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Observation of fetal brain in a rat valproate-induced autism model: a developmental neurotoxicity study
Int. J. Devl Neuroscience 27 (2009) 399–405
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Observation of fetal brain in a rat valproate-induced autism model: a developmental neurotoxicity study Makiko Kuwagata a,b,*, Tetsuo Ogawa b,c, Seiji Shioda b, Tomoko Nagata a a
Laboratory of Pathology, Division of Toxicology, Hatano Research Institute, Food and Drug Safety Center, Kanagawa, Japan Department of Anatomy I, Showa University, School of Medicine, Tokyo, Japan c Anti-aging Medicine Funded Research Labs, Showa University, School of Medicine, Tokyo, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 November 2008 Received in revised form 16 January 2009 Accepted 22 January 2009
Prenatal exposure to chemicals is well known to induce developmental abnormalities in the central nervous system of children. Developmental neurotoxicity (DNT) tests are important to identify neurotoxic agents and prevent neurodevelopmental disorders. We have investigated DNT, focusing on the fetal brain shortly after chemical exposure. To demonstrate a usefulness of a study focusing on the fetal brain in DNT tests, we assessed the fetal brain in a rat valproate-induced autism model. Rats were treated with sodium valproate (VPA, 800 mg/kg) orally on gestational day (GD) 9 or 11 (VPA9 or VPA11), and the fetal brains were examined on GD16 using immunohistochemistry for serotonin (5-HT), tyrosine hydroxylase (TH), and TuJ1 (neuron specific class III b-tubulin). Hypoplasia of the cortical plate was induced in both VPA9 and VPA11 groups. Abnormal migration of TH-positive and 5-HT neurons, possibly due to the appearance of an abnormally running nerve tract in the pons, was observed only in the VPA11 group. In addition, when we compared the incidence of these abnormalities between pregnant rats mated in our own animal facility (in-house group), and rats purchased pregnant (supplier group), the supplier group was much more sensitive, especially to the pons abnormality. Shipping stress may affect the reproducibility of VPA-induced DNT. The present results demonstrate that examination of the GD16 fetal brain was useful for detecting and characterizing abnormal development of the brain after VPA exposure. Further discussion was made with reference to the findings in children with autism. ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Valproate Autism DNT 5-HT Tyrosine hydroxylase Prenatal exposure
1. Introduction Prenatal exposure to chemicals such as alcohol, lead, PCBs and valproate (VPA), and to radiation are well known to induce developmental abnormalities in the central nervous system (CNS) of children (Carpenter, 2001; Ingram et al., 2000; Jacobson and Jacobson, 1996; Loganovsky and Loganovskaja, 2000; Mick et al., 2002; Moore et al., 2000; Rogan et al., 1988; Williams et al., 2001). Developmental neurotoxicity (DNT) tests are important to identify neurotoxic agents, leading to the prevention of neurodevelopmental disorders. Current DNT tests focus on offspring exposed to chemicals from implantation throughout lactation (OECD, 2007; US.EPA, 1998). Much labor, time and money are consumed to assess chemicals by the current DNT test guidelines. In addition, experimental conditions of the current tests are difficult to apply appropriately, and the tests may demonstrate poor reproducibility of results, even for positive control data (Crofton et al., 2004). * Corresponding author at: Laboratory of Pathology, Division of Toxicology, Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa, 227-8523, Japan. Tel.: +81 463 82 4751; fax: +81 463 82 9627. E-mail address: [email protected] (M. Kuwagata). 0736-5748/$36.00 ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2009.01.006
Against this background, we have been trying to evaluate each step of central nervous system development, such as the proliferation of neural stem cells, migration and differentiation of progenitor cells, and synaptogenesis and formation of the neural network as an alternative approach to the DNT test. Examination of these processes can be good new endpoints to detect the direct effects of chemicals on neurogenesis, and the results will support current postnatal studies. We have reported how to handle the tiny fetal brain (histological preparation of the fetal brain), and demonstrated the heterogeneity of the fetal brain after chemical exposure in the 5-bromo-20 -deoxyuridine (BrdU)-induced locomotor hyperactivity model (Kuwagata et al., 2007; Ogawa et al., 2005). To further confirm the usefulness of assessing the fetal brain, accumulating positive data is indispensable. Epidemiological and clinical reports provide opportunities to develop animal models useful for validating DNT test methods, such as the rat valproateinduced model of autism. Anticonvulsant medication during pregnancy increased the risk of autism and sodium valproate is the drug most commonly associated with autistic disorder (Rasalam et al., 2005). Following these clinical reports, several researchers reported an animal model of autism by exposing
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pregnant rats to VPA. These researches demonstrated neuron loss in the brainstem nuclei and the cerebellum (Ingram et al., 2000; Rodier et al., 1996), and behavioral alterations (Schneider and Przewlocki, 2005; Schneider et al., 2008; Stanton et al., 2007) consistent with, or similar to, reports in autistic children. Thus, this VPA-induced model is critical for validating the usefulness of assessing fetal brains in DNT tests. Previously, we have reported examination of the fetal brain at gestational day (GD) 14 in the rat autism model induced by prenatal exposure to VPA (Ogawa et al., 2007). The size of the forebrain was diminished and the development of TuJ1-positive immature neurons in the cerebral cortex was impaired. In addition, we demonstrated differences in developmental neurotoxicity between pregnant rats mated at our own animal facility (in-house group) and rats purchased pregnant (supplier group). The supplier group was much more sensitive to VPA treatment. However, the fetal brain at GD14 was too young to reveal the specific neurotoxicity induced by VPA treatment, even though delayed development of the fetal brain was observed. In the present study, following VPA treatment, we observed a more developed GD16 fetal brain, which we have evaluated in the BrdU-induced locomotor hyperactivity model (Ogawa et al., 2005). We also demonstrated here how prenatal VPA affected the development of the cerebral cortex and pons depending upon the timing of exposure, and that the rat’s condition (in-house or supplier) affected the induction of VPA-induced DNT. Furthermore, we discuss these findings with reference to the findings in children with autism. 