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Keywords:

  • autism spectrum disorder;
  • glutamatergic synapse;
  • male preponderance;
  • seizure susceptibility;
  • social interaction;
  • valproic acid

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Autism spectrum disorder (ASD) is a pervasive developmental disorder characterized by three main behavioral symptoms including social deficits, impaired communication, and stereotyped and repetitive behaviors. ASD prevalence shows gender bias to male. Prenatal exposure to valproic acid (VPA), a drug used in epilepsy and bipolar disorder, induces autistic symptoms in both human and rodents. As we reported previously, prenatally VPA-exposed animals at E12 showed impairment in social behavior without any overt reproductive toxicity. Social interactions were not significantly different between male and female rats in control condition. However, VPA-exposed male offspring showed significantly impaired social interaction while female offspring showed only marginal deficits in social interaction. Similar male inclination was observed in hyperactivity behavior induced by VPA. In addition to the ASD-like behavioral phenotype, prenatally VPA-exposed rat offspring shows crooked tail phenotype, which was not different between male and female groups. Both male and female rat showed reduced GABAergic neuronal marker GAD and increased glutamatergic neuronal marker vGluT1 expression. Interestingly, despite of the similar increased expression of vGluT1, post-synaptic marker proteins such as PSD-95 and α-CAMKII expression was significantly elevated only in male offspring. Electron microscopy showed increased number of post-synapse in male but not in female at 4 weeks of age. These results might suggest that the altered glutamatergic neuronal differentiation leads to deranged post-synaptic maturation only in male offspring prenatally exposed to VPA. Consistent with the increased post-synaptic compartment, VPA-exposed male rats showed higher sensitivity to electric shock than VPA-exposed female rats. These results suggest that prenatally VPA-exposed rats show the male preponderance of ASD-like behaviors including defective social interaction similar to human autistic patients, which might be caused by ectopic increase in glutamatergic synapses in male rats.

Abbreviations used
ADHD

attention-deficit hyperactivity disorder

ASD

autism spectrum disorder

EMB

extreme male brain

GAD

glutamic acid decarboxylase

NTDs

neural tube defects

PSD

post-synaptic density

PV

parvalbumin

SI

sociability index

SPI

social preference index

VPA

valproic acid

Autism spectrum disorder (ASD) is a prototypic pervasive developmental disorder, which results from abnormal process of brain development. ASD is generally observed before 3 years of age, and characterized by three behavioral symptoms; social deficits, impaired language and communication, and stereotyped and repetitive behaviors. In addition, substantial proportion of autistic patients displays symptoms such as abnormal sensitivities to sound or touch, aggressive behaviors (Kientz and Dunn 1997), and epilepsy (Kanner 1943). ASD shows a marked male preponderance ranging from 2.5 fold (Kim et al. 2011b) to 11 fold (Gillberg et al. 2006), similar to other early-onset antisocial disorders such as developmental language disorder and attention-deficit hyperactivity disorder (ADHD) (Zahn-Waxler et al. 2008). Consistent with these results, autistic female adolescences showed milder repetitive stereotyped behaviors, and less severe difficulties in school life than autistic male adolescences (Mandy et al. 2012). However, even with these seemingly ample evidences suggesting gender inclination to male, biological and molecular basis of gender bias has not proven yet.

Valproic acid (VPA) is a generally used anti-seizure drug to relieve symptoms of epilepsy and bipolar disorder. However, VPA exposure during early gestation induces numerous defects in human fetus including neural tube defects (Dalens et al. 1980; Bjerkedal et al. 1982) and intellectual impairments (Moore et al. 2000; Langer et al. 1994). Christianson first suggested an association between in utero VPA exposure and ASD in 1994 (Christianson et al. 1994), and rodent models as well as human cases have been widely used to investigate the effects of VPA on ASD-like symptoms as well as teratogenicity (Wegner and Nau 1991). In the past studies, several groups including us reported that prenatally VPA-exposed rats showed repetitive stereotypic-like activity (Schneider et al. 2008), impaired social interactions (Schneider and Przewlocki 2005), and decreased social preference for social novelty (Bambini-Junior et al. 2011), similar to human autistic patients. These reports positioned the prenatal VPA exposed-rats as a useful animal model of ASD (Narita et al. 2002; Ingram et al. 2000).

