**3.3. Effects of prenatal BrdU treatment and sex on rations of DOPAC/DA, HVA/DA and 5-HT**

86 Sexual Dimorphism

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).

**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.

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 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- HT metabolism**

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 the striatum (Figure 2B).

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

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 system in the striatum.

#### **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 fertility [7].

Sexual Dimorphism in Monoamine Metabolism in BrdU-Treated Rats Showing Behavioral Dopamine Hypersensitivity: An Animal Model of Schizophrenia 89

**Table 2.** A summary of sexual dimorphic effects of prenatal BrdU on the monoamine metabolism in the

The treatment with BrdU in the mid-pregnancy induced apoptotic cell death in fetal brains in rodents without sex difference. Offspring from prenatal BrdU-treatment showed prominent hyperactivity in familiar or novel environment after maturation, which was observed both in male and females. However, sexual behavior was disrupted in male offspring when they were prenatally treated with BrdU. In this animal model, sexual dimorphism in monoamine metabolism was revealed. Most obvious differences in monoamine metabolism between males and females were found in DA contents in the adult striatum; a decrease in males and an increase in females. While DA levels seem to be reduced in the frontal cortex similarly in males and females with prenatal BrdU treatment. Another sexual dimorphic effect was changes in 5-HT in the striatum and the frontal cortex. Increases in 5-HT were also found in the striatum in both sex but the magnitude in the changes were larger in males rather than in females. Increased 5-HT levels in the frontal cortex were obvious in females treated with BrdU prenetally but not in males. Therefore, prenatal BrdU treatment affects the striatal DA and 5-HT system most seriously in males. Effects of prenatal BrdU on the frontal cortical DA were moderate and the magnitude was

Results from the study of gonadectomy during prepubertal period demonstrated that the most obvious effect this manipulation was the abolishment sexual dimorphism in the effect of prenatal BrdU treatment on striatal DA. This phenomenon suggests that female-specific hormones are necessary for the development of striatal DA function in females. Female sexual hormones seem to exert a protective effect on DA neurons. In adult rodents, less neurotoxicity of 6-OHDA on midbrain DA neurons [32] and of methamphetamine on striatal DA neurons have been reported in females compared with males [33]. Furthermore, it has been suggested that susceptibility of the striatal dopaminergic system to 6-OHDA is reduced in male rats but enhanced in female rats by gonadectomy [34]. A development study indicates that gonadal hormones in female mice during the pre-pubertal period are necessary for estrogen to exert neuroprotective effects on the nigrostriatal dopaminergic system [35]. In addition, adrenalectomy accompanied with oral corticosterone replacement reduces anxiety-like behavior in male rats but it does not have significant effects on females

similar in males and females. Cortical 5-HT was changes only in females.

striatum

**4. Discussion**

**Figure 3.** DOPAC/DA, HVA/DA and 5-HIAA/5-HT ratios in male controls (Male-Control), BrdUtreated males (Male-BrdU), female controls (Female-Control) and BrdU-treated females (Female-BrdU). *P* values in applied two-way ANOVAs as factors of SEX and BrdU are incorporated in figures when results were statistically significant.


**Table 2.** A summary of sexual dimorphic effects of prenatal BrdU on the monoamine metabolism in the striatum

#### **4. Discussion**

88 Sexual Dimorphism

**Figure 3.** DOPAC/DA, HVA/DA and 5-HIAA/5-HT ratios in male controls (Male-Control), BrdUtreated males (Male-BrdU), female controls (Female-Control) and BrdU-treated females (Female-BrdU). *P* values in applied two-way ANOVAs as factors of SEX and BrdU are incorporated in figures when

results were statistically significant.

The treatment with BrdU in the mid-pregnancy induced apoptotic cell death in fetal brains in rodents without sex difference. Offspring from prenatal BrdU-treatment showed prominent hyperactivity in familiar or novel environment after maturation, which was observed both in male and females. However, sexual behavior was disrupted in male offspring when they were prenatally treated with BrdU. In this animal model, sexual dimorphism in monoamine metabolism was revealed. Most obvious differences in monoamine metabolism between males and females were found in DA contents in the adult striatum; a decrease in males and an increase in females. While DA levels seem to be reduced in the frontal cortex similarly in males and females with prenatal BrdU treatment. Another sexual dimorphic effect was changes in 5-HT in the striatum and the frontal cortex. Increases in 5-HT were also found in the striatum in both sex but the magnitude in the changes were larger in males rather than in females. Increased 5-HT levels in the frontal cortex were obvious in females treated with BrdU prenetally but not in males. Therefore, prenatal BrdU treatment affects the striatal DA and 5-HT system most seriously in males. Effects of prenatal BrdU on the frontal cortical DA were moderate and the magnitude was similar in males and females. Cortical 5-HT was changes only in females.

