**2. Methods**

82 Sexual Dimorphism

hypersensitivity.

pathology in this disease "a prefrontal hypodopaminergia and a subcortical hyperdopaminergia" [13]. We propose that prenatal BrdU-treated rats is an animal model of schizophrenia based on findings that 1) the malformation of the cerebral cortex and 2) DA

**Figure 1.** Incidence of cell death in fetal mouse brains on the embryonic day 12 after the treatment with BrdU at the embryonic day 11 without indenfying sex. Apoptotic features were inspected under Nissl stain. Data were indicated as percents of numbers of apoptotic cells; C and B in parentheses indicates control and BrdU-treated animals. Horizontal bars indicate means. Significant increases in the numbers of apoptotic cell death were indicated as asterisks: \*, \*\*; P < 0.05, 0.01, respectively vs. controls. Results suggest that apoptotic cell death occurred similarly in fetal brains between males and females because values in % cell death in BrdU-treated brains in sample of mixed males and females scattered without clustering. FCx, the frontal cortex; CCx, the central cortex; PCx, the posterior cortex; St, the striatum; Hipp, the hippocmpus; A-Th, the anterior thalamus; P-Th, the posterior thalamus; A-Hy, the anterior hypothalamus; P-Hy, the posterior thalamus; AMY, the amygdala; MB, the mamillary body; Pretect, the pretectum; MT, the mecencephalic tegmentum; SN+VTA; The sunbtantia nigra + the ventral tegmental area; SC, the superior colliculus; IC; the inferior colliculus; Isth, the inthmus; Cereb, the cerebellum.

Sexual dimorphism in animal models of mental disorders is interested theme to be explored because sexual difference has been reported in clinical features in such disorders including schizophrenia. Emergence of abnormal symptoms in human mental illnesses has been known to be relevant to the beginning of puberty and this phenomenon appears to be influenced by gonadal hormones [14]. Gender differences in the age-of-onset and prevalence of mental disorders indicate an involvement of sex hormones in schizophrenia. Schizophrenia is more likely to emerge during adolescence or shortly afterwards and the first symptoms of schizophrenia emerge earlier in men than women [15]. This is more direct evidence for the involvement of sex steroids is the observation that adult women with schizophrenia have worse psychiatric symptoms and increased rate of relapse when estrogen levels are low in premenstrual period, postparturition or postmenopausal period [16]. Furthermore, estrogen has been shown to improve recovery from acute psychotic symptoms and to reduce both positive and negative symptoms of schizophrenia [17]. In adult males with schizophrenia testosterone levels are reduced and are inversely correlated with negative symptoms [18]. Altered expression of estrogen receptor alpha putatively rendering them sex-steroid unresponsive or insensitive has been reported [19]. Blunted sex

#### **2.1. Animals, drug treatments and gonadectomy**

Sprague-Dawley rats were purchased from Charles River Laboratories (Tsukuba, Japan). They were housed in metal cages in a room in which the temperature and relative humidity were controlled at 24 ± 1°C and 50 ± 5%, respectively. Lights were turned on from 0700 to 1900 h daily, and food and tap water were freely accessed. At 11 weeks of age, female rats were cohabited overnight with males. Females with sperm in their vaginal smears were regarded as pregnant, and were randomly assigned to the control or test groups. The day when the insemination was confirmed was designated as GD 0. BrdU (Sigma, St. Louis, MO) was suspended in 0.5% sodium carboxymethyl cellulose (CMC Na) and intraperitoneally administered to the test animals at 1300 h daily on GD 9 through 15. Females in the test group received a BrdU dose of 50 mg/kg, whereas control females subjected to the same regime received 0.5% CMC Na (5 ml/kg). The dosages were based on body weight on GD 9. The day of birth was designated postnatal day 0 (PND 0). On PND 1, each litter was reduced to eight animals; four males and four females. On PND 21, all offspring were weaned. At 10 weeks of age, one male and female animal obtained from an independent litter were sacrificed by decapitation and their brains were removed between 1600 and 1800 h. Each brain was subsequently dissected on ice. All tissues were stored at –80°C until the assay. We arranged other groups to investigate the effect of gonadectomy: the BrdU/GDX and BrdU/non-GDX

groups. Female pups obtained from BrdU-treated pregnant rats were used for this investigation. Female offspring assigned to the BrdU/GDX group were gonadectomized at 21 days of age, while animals assigned to the BrdU/non-GDX group received a sham operation.