In the previous study, we reported that rat offspring prenatally exposed to VPA at embryonic day 12 show significantly impaired social interactions and vulnerability to seizure (Kim et al. 2011a). In this study, we investigated the differences in impaired sociability and social preference among VPA-exposed male and female groups and then we examined whether synapse formation as determined by western blot and electron microscopy as well as seizure susceptibility, measured by electroshock seizure threshold, show a gender differences in prenatally VPA-exposed rats.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Animals

Pregnant Sprague-Dawley rats at gestational day 5 were obtained from DaeHan BioLink (Daejeon, Korea). Rats were maintained on a 12:12-h circadian cycle with lights on at 06:00, at a constant temperature (22 ± 2°C) and humidity (55 ± 5%). Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Konkuk University, Korea. All efforts were made to minimize the number of animals as well as their suffering. Behavioral experiments were performed between 10:00 and 16:00 o'clock in dedicated test room.

Social interaction test

The social interaction test involving an unfamiliar and a familiar rat was adapted from Crawley (Crawley 2004), and performed using 4-weeks old rats as we reported previously (Kim et al. 2011a). Test rats were chosen by randomized schedule from each litter. The test took place in an environment unknown to the rat being tested, in the form of a cage with three communicating compartments. Each compartment was 23.3-cm long, 30-cm wide, and 20-cm height. Dividing walls had rectangular openings (10-cm wide and 10-cm height) allowing access into each compartment.

At the beginning of sociability test, test rat was placed in the empty central compartment. In the left compartment (stranger 1 side), a stranger rat being tested was placed under small wire cage with a radius of 5.5 cm. Stranger rats were randomly selected from control rats of same gender as test rats. In the right compartment (empty side), only a wire cage without rat was placed. After 5 min habituation period, sociability test was performed for 10 min. Sociability Index (SI) was defined as the ratio between duration of test rat in stranger 1 side and duration of test rat in empty side.

Moving on social preference test, another stranger rat (stranger 2) was added in the right compartment inside wire cage. The test rat was again placed in the empty central compartment, and social preference test was conducted for 10 min directly after the termination of the sociability test. Social preference test evaluates the initiation of social interaction (i.e. spending time) with either familiar (stranger 1) or novel (stranger 2) rat by the preference of a test rat. Social preference Index (SPI) was defined as the ratio between duration of test rat in novel side and duration of test rat in familiar side. The trace of rat movements during experiments was recorded using Ethovision software (version 3.1; Noldus information Technology, Wageningen, the Netherlands). The novel rats were also selected from control rats of same gender as test rats, and rats used as a familiar or a novel rat were not applied in social interaction tests.

Transmission electron microscopy

At 4 weeks of age, animals were perfused and fixed with 4% paraformaldehyde plus 2.0% v⁄v glutaraldehyde. After fixation, 1 mm thick sections of pre-frontal cortex were post-fixed with 1.0% osmium tetroxide for 2 h at 4°C. Specimens were embedded in Poly/Bed 812 kit (Polysciences, Warrington, PA, USA). 70 nm thin sections were double-stained with 7% uranyl acetate for 20 min followed by lead citrate for contrast staining. Ultrathin sections were cut by LEICA Ultracut UCT Ultra-microtome (Leica Microsystems, Wetzlar, Germany). The sections were observed by transmission electron microscopy (JEM-1011; JEOL, Tokyo, Japan) at the acceleration voltage of 90 kV and photographed at 10 000x and 30 000x magnification. EM photographs (10 000x) were used to count the number of post-synaptic density (PSD) in randomly selected visual fields.

Measurement of electroshock seizure threshold

Electroshock seizure threshold was measured with minor modifications with 4-weeks old rats, as we reported previously (Park et al. 2007). Briefly, seizure was evoked by a constant current stimulator, and the resulting seizure was determined by overt hind limb extension. To determine the electroshock seizure threshold, convulsive current 50 (CC50), which elicits convulsion in 50% of animals, was determined by a ‘staircase’ procedure (Browning et al. 1990) and calculated by the Litchfield–Wilcoxon II method (Litchfield and Wilcoxon 1949). Rats were given electroshocks through ear clip for 1 sec individually to determine the current-convulsion relationship.