2. Experimental procedures 2.1. Animals and chemical exposure Male and female Sprague–Dawley (Crl:CD, SD) rats, including pregnant rats, were purchased from the Japan Charles River Laboratory (Atsugi, Japan). The rats were housed in a room at the Animal Facility of Hatano Research Institute, in which the temperature and relative humidity were controlled with a 12–12 h light–dark cycle (07:00 h lights on). The rats were allowed to eat chow and drink tap water ad libitum. Animal experiments were started after an acclimation period of at least 6 days after arrival. To obtain pregnant animals (in-house group), virgin females at the age of 10–12 weeks were placed with males (1–2 females/male) overnight. The next morning, females with sperm in their vagina and/or a vaginal plug were regarded as pregnant, and that day was designated as gestational day 0 (GD0). Pregnant rats were shipped from the breeder on GD3 or GD4 (supplier group). Sodium valproate (VPA, Sigma, St. Louis, MO) was dissolved in saline immediately prior to use. VPA (800 mg/kg) was administered orally via gavage to pregnant rats on GD9 (VPA9) or GD11 (VPA11). As a vehicle control, saline (5 mL/ kg) was administered. The dosages were based on body weight on each day of exposure. The number of dams used is presented in Table 1. These dams were sacrificed at GD16. The Institutional Animal Care and Use Committee of Hatano Research Institute approved all animal care and experimental procedures. 2.2. Immunohistochemistry of fetal brain The fetuses were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (4% PFA) at 4 8C for 2 days, and weighed after fixation. Fetuses for examination were selected randomly (four fetuses per litter were selected in principle). The number of fetuses examined is shown in Table 3. Next, the specimens were embedded in 10% gelatin and coronal or sagittal sections were cut with a vibratome at a thickness of
45 mm. Every third coronal section was collected in three bottles, and every other sagittal section in two bottles. For histopathological examination, serial sections in each group were stained with Nissl. In addition, serotonin (5-HT; polyclonal 5-HT antibody developed in goats used at 1:8000 dilution; ImmunoStar, WI), tyrosine hydroxylase (TH; polyclonal TH antibody developed in sheep used at 1:1000 dilution; Pel-Freez, NA) and/or neuronal Class III b-Tubulin (TuJ1; monoclonal TuJ1 antibody used at 1:2000 dilution; COVANCE, CA) immunoreactivities were examined. After each secondary antibody was incubated with the sections, the sections were subjected to the avidin/biotin-immunoperoxidase reaction using a Vectastain ABC kit (Vector Laboratories Inc., CA) with visualization of the antigen using diaminobenzidine as a substrate. Immunohistochemistry staining was performed in a free-floating manner. 2.3. Statistical analysis All data were analyzed by SigmaStat1 (SystatSoftware Inc., Point Richmond, CA). Statistical analysis of the fetuses was carried out using the litter as the experimental unit. To analyze the effects of VPA treatment, data were analyzed by one-way ANOVA followed by Dunnett’s test, or the Kruskal–Wallis test followed by Dunn’s test. In addition, to evaluate differences between the mating places (in-house and supplier), reproductive findings, fetal viability and body weight, and the incidence of brain abnormalities were analyzed by Student’s t test (parametric data) or Mann– Whitney rank sum test (non-parametric data). A p value of less than 0.05 was considered significant.
3. Results 3.1. Fetal development at GD16 after VPA treatment The effects of reproductive findings and fetal development at GD16 after VPA treatment on GD9 or GD11 are shown in Table 1. Live fetal viability was slightly, but not significantly, decreased in both VPA9 and VPA11 groups. Fetal body weight was significantly decreased in the VPA11 group (p < 0.05). When compared between in-house and supplier groups (Table 2), only in the supplier group after VPA treatment, fetal viability decreased accompanied with an increase in the number of dead fetuses. Significant differences were detected in the VPA11 group. However, there were no effects of mating place on fetal development in the control group. 3.2. Morphological changes in fetal brain The morphology of the rat fetal brain at GD16 is shown in Fig. 1. In this developmental brain stage, the primordial brain structure had already been constructed. At this developmental stage, neural stem cells are still dividing in the neural epithelium layer, but begin to differentiate into immature neurons and migrate to developmental fate areas. 3.2.1. VPA-induced abnormality in the cerebral cortex In the control group, Nissl staining showed where immature neurons migrated and accumulated at the cortical plate (CP) (Fig. 2A). Strong TuJ1 immunoreactivity was detected in the marginal zone and the sub-plate zone across the CP, suggesting that the dendrite had extended into these areas (Fig. 2B, arrowhead). In contrast, in the VPA treatment group, CP was not observed clearly, and hypoplasia of CP was observed in both VPA9 and VPA11 groups
Table 1 Effects of valproate sodium on fetal viability and fetal body weight at gestational day 16 after VPA treatment. Groups
Number of dams
Implantations
Number of fetuses Live
Dead
Control VPA9 VPA11
14 15 16
14.4 2.3 14.5 1.8 14.8 1.3
13.9 2.6 12.5 3.3 12.9 3.9
0.5 1.0 1.9 2.4 1.9 3.4
Values represent the mean S.D. * Significantly different from the control at p < 0.05.
Live fetal viability (%)
Fetal body weight (g)
96.3 7.8 85.9 17.2 86.7 24.1
0.44 0.04 0.40 0.05 0.38 0.04*
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Table 2 Effects of mating place on reproductive findings, fetal viability and fetal body weight at gestational day 16 after VPA treatment. Group
Number of dams
Implantations
Number of fetuses Live
Dead
Live fetal viability (%)
Fetal body weight (g)
Control In-house Supplier
8 6
14.8 2.9 14.0 1.4
14.3 3.2 13.5 1.9
0.5 0.9 0.5 1.2
96.1 7.6 96.4 8.7
0.47 0.04 0.45 0.01
VPA9 In-house Supplier
7 8
15.1 1.5 13.9 1.9
13.7 2.3 11.5 3.9
1.4 1.4 2.4 3.0
90.3 9.3 82.0 22.0
0.38 0.0 0.41 0.04
VPA11 In-house Supplier
8 8
14.9 0.8 14.8 1.7
14.0 2.6 11.9 4.9
0.9 2.1 2.9 4.2*
93.8 15.0 79.7 30.1*
0.38 0.04 0.37 0.04
Values represent the mean S.D. * Significantly different from the VPA11 of the in-house group at p < 0.05.
by Nissl staining (Fig. 2C and E). TuJ1 immunoreactivity was also not distributed clearly in the marginal zone and the sub-plate zone (Fig. 2D and F). These changes were more severe in the VPA11 group than in the VPA9 group. 3.2.2. VPA-induced abnormality in the pons In the VPA11 group, a conspicuous round structure was observed in the pons (Fig. 3I), consisting of bundles immunostained with TuJ1 (Fig. 3J, arrow), whereas the control brain did not show a round structure in this area (Fig. 3A and B). In addition, some tyrosine hydroxylase immunoreactive cells were observed in this area (Fig. 3K, arrow), and 5-HT immunoreactive cells were observed around this area (Fig. 3L), indicating that abnormally migrating catecholamine (TH immunopositive) neurons were detected and the distribution of 5-HT neurons was also affected by the existence of abnormally running nerve fibers.
