a change of NR levels. For example, the NR2D gene, which contains estrogen response elements (60), could be the target of sex steroids and may contribute to the distinctive sex-specific ventilatory responses to aspartic acid. Second, subunit composition could change without any identifiable change in the total number of NRs (6), as evaluated by NR1 levels. To further explore the relationship between NRs, sex steroid hormones and their roles in control of ventilation, evaluation of other NR2 subunits and estrogen receptor-alpha and -beta protein levels in brain regions associated with control of breathing is needed.


    ACKNOWLEDGEMENTS

The authors thank Drs. T. G. Clark, K. J. Renner, and K. W. Miskimins, and many faculty and students at the University of South Dakota School of Medicine for generous help during the work.


    FOOTNOTES

This work is supported by National Center for Research Resources Grant P20 RR-15567 and a Parson's Grant.

Address for reprint requests and other correspondence: E. H. Schlenker, School of Medicine, Univ. of South Dakota, 414 E. Clark St., Vermillion, SD 57069 (E-mail: eschlenk{at}usd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published March 1, 2002;10.1152/japplphysiol.01236.2001

Received 17 December 2001; accepted in final form 27 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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