Statistical analysis

Data were expressed as the mean ± standard error of mean (SEM) and analyzed for statistical significance using one-way analysis of variance (anova) followed by Newman–Keuls test as a post hoc test. Two-way anova was used to identify VPA exposure or gender effects, or interaction between the two factors. If significant effects were found in any one of the factors, post hoc comparisons were conducted using Bonferroni's post-tests. Differences were considered statistically significant when the p-value was less than 0.05 (p < 0.05). All statistical analyses were conducted using PASW Statistics (18.0; SPSS Inc, Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Effect of VPA exposure on body weights and birth rate

In a preliminary study, the number of live birth from pregnant rat was decreased as 12.3 ± 0.89 (94.9% compared with control) when VPA is treated at embryonic day 7 and as 5.2 ± 0.67 (40.0% compared with control) at embryonic day 9.5 (data not shown). However, the number of live birth nor body weight gain of rat pups from post-natal day 2 to post-natal day 28 was not significantly different in rat pups exposed to VPA at embryonic day 12, consistent with our previous reports (Kim et al. 2011a). Because of the lack of massive teratogenic effects, we treated VPA at E12 in further studies.

Gender different effect of VPA exposure in sociability

As reported previously, VPA injection at E12 gave most consistent and robust changes in social interaction of offspring rats (Kim et al. 2011a). In this study, the sociability of rats was examined at 4 weeks of age in offspring rats born from dams treated with VPA at E12, which showed marked male inclination of impaired social interaction consistent with the results reported by other researchers (Schneider et al. 2008; Kataoka et al. 2011). In Fig. 1a, two-way anova revealed that stay duration in stranger 1 side was decreased by VPA exposure [F(1,68) = 4.88, p 0.031], but not by gender [F(1,68) = 1.26, p 0.267]. Post hoc comparisons showed that VPA-exposed males stayed less in the stranger 1 side than saline-exposed males (p < 0.01). There is a significant interaction between VPA exposure × gender [F(1,68) = 7.88, p 0.007]. Duration in central compartment was increased by VPA exposure [F(1,68) = 27.40, p < 0.001], but not by gender [F(1,68) = 2.19, p 0.145]. Post hoc comparisons showed that VPA-exposed males stayed more in the central compartment than saline-exposed males (p < 0.001). There is a significant interaction between VPA exposure × gender [F(1,68) = 13.02, p < 0.001]. Duration in empty side was not changed by VPA exposure [F(1,68) = 0.21, p 0.646] or gender [F(1,68) = 0.001, p 0.975]. There is no significant interaction between VPA exposure × gender [F(1,68) = 3.21, p 0.078].

image

Figure 1. Reduced sociability in valproic acid (VPA)-exposed male rats (a) The duration of stay in stranger 1 side, central compartment, and empty side was shown in the graph. Control male group; the time spent in stranger 1 side (320.92 ± 9.95 s), the time spent in central compartment (100.45 ± 2.84 s), the time spent in empty side (164.49 ± 10.02 s). **p < 0.01, ***p < 0.001 versus control male group; ##p < 0.01, ###p < 0.001 versus VPA-exposed female group, as revealed by post hoc Bonferroni's comparisons following two-way anova. (b) The number of entries into stranger 1 side, central compartment, and empty side was shown in the graph. Control male group; the number of entries into stranger 1 side (15 ± 1.0), the number of entries into central compartment (22.5 ± 0.9), the number of entries into empty side (11.1 ± 0.6). **p < 0.01, ***p < 0.001 versus control male group; #p < 0.05, ###p < 0.001 versus VPA-exposed female group, as revealed by post hoc Bonferroni's comparisons following two-way anova. (c) Sociability Index (SI) was calculated as the ratio of time spent in stranger 1 side over time spent in empty side. ***p < 0.001 versus control male group; #p < 0.05 versus VPA-exposed female group, as revealed by post hoc Bonferroni's comparisons following two-way anova. (d) The trace of movements during sociability tests was recorded using Ethovision software. All data are expressed as mean ± SEM. (Three male rats and three female rats were chosen from each of 6 litters. Total 18 rats per group, n = 6).