On sagittal sections, at the level of the remnant of isthmic fossa, no TuJ1-positive neural fasciculi were observed running in the mid- and hindbrain areas in the control fetal brain (Fig. 4A). TH immunohistochemistry showed a clear boundary between the mid- and hindbrain with immunopositive cells only in the area of the midbrain (Fig. 4B, dotted line). In addition, diffusing 5-HT immunoreactive cells were observed (Fig. 4C). However, in the VPA11 group, the TuJ1-positive neural fasciculus appeared abruptly in the mid- and hindbrain areas (Fig. 4G, round), indicating that the neural fasciculus followed a different route and curved regionally. Also, abnormally aggregated TuJ1-immunopositive structures were observed (Fig. 4G, arrow). In addition, the boundary of the mid- and hindbrain became fuzzy with abnormal TH-immunopositive neurons migrating to the hindbrain (Fig. 4H). Distribution of 5-HT neurons in the VPA11 group was also diminished on the ventral side (Fig. 4I).
Fig. 1. Atlas of gestational day 16 rat fetal brain (coronal sections, by Nissl staining).
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Fig. 2. Cortical development at gestational day 16 after VPA treatment on gestational day 9 or gestational day 11. Fetal brain treated with saline (CONT): (A and B); VPA treatment on gestational day 9 (VPA9): (C and D); VPA treatment on gestational day 11 (VPA11): (E and F). Fetal brain with Nissl staining: (A, C, and E), and stained for TuJ1: (B, D, and F). Formation of the cortical plate, where immature neurons are accumulating, is observed very clearly in the control group (A and B). However, this structure becomes unclear following VPA treatment on gestational day 9 (C and D) or 11 (E and F), suggesting that VPA inhibited proliferation of neural stem cells and/or migration of immature neurons. The severity of the abnormal cortical plate was not different between in-house and supplier groups among each VPA-treated group, respectively. Images show representative changes in each group.
Fig. 3. Pons of the fetal rat brain (coronal section) at gestational day 16. Fetal brain exposed to saline (CONT): (A–D); VPA treatment on gestational day 9 (VPA9): (E–H); VPA treatment on gestational day 11 (VPA11): (I–L). Fetal brain for Nissl staining: (A, E, and I), stained for TuJ1: (B, F, and J), for TH: (C, G, and K), and for 5-HT: (D, H, and L). Abnormal structure (circle in I) in the middle of the pons was detected by Nissl staining on VPA11. This abnormal structure was TuJ1 positive (arrows in J), suggesting that an abnormally running nerve tract was induced by VPA treatment. Abnormally migrating TH-positive neurons were observed in the abnormally running nerve tract (arrow in K). The distribution of 5-HT neurons was also affected by the appearance of an abnormally running nerve tract (L). No remarkable changes in the pons were detected in control and VPA9 groups. The severity of the abnormal pons was not different between in-house and supplier groups among each VPA-treated group, respectively. Images show representative changes in each group.
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Fig. 4. Boundary of midbrain and hindbrain of the fetal rat brain (sagittal section) at gestational day 16, stained for TuJ1, TH and 5-HT. Fetal brain exposed to saline (CONT): (A– C); VPA treatment on gestational day 9 (VPA9): (D–F); VPA treatment on gestational day 11 (VPA11): (G–I). Fetal brain stained for TuJ1: (A, D, and (minuscule) G); TH: (B, E, and H); 5-HT: (C, F, and I). In the VPA11 group, TuJ1-positive neural fasciculus appeared at the boundary of the mid- and hindbrain (circle in G) and abnormal aggregated TuJ1immunopositive structure was observed (arrow in G), indicating that neural fasciculus followed a different route and curved regionally. Distributions of TH (H) and 5-HTimmunopositive neurons (I) were also affected by VPA11 exposure. No remarkable changes were observed in control and VPA9 groups. The severity of changes at the boundary of the mid- and hindbrain was not different between in-house and supplier groups among each VPA-treated group, respectively. Images show representative changes in each group.
In the VPA9 group, no remarkable morphological changes were observed by coronal and saggital observations.
Table 4 Effects of mating place on the incidence of brain abnormalities at gestational day 16 after VPA treatment.
3.3. Difference in incidence of abnormality between in-house and supplier groups
Groups
The incidence of abnormality is summarized in Table 3. Hypoplasia of CP was induced in both VPA9 and VPA11 groups similarly. However, the abnormal pons was induced only in the VPA11 group. These morphological changes induced by VPA treatment were not observed in all fetuses examined. When the incidence of hypoplasia of CP was compared between in-house and supplier groups (Table 4), the incidence was higher in the supplier than in the in-house group regardless of VPA treatment day (VPA9 treatment is more sensitive, p < 0.001). The incidence of dysplasia of the pons was markedly higher in the
Table 3 Incidence of brain abnormalities at gestational day 16 after VPA treatment. Groups
Control VPA9 VPA11
No. of dams
14 15 16
No. of fetuses examined
Incidence of morphological abnormalities (%)
Per litter
Total
Hypoplasia of cortical plate
Dysplasia of pons
4–5 4–5 3–4
60 64 63
0 84.2 21.7** 85.9 22.3**
0 0 60.9 45.6**
Values represent the mean S.D. ** Significantly different from the control at p < 0.01.
No. of dams
No. of fetuses examined
Incidence of morphological abnormalities (%)
Per litter
Total
Hypoplasia of cortical plate
Control In-house Supplier
7 7
4–5 4–5
30 30
VPA9 In-house Supplier
7 8
4–5 4–5
30 34
73.6 22.6 91.7 17.8a
VPA11 In-house Supplier
8 8
3–4 3–4
30 33
81.3 25.9 90.6 30.1
0 0
Dysplasia of pons
0 0
0 0
37.5 51.8 87.5 23.1b
Values represent the mean S.D. a Significantly different from the VPA9 of the in-house group at p < 0.001. b Significantly different from the VPA11 of the in-house group at p < 0.001.