Sexual Dimorphism in Monoamine Metabolism in BrdU-Treated Rats Showing Behavioral Dopamine Hypersensitivity: An Animal Model of Schizophrenia 85

cages of BrdU-treated animals was also elevated both in males and females but this hyperactivity was found in the dark cycle but not in the light cycle. The characteristic hyperactivity in the open-field induced by prenatal BrdU treatment was challenged to be influenced with dopaminergic, serotonergic and noradrenergic agents; methylphenidate (DA agonist), SCH23390 (DA D1 receptor antagonist), sulpiride (DA D2 receptor antagonist), NAN190 (5-HT1A receptor antagonist), ketanserin (5-HT2A receptor antagonist), paroxetine (selective serotonin reuptake inhibitor) and desipramine (noradrenaline reuptake inhibitor) [5, 6]. The data were obtained in male rats. Methylphenidate facilitated hyperlocomotion shown in BrdU-treated rats dose-dependently. The dose of methylphenidate (1 mg/kg) that did not stimulate locomotion in controls elevated locomotor activity of BrdU-treated rats [5, 8]. SCH23390 and NAN190 decreased activity in BrdU-treated rats but similar changes were also found in control rats. Effects of sulpiride and ketanserin on hyperactivity of the BrdUtreated rats were not certain. However, paroxetine and desipramine suppressed hyperlocomotion of BrdU-treated rats dose-dependently without influencing the activity of controls [5]. These results suggest that BrdU exposed to fetus induced similar behavioral phenotype in male and female animals. The BrdU-treated animals seem to show behavioral hypersensitivity to a DA agonist. Inhibition of reuptake of 5-HT or noradrenaline seems to suppress locomotor activity specifically in BrdU-treated rats rather than antagonists to DA

**Table 1.** Behavioral and pharmacological aspects of prenatally BrdU-treated rats

**3.2. Effects of prenatal BrdU treatment on DA and 5-HT and their metabolites in** 

In the frontal cortex, brain contents of 5-HT and 5-HIAA seem to elevate in BrdU-treated females but not in BrdU-treated males (Figure 2A). In the striatum, DA and DOPAC contents significantly decreased in BrdU-treated males while DA contents significantly elevated and DOPAC contents showed control levels in BrdU-treated females (Figure 2B).

or 5-HT receptors.

**male and female offsprings** 

### **2.2. Biochemical measurements**

DA, 5-HT and their major metabolites, dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA), and 5-hydroxy-3-indolacetic acid (5-HIAA) were determined by reversephase high-performance liquid chromatography with electrochemical detection (HPLC-ECD) as previously described [31]. Briefly, the brain tissues were homogenized in 0.1 M perchloric acid containing 1 mM EDTA and 2 mM Na2S2Os. Chloroform was added, and the mixture was then centrifuged at 11752 x *g* for 30 min at 4°C. The supernatant was removed and injected into the HPLC system. The HPLC system consisted of an EP-300 pump (Eicom Co., Kyoto, Japan), an ODS C18 reverse-phase column (Eicompak MA-5ODS, 4.5• x 150 mm; Eicom Co.) and an ECD-300 electrochemical detector (Eicom Co.) with a graphite working electrode maintained at +0.7 V with respect to an Ag/AgCl reference electrode. The mobile phase was 0.035 M sodium acetate–0.05 M citric acid (pH=3.9) containing 1.1 mM octanesulfonate, 8.3 mM EDTA, and 15% methanol (v/v). As indices of DA turnovers, DOPAC/DA and HVA/DA ratios were calculated. As an index of 5-HT turnover, 5-HIAA/5-HT ratio was calculated.

#### **2.3. Evaluation of sexual behavior in offsprings**

At 15 weeks of age, male animals were presented with an ovariectomized female brought into sexual receptivity by sequential treatment with estradiol benzoate and followed progesterone. Musculine sexual behavior was evaluated in the numbers of mounts and latency to the first mount. After this observation, each male was cohabited with an treated female to evaluate fertility.

### **2.4. Statistical analysis**

Data are shown as means ± S.E.M. Student's *t*-tests and one-way ANOVA were applied for comparisons between two groups and three groups, respectively. Monoamine contents were formulated into percent values of controls before analysis for differences between Male-BrdU, Female-BrdU and Female-BrdU-GDX because each group had independent controls. Post-hoc Tukey/Kramer tests was performed accompanied with one-way ANOVAs. Twoway ANOVAs were applied to analyze two independent factors (Sex and BrdU) simultaneously. *P* values less than 0.05 were considered to be statistically significant.