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In Fig. 1b, entries into stranger 1 side was not changed by VPA exposure [F(1,68) = 3.77, p 0.057] or gender [F(1,68) = 0.54, p 0.466]. In post hoc comparisons, VPA-exposed males entered less into the stranger 1 side than saline-exposed males (p < 0.01). There is a significant interaction between VPA exposure × gender [F(1,68) = 4.84, p 0.032]. Entries into central compartment were increased by VPA exposure [F(1,68) = 9.24, p 0.004] and gender [F(1, 68) = 24.01, p < 0.001]. Post hoc comparisons showed that VPA-exposed males entered more often into the central compartment than saline-exposed males (p < 0.001), but VPA-exposed females did not. There is a significant interaction between VPA exposure × gender [F(1,68) = 31.17, p < 0.001]. Entries into empty side was not changed by VPA exposure [F(1,68) = 0.36, p 0.548] or gender [F(1,68) = 0.19, p 0.668], and there is no significant interaction between VPA exposure × gender [F(1,68) = 2.15, p 0.148].

Sociability Index (SI) was significantly decreased by VPA exposure [F(1,68) = 8.15, p 0.006], but not by gender [F(1,68) = 3.54, p 0.065]. Post hoc comparisons showed that VPA-exposed males had lower SI than saline-exposed males (p < 0.001) and there is a significant interaction between VPA exposure × gender [F(1,68) = 6.07, p 0.017]. The actual track of the movements of each experimental group was shown in Fig. 1d. These results suggest that prenatally VPA-exposed male rats display less approaches to a conspecific stranger rat as compared with empty wire cage than control male rats and VPA-exposed female rats, whereas prenatally VPA-exposed female rats did not show a significant difference from control female rats.

Gender different effect of VPA exposure in social preference

Next, we evaluated the curiosity to novel animal using social preference test. In Fig. 2a, stay duration in familiar side (stranger 1) was increased by VPA exposure [F(1,68) = 76.07, p < 0.001] and gender [F(1,68) = 27.53, p < 0.001]. Post hoc comparisons showed that VPA-exposed males stayed more in the familiar side than saline-exposed males (p < 0.001), but VPA-exposed females did not as compared with control females. There is a significant interaction between VPA exposure × gender [F(1,68) = 38.41, p < 0.001]. Duration in central compartment was increased by VPA exposure [F(1,68) = 6.85, p 0.011] and gender [F(1,68) = 13.79, p < 0.001]. Post hoc comparisons showed that VPA-exposed males stayed more in the central compartment than saline-exposed males (p < 0.01), but VPA-exposed females did not. There is a significant interaction between VPA exposure × gender [F(1,68) = 5.20, p 0.026]. Duration in novel side (stranger 2) was decreased by VPA exposure [F(1,68) = 130.38, p < 0.001] and gender [F(1,68) = 43.33, p < 0.001]. Post hoc comparisons showed that VPA-exposed males stayed less in the novel side than saline-exposed males (p < 0.001), and VPA-exposed females stayed less in the novel side than saline-exposed females (p < 0.01). There is a significant interaction between VPA exposure × gender [F(1,68) = 46.71, p < 0.001].