supplier than in the in-house group (p < 0.001). However, the severity of these abnormalities was not different between in-house and supplier groups among each VPA-treated group, respectively. 4. Discussion In the present study, we examined the fetal brain at GD16 following VPA treatment on GD9 or GD11. We detected abnormal development of the cortical plate, the appearance of an abnormally
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running nerve tract at the pons, and abnormal migration and/or distribution of TH-positive and 5-HT neurons. These changes were dependent on the timing of VPA treatment. Hypoplasia of CP was detected after both VPA treatments on GD9 and GD11, but the degree of change following GD11 treatment was more severe than after GD9 treatment. Abnormal structures in the pons, including the disorganized neural fasciculus, and different migration patterns of 5-HT or TH immunopositive neurons, were only detected with GD11 treatment. Epidemiological studies and case reports are useful for investigating the critical window of chemically related autism. Timing of the impact, which increases the risk, can be identified by questioning mothers or deduced from accompanying somatic defects in affected children. Children with prenatal exposure to VPA exhibit dysmorphic features (e.g., neural tube defects, congenital heart disease, craniofacial abnormalities, abnormally shaped or posteriorly rotated ears, genital abnormalities, and limb defects), which are indicative of injury around the time of neural tube closure (Rasalam et al., 2005). This window corresponds to GD11 in the rat. The present fetal observation succeeded in demonstrating VPA-induced abnormality in the pons following exposure in this critical window (GD11). Further studies on the differences between VPA9 and VPA11 may suggest why autistic disorders show a spectrum of induction. In the pons, a disorganized neural fasciculus was detected only with GD11 treatment. According to assessments of coronal and sagittal serial sections in this area, the fasciculus was driven into a subtly different pathway (close to midline) and curved in the residual of the isthmic fossa focally, resulting in a disturbance of migration and/or distribution of catecholamine neurons. This abnormal running pathway may affect the function of brain nerves. A clinicopathological study (Bailey et al., 1998) also demonstrated abnormal tracts running through the pontine tegmentum. We should trace the development of this abnormal running fasciculus at a later developmental stage. In addition, different distribution patterns in 5-HT and TH immunopositive neurons were observed in the pons. In normal development, 5-HT neurons were distributed along the midline in this area (dorsal and median raphe nuclei). However, 5-HT neurons existed outside of the disorganized neural fasciculus, which existed on the midline. 5-HT has been suggested to have roles in not only neurotransmission, but also in brain development. 5-HT regulates neurogenesis, dendritic elaboration, synaptogenesis, and the organization of the cortex (Azmitia, 2001; Bennett-Clarke et al., 1994; Cases et al., 1995; Gross et al., 2002; Hendricks et al., 2003; Osterheld-Haas and Hornung, 1996; Whitaker-Azmitia, 2001; Yan et al., 1997). Even though further investigation at later stages is needed, early abnormality of the 5HT system might be followed by postnatal abnormalities, such as abnormal 5-HT concentrations, and abnormal neural network formations in autistic children. TH-immunopositive neurons were detected in the abnormally running tract and across the boundary between the mid- and hindbrain, indicating that VPA treatment on GD11 also induced abnormal migration of TH-positive neurons. Investigation of peripheral catecholamine markers such as plasma and urinary concentration in children with autism has not concluded the contribution of a catecholamine nervous system in autism (Croonenberghs et al., 2000; Makkonen et al., 2008). However, dopamine has been shown to have a role in the development of neurons in the cerebral cortex such as GABA neurons (Crandall et al., 2007; Ohtani et al., 2003). Mesocortical DA modulates frontal cortex function involved in mediating functions such as behavioral inhibition, attentional processes, and working memory (Sullivan and Brake, 2003). Therefore, this abnormal development of catecholamine neurons may lead to the abnormal behaviors reported in this rat model.
In the cortex, VPA treatments on both GD9 and GD11 affected development of the cortical plate in the cerebral cortex accompanied by obscure formation of the sub-plate, suggesting that VPA inhibited the proliferation of neural stem cells and/or migration of immature neurons in the cerebral cortex, and the extension of dendrites. In the clinicopathologic study, cortical dysgenesis was detected in post-mortem brains of autistic subjects with mental handicaps (Bailey et al., 1998). Recently, Nakasato et al. (2008) has reported that prenatal exposure to VPA-induced locomotor hyperactivity. The relationship between the dysgenesis of CP in the fetal brain and hyperactivity after birth might be revealed. We have already detected a similar fetal-brain finding in a locomotor hyperactivity model induced by prenatal treatment with BrdU (Kuwagata et al., 2006). As described in Section 3, the morphological changes did not occur in all fetuses examined in this study. The results obtained may suggest that VPA toxicity depends on the genetic background. In the field of toxicology, outbred strain rodents, such as Sprague– Dawley rats, are usually used to avoid a genetic background bias. When inbred strains are investigated, in the case of VPA-induced exencephaly, one inbred strain, C57BL/6NCr1BR, is completely insensitive to VPA while another inbred strain, SWV, is clearly sensitive, showing an incidence of 20%, suggesting dependence on a genetic background (Naruse et al., 1988). Thus, using inbred strains has the risk of completely failing to detect toxicity. On the other hand, using outbred strains with a broader genetic background definitely decreases this risk, even if the incidence of toxic effects detected is lower than in more sensitive inbred strains. It should also be noted that even in humans the incidence of VPA-induced malformations after maternal medication is 10– 30% and half of all children with VPA syndrome have some autistic features (Battino et al., 1992; Lindhout et al., 1992; Moore et al., 2000; Omtzigt et al., 1992). We have previously examined the fetal brain at GD14 and reported differences in the DNT induced by VPA between pregnant rats mated at our own animal facility (in-house group) and rats purchased pregnant (supplier group) (Ogawa et al., 2007). However, the GD14 fetal brain was too young to reveal the specific VPA-induced neurotoxicity. The effect of the mating place was observed in only the reproductive findings, but not on neural development. In the present examination of a more developed GD16 fetal brain, we detected an abnormal pons and/ or dysplasia of CP after VPA treatment. Furthermore, the incidence of these fetal brain abnormalities was increased in the supplier group. Thus, examination of the fetal brain at GD16 clearly exhibited more brain development-specific VPA-induced toxicity and the effect of mating place on VPA-induced DNT. Although many studies of DNT use pregnant rodents mated at the supplier, early pregnant stress such as shipping and environmental changes may be one of the factors affecting the reproducibility of DNT tests. From the results obtained in this study, this VPA-induced animal model can reproduce at least part of the pathological findings reported in idiopathic autism, and postnatal findings in the animal model. Examination of the GD16 fetal brain shortly after chemical exposure is useful to evaluate early neurodevelopmental processes and to support current postnatal data. Further accumulation of fetal data related to postnatal abnormalities will make it possible to extrapolate postnatal abnormalities from fetal observation in the evaluation of potential toxins. Acknowledgements We thank Mrs. Chie Sakurai for her technical support. This research was supported by a grant for the Long-range Research Initiative from the Japan Chemical Industry Association.