Results from the study of gonadectomy during prepubertal period demonstrated that the most obvious effect this manipulation was the abolishment sexual dimorphism in the effect of prenatal BrdU treatment on striatal DA. This phenomenon suggests that female-specific hormones are necessary for the development of striatal DA function in females. Female sexual hormones seem to exert a protective effect on DA neurons. In adult rodents, less neurotoxicity of 6-OHDA on midbrain DA neurons [32] and of methamphetamine on striatal DA neurons have been reported in females compared with males [33]. Furthermore, it has been suggested that susceptibility of the striatal dopaminergic system to 6-OHDA is reduced in male rats but enhanced in female rats by gonadectomy [34]. A development study indicates that gonadal hormones in female mice during the pre-pubertal period are necessary for estrogen to exert neuroprotective effects on the nigrostriatal dopaminergic system [35]. In addition, adrenalectomy accompanied with oral corticosterone replacement reduces anxiety-like behavior in male rats but it does not have significant effects on females

[36]. A study of rhesus macaques has demonstrated that intact animals display less prepulse inhibition than animals given prepubertal castration [28]. This study also reveals that testosterone levels are correlated with tyrosine hydroxylase levels in the putamen among intact animals, suggesting the attenuation of PPI by gonadal sex hormones is mediated by dopaminergic activity in striatal regions. In addition, methamphetamine increases latent inhibition in male rats while this agent decreases this behavior in female rats, suggesting that presynaptic dopaminergic function shows a sex difference [37]. Hence, striatal DA function, especially presynaptic DA function may be different intrinsically between males and females.

Sexual Dimorphism in Monoamine Metabolism in BrdU-Treated Rats Showing Behavioral Dopamine Hypersensitivity: An Animal Model of Schizophrenia 91

presynaptic, which affects DA synthesis capacity, baseline synaptic DA levels, and DA release although a primary target of current antipsychotic drugs is blockade of DA D2/D3 receptors [45]. Higher DA concentration in the associative striatum in schizophrenia has been shown in a PET study using [11C]raclopride, and this result suggests that elevated subcortical DA function adversely affect performance of the dorsolateral prefrontal cortex in patients [46]. 18F-dopa uptake into the associative striatum is elevated in patients with prodromal symptoms of schizophrenia. This finding using PET indicates that DA overactivity in individuals with prodoromal psychotic symptoms [47]. This study also shows that striatal subdivision is negatively related to verbal fluency performance, but this is not the case for the limbic subdivision. Verbal fluency depends on prefrontal function [48]. The associative striatum regulates information flow to and from the prefrontal cortex [49, 50]. These findings provide a plausible mechanistic link between striatal dopaminergic dysfunction and prefrontal or executive dysfunction in schizophrenia. In addition, 5-HT2c receptor antagonist increased incentive motivation in an animal model of the negativesymptoms of schizophrenia that was produced by increasing striatal-specific DA D2 receptor density [51]. These data suggests a possibility that the primary focus of pathology of schizophrenia is the striatum, which includes abnormal presynatic DA function, accompanied GABAergic and 5-HT dysfunction and parallel existence of aberrance in the

Although further investigation is needed, this BrdU-animal could be a possible animal model for schizophrenia given that it includes abnormal presynaptic striatal DA function with sexual dimorphism and frontal cortical dysfunction relating to DA hypersensitivity.

*Hatano Research Institute, Food and Drug Safety Center, Toxicology Division, Kanagawa, Japan* 

[1] Morris, S. M. (1991): The genetic toxicology of 5-bromodeoxyuridine in mammalian cells. *Mutat Res*, Vol. 258, No. 2,pp.161-188, 0027-5107 (Print) 0027-5107 (Linking) [2] Kolb, B., Pederson, B., Ballermann, M., Gibb, R.& Whishaw, I. Q. (1999): Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities. *J Neurosci*, Vol. 19, No. 6,pp.2337-2346, 0270-6474 (Print) 0270-

[3] Kuwagata, M., Ogawa, T., Nagata, T.& Shioda, S. (2007): The evaluation of early embryonic neurogenesis after exposure to the genotoxic agent 5-bromo-2'-deoxyuridine in mice. *Neurotoxicology*, Vol. 28, No. 4,pp.780-789, 0161-813X (Print) 0161-813X

*Showa University School of Medicine, Department of Anatomy 1,Tokyo, Japan* 

prefrontal cortical function.