image

Figure 2. Reduced social preference in valproic acid (VPA)-exposed male rats (a) The duration of stay in familiar side (stranger 1), central compartment, and novel side (stranger 2) was shown in the graph. Control male group; the time spent in familiar side (265.67 ± 10.91 s), the time spent in central compartment (119.24 ± 8.44 s), the time spent in novel side (191.17 ± 7.39 s). **p < 0.01, ***p < 0.001 versus control male group; #p < 0.05, ###p < 0.001 versus VPA-exposed female group; α< 0.01 versus control female group, as revealed by post hoc Bonferroni's comparisons following two-way anova. (b) The number of entries into familiar side (stranger 1), central compartment, and novel side (stranger 2) was shown in the graph. Control male group; the number of entries into familiar side (14 ± 0.5), the number of entries into central compartment (23.1 ± 0.8), the number of entries into novel side (10.8 ± 0.8). ***p < 0.001 versus control male group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus VPA-exposed female group, as revealed by post hoc Bonferroni's comparisons following two-way anova. (c) Social preference Index (SPI) was calculated as the ratio of time spent in novel side over time spent in familiar side. ***p < 0.001 versus control male group; ##p < 0.01 versus VPA-exposed female group, as revealed by post hoc Bonferroni's comparisons following two-way anova. (d) The trace of movements during social preference tests was recorded using Ethovision software. All data are expressed as mean ± SEM. (Three male rats and three female rats were chosen from each of 6 litters. Total 18 rats per group, n = 6).

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In Fig. 2b, the number of entries into familiar side (stranger 1) was increased by VPA exposure [F(1,68) = 30.64, p < 0.001] and gender [F(1,68) = 19.94, p < 0.001]. Post hoc comparisons showed that VPA-exposed males entered more frequently into the familiar side than saline-exposed males (p < 0.001), but VPA-exposed females did not. There is a significant interaction between VPA exposure × gender [F(1,68) = 8.81, p 0.004]. Entries into central compartment was increased by VPA exposure [F(1,68) = 13.42, p < 0.001] and gender [F(1,68) = 22.52, p < 0.001]. Post hoc comparisons showed that VPA-exposed males entered more often into the central compartment than saline-exposed males (p < 0.001), but VPA-exposed females did not. There is a significant interaction between VPA exposure × gender [F(1,68) = 16.19, p < 0.001]. Entries into novel side (stranger 2) were decreased by VPA exposure [F(1,68) = 16.30, p < 0.001] and gender [F(1,68) = 14.85, p < 0.001]. Post hoc comparisons showed that VPA-exposed males entered less into the novel side than saline-exposed males (p < 0.001), but VPA-exposed females did not. There is a significant interaction between VPA exposure × gender [F(1,68) = 5.69, p 0.0202].

Social preference Index (SPI) was significantly decreased by VPA exposure [F(1,68) = 38.42, p < 0.001] and gender [F(1,68) = 6.32, p 0.018]. Post hoc comparisons showed that VPA-exposed males had lower SI than saline-exposed males (p < 0.001), but VPA-exposed females did not. There is a significant interaction between VPA exposure × gender [F(1,68) = 11.58, p 0.002]. The actual track of the movements of each experimental group was shown in Fig. 2d. These results suggest that prenatally VPA-exposed male rats displayed more approaches to familiar rats and less approaches to the socially novel rats than control males and VPA-exposed female rats, whereas VPA-exposed female rats did not show any difference from control females. Prenatal VPA exposure induced more vulnerability in male group on social interactions compared with female group, similar to human autistic patients.

Effect of VPA exposure on the crooked tail phenotype and motor function

Neural tube defects (NTDs) may result from genetic mutations or exposure to teratogen during gestation. Crooked tail is one of the mildest forms of NTD subtype phenomena in rodents including rats and mice. Although no major teratogenic effects were observed in VPA-treated rat pups, 79% rat pups exposed to VPA at E12 showed crooked tail phenotype, which is consistent with the previous report (Foley et al. 2012). VPA exposure at E9.5 produced more severe tail malformation phenotype including unformed or very short tails. In contrast to VPA-induced aberration in social behaviors, there was no gender difference in the occurrence of crooked tail in rat offspring exposed to VPA at E12 (Figure S1a). To test whether abnormal formation of tail might result in the developmental and functional defects of motor system, we evaluated the motor coordination of VPA-exposed rats using rota-rod test. Figure S1b showed that prenatal VPA exposure did not influence the falling latency and frequency of rats during rota-rod test.