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Observation of fetal brain in a rat valproate-induced autism model: a developmental neurotoxicity study Makiko Kuwagata a,b,*, Tetsuo Ogawa b,c, Seiji Shioda b, Tomoko Nagata a a
Laboratory of Pathology, Division of Toxicology, Hatano Research Institute, Food and Drug Safety Center, Kanagawa, Japan Department of Anatomy I, Showa University, School of Medicine, Tokyo, Japan c Anti-aging Medicine Funded Research Labs, Showa University, School of Medicine, Tokyo, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 November 2008 Received in revised form 16 January 2009 Accepted 22 January 2009
Prenatal exposure to chemicals is well known to induce developmental abnormalities in the central nervous system of children. Developmental neurotoxicity (DNT) tests are important to identify neurotoxic agents and prevent neurodevelopmental disorders. We have investigated DNT, focusing on the fetal brain shortly after chemical exposure. To demonstrate a usefulness of a study focusing on the fetal brain in DNT tests, we assessed the fetal brain in a rat valproate-induced autism model. Rats were treated with sodium valproate (VPA, 800 mg/kg) orally on gestational day (GD) 9 or 11 (VPA9 or VPA11), and the fetal brains were examined on GD16 using immunohistochemistry for serotonin (5-HT), tyrosine hydroxylase (TH), and TuJ1 (neuron specific class III b-tubulin). Hypoplasia of the cortical plate was induced in both VPA9 and VPA11 groups. Abnormal migration of TH-positive and 5-HT neurons, possibly due to the appearance of an abnormally running nerve tract in the pons, was observed only in the VPA11 group. In addition, when we compared the incidence of these abnormalities between pregnant rats mated in our own animal facility (in-house group), and rats purchased pregnant (supplier group), the supplier group was much more sensitive, especially to the pons abnormality. Shipping stress may affect the reproducibility of VPA-induced DNT. The present results demonstrate that examination of the GD16 fetal brain was useful for detecting and characterizing abnormal development of the brain after VPA exposure. Further discussion was made with reference to the findings in children with autism. ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Valproate Autism DNT 5-HT Tyrosine hydroxylase Prenatal exposure
1. Introduction Prenatal exposure to chemicals such as alcohol, lead, PCBs and valproate (VPA), and to radiation are well known to induce developmental abnormalities in the central nervous system (CNS) of children (Carpenter, 2001; Ingram et al., 2000; Jacobson and Jacobson, 1996; Loganovsky and Loganovskaja, 2000; Mick et al., 2002; Moore et al., 2000; Rogan et al., 1988; Williams et al., 2001). Developmental neurotoxicity (DNT) tests are important to identify neurotoxic agents, leading to the prevention of neurodevelopmental disorders. Current DNT tests focus on offspring exposed to chemicals from implantation throughout lactation (OECD, 2007; US.EPA, 1998). Much labor, time and money are consumed to assess chemicals by the current DNT test guidelines. In addition, experimental conditions of the current tests are difficult to apply appropriately, and the tests may demonstrate poor reproducibility of results, even for positive control data (Crofton et al., 2004). * Corresponding author at: Laboratory of Pathology, Division of Toxicology, Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa, 227-8523, Japan. Tel.: +81 463 82 4751; fax: +81 463 82 9627. E-mail address: [email protected] (M. Kuwagata). 0736-5748/$36.00 ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2009.01.006
Against this background, we have been trying to evaluate each step of central nervous system development, such as the proliferation of neural stem cells, migration and differentiation of progenitor cells, and synaptogenesis and formation of the neural network as an alternative approach to the DNT test. Examination of these processes can be good new endpoints to detect the direct effects of chemicals on neurogenesis, and the results will support current postnatal studies. We have reported how to handle the tiny fetal brain (histological preparation of the fetal brain), and demonstrated the heterogeneity of the fetal brain after chemical exposure in the 5-bromo-20 -deoxyuridine (BrdU)-induced locomotor hyperactivity model (Kuwagata et al., 2007; Ogawa et al., 2005). To further confirm the usefulness of assessing the fetal brain, accumulating positive data is indispensable. Epidemiological and clinical reports provide opportunities to develop animal models useful for validating DNT test methods, such as the rat valproateinduced model of autism. Anticonvulsant medication during pregnancy increased the risk of autism and sodium valproate is the drug most commonly associated with autistic disorder (Rasalam et al., 2005). Following these clinical reports, several researchers reported an animal model of autism by exposing
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pregnant rats to VPA. These researches demonstrated neuron loss in the brainstem nuclei and the cerebellum (Ingram et al., 2000; Rodier et al., 1996), and behavioral alterations (Schneider and Przewlocki, 2005; Schneider et al., 2008; Stanton et al., 2007) consistent with, or similar to, reports in autistic children. Thus, this VPA-induced model is critical for validating the usefulness of assessing fetal brains in DNT tests. Previously, we have reported examination of the fetal brain at gestational day (GD) 14 in the rat autism model induced by prenatal exposure to VPA (Ogawa et al., 2007). The size of the forebrain was diminished and the development of TuJ1-positive immature neurons in the cerebral cortex was impaired. In addition, we demonstrated differences in developmental neurotoxicity between pregnant rats mated at our own animal facility (in-house group) and rats purchased pregnant (supplier group). The supplier group was much more sensitive to VPA treatment. However, the fetal brain at GD14 was too young to reveal the specific neurotoxicity induced by VPA treatment, even though delayed development of the fetal brain was observed. In the present study, following VPA treatment, we observed a more developed GD16 fetal brain, which we have evaluated in the BrdU-induced locomotor hyperactivity model (Ogawa et al., 2005). We also demonstrated here how prenatal VPA affected the development of the cerebral cortex and pons depending upon the timing of exposure, and that the rat’s condition (in-house or supplier) affected the induction of VPA-induced DNT. Furthermore, we discuss these findings with reference to the findings in children with autism. 