## **3. Results**

#### **3.1. Behavioral and pharmacological aspects of prenatally BrdU-treated rats**

Behavioral and pharmacological aspects of prenatally BrdU-treated rats are summarized in Table 1. Spontaneous locomotor activity in the open-field was elevated both in male and female BrdU-treated rats when it was measured for 3 and 60 min [5, 8]. Activity in home cages of BrdU-treated animals was also elevated both in males and females but this hyperactivity was found in the dark cycle but not in the light cycle. The characteristic hyperactivity in the open-field induced by prenatal BrdU treatment was challenged to be influenced with dopaminergic, serotonergic and noradrenergic agents; methylphenidate (DA agonist), SCH23390 (DA D1 receptor antagonist), sulpiride (DA D2 receptor antagonist), NAN190 (5-HT1A receptor antagonist), ketanserin (5-HT2A receptor antagonist), paroxetine (selective serotonin reuptake inhibitor) and desipramine (noradrenaline reuptake inhibitor) [5, 6]. The data were obtained in male rats. Methylphenidate facilitated hyperlocomotion shown in BrdU-treated rats dose-dependently. The dose of methylphenidate (1 mg/kg) that did not stimulate locomotion in controls elevated locomotor activity of BrdU-treated rats [5, 8]. SCH23390 and NAN190 decreased activity in BrdU-treated rats but similar changes were also found in control rats. Effects of sulpiride and ketanserin on hyperactivity of the BrdUtreated rats were not certain. However, paroxetine and desipramine suppressed hyperlocomotion of BrdU-treated rats dose-dependently without influencing the activity of controls [5]. These results suggest that BrdU exposed to fetus induced similar behavioral phenotype in male and female animals. The BrdU-treated animals seem to show behavioral hypersensitivity to a DA agonist. Inhibition of reuptake of 5-HT or noradrenaline seems to suppress locomotor activity specifically in BrdU-treated rats rather than antagonists to DA or 5-HT receptors.

84 Sexual Dimorphism

**2.2. Biochemical measurements** 

groups. Female pups obtained from BrdU-treated pregnant rats were used for this investigation. Female offspring assigned to the BrdU/GDX group were gonadectomized at 21 days of age, while animals assigned to the BrdU/non-GDX group received a sham operation.

DA, 5-HT and their major metabolites, dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA), and 5-hydroxy-3-indolacetic acid (5-HIAA) were determined by reversephase high-performance liquid chromatography with electrochemical detection (HPLC-ECD) as previously described [31]. Briefly, the brain tissues were homogenized in 0.1 M perchloric acid containing 1 mM EDTA and 2 mM Na2S2Os. Chloroform was added, and the mixture was then centrifuged at 11752 x *g* for 30 min at 4°C. The supernatant was removed and injected into the HPLC system. The HPLC system consisted of an EP-300 pump (Eicom Co., Kyoto, Japan), an ODS C18 reverse-phase column (Eicompak MA-5ODS, 4.5• x 150 mm; Eicom Co.) and an ECD-300 electrochemical detector (Eicom Co.) with a graphite working electrode maintained at +0.7 V with respect to an Ag/AgCl reference electrode. The mobile phase was 0.035 M sodium acetate–0.05 M citric acid (pH=3.9) containing 1.1 mM octanesulfonate, 8.3 mM EDTA, and 15% methanol (v/v). As indices of DA turnovers, DOPAC/DA and HVA/DA ratios were

At 15 weeks of age, male animals were presented with an ovariectomized female brought into sexual receptivity by sequential treatment with estradiol benzoate and followed progesterone. Musculine sexual behavior was evaluated in the numbers of mounts and latency to the first mount. After this observation, each male was cohabited with an treated

Data are shown as means ± S.E.M. Student's *t*-tests and one-way ANOVA were applied for comparisons between two groups and three groups, respectively. Monoamine contents were formulated into percent values of controls before analysis for differences between Male-BrdU, Female-BrdU and Female-BrdU-GDX because each group had independent controls. Post-hoc Tukey/Kramer tests was performed accompanied with one-way ANOVAs. Twoway ANOVAs were applied to analyze two independent factors (Sex and BrdU)

simultaneously. *P* values less than 0.05 were considered to be statistically significant.

**3.1. Behavioral and pharmacological aspects of prenatally BrdU-treated rats** 

Behavioral and pharmacological aspects of prenatally BrdU-treated rats are summarized in Table 1. Spontaneous locomotor activity in the open-field was elevated both in male and female BrdU-treated rats when it was measured for 3 and 60 min [5, 8]. Activity in home

calculated. As an index of 5-HT turnover, 5-HIAA/5-HT ratio was calculated.