**Author details** 

Makiko Kuwagata

**5. References** 

6474 (Linking)

(Linking)

Katsumasa Muneoka

The reduced DA and DOPAC in the striatum can be interpreted as a decreased total DA contents in presynaptic DA terminals that imply decrement of the number in DA presynaptic terminals or reduced DA synthesis rates. Ineffectiveness of DA receptor antagonists on hyperlocomotion found in BrdU-rats also supports abnormality in presynaptic function rather than postsynaptic DA receptors. In rodent, DA agonists usually facilitate locomotor activity. This study indicated opposite changes in striatal DA between males and females as an effect of prenatal BrdU while hyperlocomotion was obviously detected in both sex. Hence, the hyperlocomotion may be attributed to DA abnormality in the frontal cortex because the change was same in males and females.

Schizophrenia includes multiple pathology in brain functions. Striatal dysfunction is thought to be a fundamental element in schizophrenia [13]. A study using functional magnetic resonance imaging (fMRI) in schizophrenic patients has demonstrated that increased coherent intrinsic activity in the dorsal striatum during psychosis is predictive for delusion and hallucination and increased activity during psychotic remission in the ventral striatum is predictive for blunted affect and emotional withdrawal [38]. A positron emission tomography (PET) study has indicated an increased DA D2/D3 receptor density in a restricted area in the striatum [39]. A double-blind PET study has indicated that D2 blockade in the striatum predicts antipsychotic response better than frontal, temporal, thalamic occupancy [40]. In addition, an involvement of the striatum in the cognitive impairment in schizophrenia has been proposed [41]. Furthermore, a study using recent molecular technique has shown that D2 receptor overexpression in the striatum results in a functional deficit in the GABAergic system and this result suggests that the postulated deficit in GABAergic function in schizophrenia could be secondary to alterations in the striatum DA system [42].

It is hypothesized that psychosis is viewed as a process of aberrant salience [43] and a central role of DA is to mediate the salience of environmental events an internal representations [44]. A study using resting-state functional MRI has indicated increased that coherent intrinsic activity in the dorsal striatum during psychosis is predictive for delusion and hallucination, and that increased activity during psychotic remission in the ventral striatum is predictive for blunted affect and emotional withdrawal [38]. A meta-analysis of imaging studies using PET or single-photon emission computed tomography (SPECT) has indicated that the locus of the largest dopaminergic abnormality in schizophrenia is presynaptic, which affects DA synthesis capacity, baseline synaptic DA levels, and DA release although a primary target of current antipsychotic drugs is blockade of DA D2/D3 receptors [45]. Higher DA concentration in the associative striatum in schizophrenia has been shown in a PET study using [11C]raclopride, and this result suggests that elevated subcortical DA function adversely affect performance of the dorsolateral prefrontal cortex in patients [46]. 18F-dopa uptake into the associative striatum is elevated in patients with prodromal symptoms of schizophrenia. This finding using PET indicates that DA overactivity in individuals with prodoromal psychotic symptoms [47]. This study also shows that striatal subdivision is negatively related to verbal fluency performance, but this is not the case for the limbic subdivision. Verbal fluency depends on prefrontal function [48]. The associative striatum regulates information flow to and from the prefrontal cortex [49, 50]. These findings provide a plausible mechanistic link between striatal dopaminergic dysfunction and prefrontal or executive dysfunction in schizophrenia. In addition, 5-HT2c receptor antagonist increased incentive motivation in an animal model of the negativesymptoms of schizophrenia that was produced by increasing striatal-specific DA D2 receptor density [51]. These data suggests a possibility that the primary focus of pathology of schizophrenia is the striatum, which includes abnormal presynatic DA function, accompanied GABAergic and 5-HT dysfunction and parallel existence of aberrance in the prefrontal cortical function.

Although further investigation is needed, this BrdU-animal could be a possible animal model for schizophrenia given that it includes abnormal presynaptic striatal DA function with sexual dimorphism and frontal cortical dysfunction relating to DA hypersensitivity.