2. Experimental procedures 2.1. Animals and chemical exposure Male and female Sprague–Dawley (Crl:CD, SD) rats, including pregnant rats, were purchased from the Japan Charles River Laboratory (Atsugi, Japan). The rats were housed in a room at the Animal Facility of Hatano Research Institute, in which the temperature and relative humidity were controlled with a 12–12 h light–dark cycle (07:00 h lights on). The rats were allowed to eat chow and drink tap water ad libitum. Animal experiments were started after an acclimation period of at least 6 days after arrival. To obtain pregnant animals (in-house group), virgin females at the age of 10–12 weeks were placed with males (1–2 females/male) overnight. The next morning, females with sperm in their vagina and/or a vaginal plug were regarded as pregnant, and that day was designated as gestational day 0 (GD0). Pregnant rats were shipped from the breeder on GD3 or GD4 (supplier group). Sodium valproate (VPA, Sigma, St. Louis, MO) was dissolved in saline immediately prior to use. VPA (800 mg/kg) was administered orally via gavage to pregnant rats on GD9 (VPA9) or GD11 (VPA11). As a vehicle control, saline (5 mL/ kg) was administered. The dosages were based on body weight on each day of exposure. The number of dams used is presented in Table 1. These dams were sacrificed at GD16. The Institutional Animal Care and Use Committee of Hatano Research Institute approved all animal care and experimental procedures. 2.2. Immunohistochemistry of fetal brain The fetuses were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (4% PFA) at 4 8C for 2 days, and weighed after fixation. Fetuses for examination were selected randomly (four fetuses per litter were selected in principle). The number of fetuses examined is shown in Table 3. Next, the specimens were embedded in 10% gelatin and coronal or sagittal sections were cut with a vibratome at a thickness of
45 mm. Every third coronal section was collected in three bottles, and every other sagittal section in two bottles. For histopathological examination, serial sections in each group were stained with Nissl. In addition, serotonin (5-HT; polyclonal 5-HT antibody developed in goats used at 1:8000 dilution; ImmunoStar, WI), tyrosine hydroxylase (TH; polyclonal TH antibody developed in sheep used at 1:1000 dilution; Pel-Freez, NA) and/or neuronal Class III b-Tubulin (TuJ1; monoclonal TuJ1 antibody used at 1:2000 dilution; COVANCE, CA) immunoreactivities were examined. After each secondary antibody was incubated with the sections, the sections were subjected to the avidin/biotin-immunoperoxidase reaction using a Vectastain ABC kit (Vector Laboratories Inc., CA) with visualization of the antigen using diaminobenzidine as a substrate. Immunohistochemistry staining was performed in a free-floating manner. 2.3. Statistical analysis All data were analyzed by SigmaStat1 (SystatSoftware Inc., Point Richmond, CA). Statistical analysis of the fetuses was carried out using the litter as the experimental unit. To analyze the effects of VPA treatment, data were analyzed by one-way ANOVA followed by Dunnett’s test, or the Kruskal–Wallis test followed by Dunn’s test. In addition, to evaluate differences between the mating places (in-house and supplier), reproductive findings, fetal viability and body weight, and the incidence of brain abnormalities were analyzed by Student’s t test (parametric data) or Mann– Whitney rank sum test (non-parametric data). A p value of less than 0.05 was considered significant.
3. Results 3.1. Fetal development at GD16 after VPA treatment The effects of reproductive findings and fetal development at GD16 after VPA treatment on GD9 or GD11 are shown in Table 1. Live fetal viability was slightly, but not significantly, decreased in both VPA9 and VPA11 groups. Fetal body weight was significantly decreased in the VPA11 group (p < 0.05). When compared between in-house and supplier groups (Table 2), only in the supplier group after VPA treatment, fetal viability decreased accompanied with an increase in the number of dead fetuses. Significant differences were detected in the VPA11 group. However, there were no effects of mating place on fetal development in the control group. 3.2. Morphological changes in fetal brain The morphology of the rat fetal brain at GD16 is shown in Fig. 1. In this developmental brain stage, the primordial brain structure had already been constructed. At this developmental stage, neural stem cells are still dividing in the neural epithelium layer, but begin to differentiate into immature neurons and migrate to developmental fate areas. 3.2.1. VPA-induced abnormality in the cerebral cortex In the control group, Nissl staining showed where immature neurons migrated and accumulated at the cortical plate (CP) (Fig. 2A). Strong TuJ1 immunoreactivity was detected in the marginal zone and the sub-plate zone across the CP, suggesting that the dendrite had extended into these areas (Fig. 2B, arrowhead). In contrast, in the VPA treatment group, CP was not observed clearly, and hypoplasia of CP was observed in both VPA9 and VPA11 groups
Table 1 Effects of valproate sodium on fetal viability and fetal body weight at gestational day 16 after VPA treatment. Groups
Number of dams
Implantations
Number of fetuses Live
Dead
Control VPA9 VPA11
14 15 16
14.4 2.3 14.5 1.8 14.8 1.3
13.9 2.6 12.5 3.3 12.9 3.9
0.5 1.0 1.9 2.4 1.9 3.4
Values represent the mean S.D. * Significantly different from the control at p < 0.05.
Live fetal viability (%)
Fetal body weight (g)
96.3 7.8 85.9 17.2 86.7 24.1
0.44 0.04 0.40 0.05 0.38 0.04*
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Table 2 Effects of mating place on reproductive findings, fetal viability and fetal body weight at gestational day 16 after VPA treatment. Group
Number of dams
Implantations
Number of fetuses Live
Dead
Live fetal viability (%)
Fetal body weight (g)
Control In-house Supplier
8 6
14.8 2.9 14.0 1.4
14.3 3.2 13.5 1.9
0.5 0.9 0.5 1.2
96.1 7.6 96.4 8.7
0.47 0.04 0.45 0.01
VPA9 In-house Supplier
7 8
15.1 1.5 13.9 1.9
13.7 2.3 11.5 3.9
1.4 1.4 2.4 3.0
90.3 9.3 82.0 22.0
0.38 0.0 0.41 0.04
VPA11 In-house Supplier
8 8
14.9 0.8 14.8 1.7
14.0 2.6 11.9 4.9
0.9 2.1 2.9 4.2*
93.8 15.0 79.7 30.1*
0.38 0.04 0.37 0.04
Values represent the mean S.D. * Significantly different from the VPA11 of the in-house group at p < 0.05.
by Nissl staining (Fig. 2C and E). TuJ1 immunoreactivity was also not distributed clearly in the marginal zone and the sub-plate zone (Fig. 2D and F). These changes were more severe in the VPA11 group than in the VPA9 group. 3.2.2. VPA-induced abnormality in the pons In the VPA11 group, a conspicuous round structure was observed in the pons (Fig. 3I), consisting of bundles immunostained with TuJ1 (Fig. 3J, arrow), whereas the control brain did not show a round structure in this area (Fig. 3A and B). In addition, some tyrosine hydroxylase immunoreactive cells were observed in this area (Fig. 3K, arrow), and 5-HT immunoreactive cells were observed around this area (Fig. 3L), indicating that abnormally migrating catecholamine (TH immunopositive) neurons were detected and the distribution of 5-HT neurons was also affected by the existence of abnormally running nerve fibers.