**2.3. Evaluation of sexual behavior in offsprings** 

female to evaluate fertility.

**2.4. Statistical analysis** 

**3. Results**


**Table 1.** Behavioral and pharmacological aspects of prenatally BrdU-treated rats

#### **3.2. Effects of prenatal BrdU treatment on DA and 5-HT and their metabolites in male and female offsprings**

In the frontal cortex, brain contents of 5-HT and 5-HIAA seem to elevate in BrdU-treated females but not in BrdU-treated males (Figure 2A). In the striatum, DA and DOPAC contents significantly decreased in BrdU-treated males while DA contents significantly elevated and DOPAC contents showed control levels in BrdU-treated females (Figure 2B). BrdU-treated male rats shows prominent increases in striatal 5-HT and 5-HIAA while 5-HT levels were mildly elevated and 5-HIAA levels were comparable to controls' values in BrdUtreated females (Figure 2B). Significant reductions in DA and a significant reduction and a tendency to decrease in DOPAC contents were found in the hypothalamus of male and female BrdU-treated rats (Figure 2C). There were a significant reduction in HVA in females and a significant reduction in 5-HIAA in males in BrdU-treated animals in the midbrain (Figure 2D).

Sexual Dimorphism in Monoamine Metabolism in BrdU-Treated Rats Showing Behavioral Dopamine Hypersensitivity: An Animal Model of Schizophrenia 87

**3.3. Effects of prenatal BrdU treatment and sex on rations of DOPAC/DA,** 

brain regions seem to show intrinsic sexual differences in DA and 5-HT turnovers.

**3.4. Sexual dimorphism in the effects of prenatal BrdU treatment on DA and 5-**

**3.5. Effects of prepubertal gonadectomy of BrdU-treated females on striatal** 

Results were summarized in Table 2. No statistical difference in DA and DOPAC levels were found in the BrdU-treated females that gave gonadectomy prepubertally compared with the BruU-treated male and female rats without gonadectomy although marked differences in BrdU-induced changes were found in DA and DOPAC levels between male and female groups (Figure 2B). Significant differences in 5-HT and 5-HIAA levels were found in the BrdU-treated females with gonadectomy compared with the BrdU-treated males (Figure 2B), which were similar changes to the BrdU-treated females without gonadectomy. These results indicate abolishment of sexual dimorphism by prepubertal gonadectomy in effects of prenatal BrdU treatment on the DA system but not in the 5-HT

**3.6. Disruption of sexual behavior in male offsprings from BrdU-treated dams** 

Males in the BrdU group showed the significantly lowered number of mounts and aberrant latency of the first amount, which resulted in a significant decrease in the copulation and

Statistical analysis indicated significant sexual dimorphic effects of prenatal BrdU on monoamines in the frontal cortex and the striatum but not in the hypothalamus and midbrain. There were significant differences in percent changes of 5-HT and 5-HIAA compared with control levels between male and female offspring in the frontal cortex (Figure 2A). In the striatum, significant differences in percent changes of DA, DOPAC and 5-HIAA compared with control levels were detected between male and female offspring in

There was no significant effect of BrdU or Sex on DOPAC/DA, HVA/DA or 5-HT in the frontal cortex (Figure 3A). In the striatum and the midbrain, significant effects of Sex on all measured turnover ratios and a significant effect of BrdU on 5-HIAA/5-HT ratio were detected (Figure 3B and D). There were significant effects of Sex on DOPAC/DA and 5- HIAA/5-HT and a significant effect of BrdU on 5-HIAA/5-HT in the hypothalamus (Figure 3C). Significant interaction of the two independent factors Sex and BrdU was detected in any measured values. There data suggest that prenatal BrdU affects 5-HT turnovers in the striatum, hypothalamus and midbrain in male and female offsprings in same manner. These

**HVA/DA and 5-HT** 

**HT metabolism** 

the striatum (Figure 2B).

system in the striatum.

fertility [7].

**monoamines** 

**Figure 2.** Changes in tissue contents of monoamines and their metabolites in prenatally BrdU-treated rats. Data are indicated as percent values of controls. Statistical significance in difference between controls and BrdU-treated animals is indicated as symbols located at the top of columns; #, *P* < 0.05 vs. male controls; ##, *P* < 0.01 vs. male controls; §, *P* < 0.05 vs. female controls. Statistical significance in percent values between BrdU-treated males (Male-BrdU), BrdU-treated females (Female-BrdU) and BrdU-treated females that are given gonadectomy (Female-BrdU-GDX) is indicated as asterisks located between columns; \*, *P* < 0.05; \*\*, *P* < 0.01.