Gender different effect of VPA exposure on locomotor activity

Exposure to VPA also showed increased activity in open-field locomotor test, which is significant only in male rats (Figure S2). Moved distance was increased by VPA exposure [F(1,68) = 4.95, p 0.031], not by gender [F(1,68) = 0.002, p 0.97]. Post hoc comparisons showed that VPA-exposed males moved more in the open field than saline-exposed males (p < 0.05). There is no significant interaction between VPA exposure × gender [F(1,68) = 1.87, p 0.178]. Moved duration was also increased by VPA exposure [F(1,68) = 12.94, p < 0.001], not by gender [F(1,68) = 0.42, p 0.52]. Post hoc comparisons showed that VPA-exposed males moved more in the open field than saline-exposed males (p < 0.05). There is no significant interaction between VPA exposure × gender [F(1,68) = 0.69, p 0.412]. These results suggest that gender difference in increased locomotor activity is not statistically significant, albeit VPA-exposed male rats displayed more locomotive activity than VPA-exposed female rats.

Gender difference of synaptic maturation in prenatally VPA-exposed rats

Next, we investigated whether aberrant synaptic maturation underlies in gender specific manner in the VPA-induced rat model of ASD. Cerebral cortex and hippocampus were analyzed by western blot. Expression of post-synaptic proteins was increased in the cortex of VPA-exposed male rat offspring (Fig. 3a). PSD-95 expression was increased by VPA exposure [F(1,8) = 42.45, p 0.0002] and Gender [F(1,8) = 19.09, p 0.0024]. Post hoc comparisons showed that PSD-95 expression in the VPA-exposed male rat was higher than that of VPA-exposed female rat (p < 0.001). There is a significant interaction between VPA exposure × gender [F(1,8) = 23.26, p 0.0013]. α-CaMKII expression was also increased by VPA exposure [F(1,8) = 101.9, p < 0.0001] and Gender [F(1,8) = 42.85, p < 0.0001]. Post hoc comparisons showed that α-CaMKII expression in the cortex of VPA-exposed male was higher than that of VPA-exposed female (p < 0.001). There is a significant interaction between VPA exposure × gender [F(1,8) = 43.04, p 0.0002]. However, proteins predominantly localized in the somatic or pre-synaptic regions were similarly changed in the cortex of male and female by pre-natal VPA exposure (Fig. 3a). Excitatory neuronal marker vGluT1 expression was increased by VPA exposure [F(1,8) = 77.80, p < 0.0001], not by Gender [F(1,8) = 4.319, p 0.0713]. There is no significant interaction between VPA exposure × gender [F(1,8) = 0.8022, p 0.3966]. Pre-synaptic protein synaptophysin expression was increased by VPA exposure [F(1,8) = 146.4, p < 0.0001], not by Gender [F(1,8) = 0.5658, p 0.4735]. There is no significant interaction between VPA exposure × gender [F(1,8) = 3.613, p 0.0939]. Inhibitory neuronal marker GAD67 expression was decreased by VPA exposure [F(1,8) = 39.64, p 0.0002], not by Gender [F(1,8) = 0.1148, p 0.7435]. There is no significant interaction between VPA exposure × gender [F(1,8) = 1.744, p 0.2231]. These results suggest that pre-natal exposure to VPA increased excitatory neuronal differentiation while reducing inhibitory neuronal differentiation both in male and female rat offspring, but post-synaptic changes in the cortex are mostly confined to male rat offspring.

image

Figure 3. Aberrant excitatoty/inhibitory neuronal development in valproic acid (VPA)-exposed male rats (a) Post-/pre-synaptic protein levels in the cortex. All data are expressed as mean ± SEM (n = 3). ***p < 0.001 versus control group; ###p < 0.001 versus VPA-exposed male group, as revealed by post hoc Bonferroni's comparisons following two-way anova. (b) Post-/pre-synaptic protein levels in the hippocampus. All data are expressed as mean ± SEM (n = 3). **p < 0.01, ***p < 0.001 versus control group; #p < 0.05, ###p < 0.001 versus VPA-exposed male group, as revealed by post hoc Bonferroni's comparisons following two-way anova. (c) Excessive post-synaptic development in VPA-exposed male rats. Post-synaptic density was distinguished by the presence of an electron-dense band. Scale bar; 2000 nm. In lower panel, magnified image was shown with arrowheads which mark post-synaptic density. All data are expressed as mean ± SEM. (Two male rats and two female rats were chosen from each of 3 litters. Total six rats per group, n = 3). ***p < 0.001 versus. control male group; ###p < 0.001 versus. VPA-exposed male group, as revealed by post hoc Bonferroni's comparisons following two-way anova.