On sagittal sections, at the level of the remnant of isthmic fossa, no TuJ1-positive neural fasciculi were observed running in the mid- and hindbrain areas in the control fetal brain (Fig. 4A). TH immunohistochemistry showed a clear boundary between the mid- and hindbrain with immunopositive cells only in the area of the midbrain (Fig. 4B, dotted line). In addition, diffusing 5-HT immunoreactive cells were observed (Fig. 4C). However, in the VPA11 group, the TuJ1-positive neural fasciculus appeared abruptly in the mid- and hindbrain areas (Fig. 4G, round), indicating that the neural fasciculus followed a different route and curved regionally. Also, abnormally aggregated TuJ1-immunopositive structures were observed (Fig. 4G, arrow). In addition, the boundary of the mid- and hindbrain became fuzzy with abnormal TH-immunopositive neurons migrating to the hindbrain (Fig. 4H). Distribution of 5-HT neurons in the VPA11 group was also diminished on the ventral side (Fig. 4I).
Fig. 1. Atlas of gestational day 16 rat fetal brain (coronal sections, by Nissl staining).
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Fig. 2. Cortical development at gestational day 16 after VPA treatment on gestational day 9 or gestational day 11. Fetal brain treated with saline (CONT): (A and B); VPA treatment on gestational day 9 (VPA9): (C and D); VPA treatment on gestational day 11 (VPA11): (E and F). Fetal brain with Nissl staining: (A, C, and E), and stained for TuJ1: (B, D, and F). Formation of the cortical plate, where immature neurons are accumulating, is observed very clearly in the control group (A and B). However, this structure becomes unclear following VPA treatment on gestational day 9 (C and D) or 11 (E and F), suggesting that VPA inhibited proliferation of neural stem cells and/or migration of immature neurons. The severity of the abnormal cortical plate was not different between in-house and supplier groups among each VPA-treated group, respectively. Images show representative changes in each group.
Fig. 3. Pons of the fetal rat brain (coronal section) at gestational day 16. Fetal brain exposed to saline (CONT): (A–D); VPA treatment on gestational day 9 (VPA9): (E–H); VPA treatment on gestational day 11 (VPA11): (I–L). Fetal brain for Nissl staining: (A, E, and I), stained for TuJ1: (B, F, and J), for TH: (C, G, and K), and for 5-HT: (D, H, and L). Abnormal structure (circle in I) in the middle of the pons was detected by Nissl staining on VPA11. This abnormal structure was TuJ1 positive (arrows in J), suggesting that an abnormally running nerve tract was induced by VPA treatment. Abnormally migrating TH-positive neurons were observed in the abnormally running nerve tract (arrow in K). The distribution of 5-HT neurons was also affected by the appearance of an abnormally running nerve tract (L). No remarkable changes in the pons were detected in control and VPA9 groups. The severity of the abnormal pons was not different between in-house and supplier groups among each VPA-treated group, respectively. Images show representative changes in each group.
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Fig. 4. Boundary of midbrain and hindbrain of the fetal rat brain (sagittal section) at gestational day 16, stained for TuJ1, TH and 5-HT. Fetal brain exposed to saline (CONT): (A– C); VPA treatment on gestational day 9 (VPA9): (D–F); VPA treatment on gestational day 11 (VPA11): (G–I). Fetal brain stained for TuJ1: (A, D, and (minuscule) G); TH: (B, E, and H); 5-HT: (C, F, and I). In the VPA11 group, TuJ1-positive neural fasciculus appeared at the boundary of the mid- and hindbrain (circle in G) and abnormal aggregated TuJ1immunopositive structure was observed (arrow in G), indicating that neural fasciculus followed a different route and curved regionally. Distributions of TH (H) and 5-HTimmunopositive neurons (I) were also affected by VPA11 exposure. No remarkable changes were observed in control and VPA9 groups. The severity of changes at the boundary of the mid- and hindbrain was not different between in-house and supplier groups among each VPA-treated group, respectively. Images show representative changes in each group.
In the VPA9 group, no remarkable morphological changes were observed by coronal and saggital observations.
Table 4 Effects of mating place on the incidence of brain abnormalities at gestational day 16 after VPA treatment.
3.3. Difference in incidence of abnormality between in-house and supplier groups
Groups
The incidence of abnormality is summarized in Table 3. Hypoplasia of CP was induced in both VPA9 and VPA11 groups similarly. However, the abnormal pons was induced only in the VPA11 group. These morphological changes induced by VPA treatment were not observed in all fetuses examined. When the incidence of hypoplasia of CP was compared between in-house and supplier groups (Table 4), the incidence was higher in the supplier than in the in-house group regardless of VPA treatment day (VPA9 treatment is more sensitive, p < 0.001). The incidence of dysplasia of the pons was markedly higher in the
Table 3 Incidence of brain abnormalities at gestational day 16 after VPA treatment. Groups
Control VPA9 VPA11
No. of dams
14 15 16
No. of fetuses examined
Incidence of morphological abnormalities (%)
Per litter
Total
Hypoplasia of cortical plate
Dysplasia of pons
4–5 4–5 3–4
60 64 63
0 84.2 21.7** 85.9 22.3**
0 0 60.9 45.6**
Values represent the mean S.D. ** Significantly different from the control at p < 0.01.
No. of dams
No. of fetuses examined
Incidence of morphological abnormalities (%)
Per litter
Total
Hypoplasia of cortical plate
Control In-house Supplier
7 7
4–5 4–5
30 30
VPA9 In-house Supplier
7 8
4–5 4–5
30 34
73.6 22.6 91.7 17.8a
VPA11 In-house Supplier
8 8
3–4 3–4
30 33
81.3 25.9 90.6 30.1
0 0
Dysplasia of pons
0 0
0 0
37.5 51.8 87.5 23.1b
Values represent the mean S.D. a Significantly different from the VPA9 of the in-house group at p < 0.001. b Significantly different from the VPA11 of the in-house group at p < 0.001.
supplier than in the in-house group (p < 0.001). However, the severity of these abnormalities was not different between in-house and supplier groups among each VPA-treated group, respectively. 4. Discussion In the present study, we examined the fetal brain at GD16 following VPA treatment on GD9 or GD11. We detected abnormal development of the cortical plate, the appearance of an abnormally
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running nerve tract at the pons, and abnormal migration and/or distribution of TH-positive and 5-HT neurons. These changes were dependent on the timing of VPA treatment. Hypoplasia of CP was detected after both VPA treatments on GD9 and GD11, but the degree of change following GD11 treatment was more severe than after GD9 treatment. Abnormal structures in the pons, including the disorganized neural fasciculus, and different migration patterns of 5-HT or TH immunopositive neurons, were only detected with GD11 treatment. Epidemiological studies and case reports are useful for investigating the critical window of chemically related autism. Timing of the impact, which increases the risk, can be identified by questioning mothers or deduced from accompanying somatic defects in affected children. Children with prenatal exposure to VPA exhibit dysmorphic features (e.g., neural tube defects, congenital heart disease, craniofacial abnormalities, abnormally shaped or posteriorly rotated ears, genital abnormalities, and limb defects), which are indicative of injury around the time of neural tube closure (Rasalam et al., 2005). This window corresponds to GD11 in the rat. The present fetal observation succeeded in demonstrating VPA-induced abnormality in the pons following exposure in this critical window (GD11). Further studies on the differences between VPA9 and VPA11 may suggest why autistic disorders show a spectrum of induction. In the pons, a disorganized neural fasciculus was detected only with GD11 treatment. According to assessments of coronal and sagittal serial sections in this area, the fasciculus was driven into a subtly different pathway (close to midline) and curved in the residual of the isthmic fossa focally, resulting in a disturbance of migration and/or distribution of catecholamine neurons. This abnormal running pathway may affect the function of brain nerves. A clinicopathological study (Bailey et al., 1998) also demonstrated abnormal tracts running through the pontine tegmentum. We should trace the development of this abnormal running fasciculus at a later developmental stage. In addition, different distribution patterns in 5-HT and TH immunopositive neurons were observed in the pons. In normal development, 5-HT neurons were distributed along the midline in this area (dorsal and median raphe nuclei). However, 5-HT neurons existed outside of the disorganized neural fasciculus, which existed on the midline. 5-HT has been suggested to have roles in not only neurotransmission, but also in brain development. 5-HT regulates neurogenesis, dendritic elaboration, synaptogenesis, and the organization of the cortex (Azmitia, 2001; Bennett-Clarke et al., 1994; Cases et al., 1995; Gross et al., 2002; Hendricks et al., 2003; Osterheld-Haas and Hornung, 1996; Whitaker-Azmitia, 2001; Yan et al., 1997). Even though further investigation at later stages is needed, early abnormality of the 5HT system might be followed by postnatal abnormalities, such as abnormal 5-HT concentrations, and abnormal neural network formations in autistic children. TH-immunopositive neurons were detected in the abnormally running tract and across the boundary between the mid- and hindbrain, indicating that VPA treatment on GD11 also induced abnormal migration of TH-positive neurons. Investigation of peripheral catecholamine markers such as plasma and urinary concentration in children with autism has not concluded the contribution of a catecholamine nervous system in autism (Croonenberghs et al., 2000; Makkonen et al., 2008). However, dopamine has been shown to have a role in the development of neurons in the cerebral cortex such as GABA neurons (Crandall et al., 2007; Ohtani et al., 2003). Mesocortical DA modulates frontal cortex function involved in mediating functions such as behavioral inhibition, attentional processes, and working memory (Sullivan and Brake, 2003). Therefore, this abnormal development of catecholamine neurons may lead to the abnormal behaviors reported in this rat model.
In the cortex, VPA treatments on both GD9 and GD11 affected development of the cortical plate in the cerebral cortex accompanied by obscure formation of the sub-plate, suggesting that VPA inhibited the proliferation of neural stem cells and/or migration of immature neurons in the cerebral cortex, and the extension of dendrites. In the clinicopathologic study, cortical dysgenesis was detected in post-mortem brains of autistic subjects with mental handicaps (Bailey et al., 1998). Recently, Nakasato et al. (2008) has reported that prenatal exposure to VPA-induced locomotor hyperactivity. The relationship between the dysgenesis of CP in the fetal brain and hyperactivity after birth might be revealed. We have already detected a similar fetal-brain finding in a locomotor hyperactivity model induced by prenatal treatment with BrdU (Kuwagata et al., 2006). As described in Section 3, the morphological changes did not occur in all fetuses examined in this study. The results obtained may suggest that VPA toxicity depends on the genetic background. In the field of toxicology, outbred strain rodents, such as Sprague– Dawley rats, are usually used to avoid a genetic background bias. When inbred strains are investigated, in the case of VPA-induced exencephaly, one inbred strain, C57BL/6NCr1BR, is completely insensitive to VPA while another inbred strain, SWV, is clearly sensitive, showing an incidence of 20%, suggesting dependence on a genetic background (Naruse et al., 1988). Thus, using inbred strains has the risk of completely failing to detect toxicity. On the other hand, using outbred strains with a broader genetic background definitely decreases this risk, even if the incidence of toxic effects detected is lower than in more sensitive inbred strains. It should also be noted that even in humans the incidence of VPA-induced malformations after maternal medication is 10– 30% and half of all children with VPA syndrome have some autistic features (Battino et al., 1992; Lindhout et al., 1992; Moore et al., 2000; Omtzigt et al., 1992). We have previously examined the fetal brain at GD14 and reported differences in the DNT induced by VPA between pregnant rats mated at our own animal facility (in-house group) and rats purchased pregnant (supplier group) (Ogawa et al., 2007). However, the GD14 fetal brain was too young to reveal the specific VPA-induced neurotoxicity. The effect of the mating place was observed in only the reproductive findings, but not on neural development. In the present examination of a more developed GD16 fetal brain, we detected an abnormal pons and/ or dysplasia of CP after VPA treatment. Furthermore, the incidence of these fetal brain abnormalities was increased in the supplier group. Thus, examination of the fetal brain at GD16 clearly exhibited more brain development-specific VPA-induced toxicity and the effect of mating place on VPA-induced DNT. Although many studies of DNT use pregnant rodents mated at the supplier, early pregnant stress such as shipping and environmental changes may be one of the factors affecting the reproducibility of DNT tests. From the results obtained in this study, this VPA-induced animal model can reproduce at least part of the pathological findings reported in idiopathic autism, and postnatal findings in the animal model. Examination of the GD16 fetal brain shortly after chemical exposure is useful to evaluate early neurodevelopmental processes and to support current postnatal data. Further accumulation of fetal data related to postnatal abnormalities will make it possible to extrapolate postnatal abnormalities from fetal observation in the evaluation of potential toxins. Acknowledgements We thank Mrs. Chie Sakurai for her technical support. This research was supported by a grant for the Long-range Research Initiative from the Japan Chemical Industry Association.
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