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In the hippocampus, post-synaptic proteins were also increased in the VPA-exposed male offspring (Fig. 3b). PSD-95 expression was increased by VPA exposure [F(1,8) = 99.83, p < 0.0001], not by Gender [F(1,8) = 4.798, p 0.0599]. Post hoc comparisons showed that PSD-95 expression in the VPA-exposed male was higher than that of VPA-exposed female (p < 0.05). There is a significant interaction between VPA exposure × gender [F(1,8) = 9.052, p 0.0168]. α-CaMKII expression was increased by VPA exposure [F(1,8) = 349.1, p < 0.0001] and Gender [F(1,8) = 8.418, p 0.0198]. Post hoc comparisons showed that α-CaMKII expression in the hippocampus of VPA-exposed male was higher than that of VPA-exposed female (p < 0.01). There is a significant interaction between VPA exposure × gender [F(1,8) = 18.16, p 0.0028]. vGluT1 and synaptophysin were increased in both male and female by VPA exposure (Fig. 3b). vGluT1 expression was increased by VPA exposure [F(1,8) = 85.76, p < 0.0001] but not by Gender [F(1,8) = 0.3395, p 0.5762]. There is no significant interaction between VPA exposure × gender [F(1,8) = 1.629, p 0.2377]. Synaptophysin expression was increased by VPA exposure [F(1,8) = 362.4, p < 0.0001] but not by Gender [F(1,8) = 2.883, p 0.1280]. There is no significant interaction between VPA exposure × gender [F(1,8) = 0.3633, p 0.5634]. GAD67 expression was decreased in the VPA-exposed male and female offspring, which is more pronounce in male offspring. GAD67 expression was decreased by VPA exposure [F(1,8) = 22.42, p 0.0015], but not by Gender [F(1,8) = 0.5901, p 0.4645]. Post hoc comparisons showed that GAD67 expression in the VPA-exposed male was lower than that of VPA-exposed female (p < 0.05). There is a significant interaction between VPA exposure × gender [F(1,8) = 11.85, p 0.0088].

Next, we investigated the gross appearance of post-synaptic compartment by electron microscopy. As shown in Fig. 3c, we found that the number of post-synaptic compartment was increased by VPA exposure [F(1,76) = 12.21, p 0.0008], not by Gender [F(1,76) = 0.092, p 0.7621]. And there is a significant interaction between VPA exposure × gender [F(1,76) = 5.056, p 0.0274]. Post hoc comparisons showed that post-synaptic increment in VPA-exposed male was higher than that of VPA-exposed female (p < 0.001). Taken together, these results suggest that pre-natal VPA exposure induces imbalance of excitatory/inhibitory neuronal differentiation both male and female rat but specifically increases post-synaptic development of excitatory glutamatergic neuron in the brain of male offspring.

Gender difference of electroshock seizure threshold in prenatally VPA-exposed rats

Increased excitatory tone may affect excitability of the brain, which is often increased in ASD brain. We next examined the effects of VPA exposure on the electroshock seizure threshold (Fig. 4). To measure the electroshock seizure threshold, we calculated the convulsive current 50 (CC50) using the Litchfield–Wilcoxon II method, which is an estimate to induce convulsion in 50% of animals. Electroshock seizure thresholds of the VPA-exposed male group were significantly lower than those of the control male group (p < 0.001) and VPA-exposed female group (p < 0.001). These results suggested that VPA-exposed rat model of ASD also show male preference in terms of reduced inhibitory function of the brain.

Increased excitatory tone may affect excitability of the brain, which is often increased in ASD brain. We next examined the effects of VPA exposure on the electroshock seizure threshold (Fig. 3). To measure the electroshock seizure threshold, we calculated the convulsive current 50 (CC50) using the Litchfield–Wilcoxon II method, which is an estimate to induce convulsion in 50% of animals. Electroshock seizure thresholds of the VPA-exposed male group were significantly lower than those of the control male group (p < 0.001) and VPA-exposed female group (p < 0.001). These results suggested that VPA-exposed rat model of ASD also show male preference in terms of reduced inhibitory function of the brain.

image

Figure 4. Increased sensitivity to electroshock in valproic acid (VPA)-exposed male rats. Measurement of electroshock seizure threshold was performed as described in 'Materials and methods'. CC50 of VPA-exposed male rats was significantly lower than that of control group (***p < 0.001), and VPA-exposed female rats (###p < 0.001). 24 male rats and 24 female rats from 6 litters were used for the determination of CC50. Table showed actual value of CC50 with upper and lower confidence limits.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

In the early development, it was well known that teratogenic substances including VPA induced gender-dependent behavioral abnormalities in rodents (Vorhees 1987a; Roegge et al. 2000; Geller et al. 2001; Schneider et al. 2008; Kataoka et al. 2011). In this study, we injected 400 mg/kg of sodium VPA to pregnant rats at E12. Because exposure to VPA at earlier time points in high dose may produce severe neural tube defects, E12 is the ideal time point to induce abnormal neuronal differentiation withholding the gross structural abnormalities. The number of live birth was decreased in rats treated with VPA at E9.5 and the survived rat offspring showed severe malformation of tails such as unformed or short tails in 7.7% of offspring (Figure S1a). In contrast, VPA exposure at E12 produced significant defects in social behavior and induced only mild abnormalities in tail morphology, without any other signs of teratogenicity. These results suggest that crooked tail might be the mildest form of neural tube defects (Vorhees 1987b), which may overlap with the critical time periods for the differentiation of neuronal circuitry for normal social behaviors. Although crooked tail phenotype occurred equally in both male and female rats by VPA exposure (Figure S1a), aberration in post-synaptic density, impairments of social interactions and sensitivity to electroshock were only shown in VPA-exposed male rats, suggesting the crooked tail formation and the autism-like symptoms may have distinct underlying causes or pathophysiological mechanisms, which might provide an opportunity for comparative studies on the functional (including behavioral) and structural determinants of gender specific abnormal neural development in pre-natal VPA model of ASD.

Many researchers suggested various risk factors of male preponderance in ASD, and tried to conceptualize gender differences in ASD as a mind theory. Excessive fetal testosterone, X chromosome-linked abnormalities, Y chromosome-linked abnormalities, and de novo copy number variations are all suggested as causes of male preponderance in ASD, but these hypothesis fail to explain gender differences in ASD clearly (Baron-Cohen et al. 2011). Male specificity in ASD has been explained by concepts ranging from the empathizing-systemizing (E-S) theory to the extreme male brain (EMB) theory. According to the E-S theory, ASD patients are explained as having lower empathizing function and average or higher systemizing function (Baron-Cohen 2010). The EMB theory, first informally suggested by Hans Asperger in 1944 (Asperger 1944; Baron-Cohen 2002), was extended from the E-S theory. In the EMB theory, females perform empathizing function better, males perform systemizing function better, and autism can be seen as an extreme of the typical male profile (Baron-Cohen 2010). However, there is still no clear explanation how male preponderance in ASD occurs.

Recently, a sex-specific multiple-threshold model has been suggested in which females need to carry more genetic liability than males to develop ASD (Szatmari et al. 2012). In this model, hitherto unknown protective factors must exist to account for that higher threshold in female. A recent report of rare autosomal SHANK1 deletions associated with ASD or the broader autism phenotype that is limited to males but not to females provides a support for sex-specific multiple-threshold model (Sato et al. 2012). SHANK1 is one of the key post-synaptic scaffolding proteins and it is intriguing that we also found major gender difference in post-synaptic protein expression and morphology in pre-natal VPA exposure model of ASD. Whether our model also shows defects in SHANK1 expression and the SHANK1 mutation in human represents male-specific defects in organization and function of post-synaptic compartment in human, as well as post-synaptic disorganization may lies at the heart of ASD pathophysiological processes should be investigated further in the future. Besides, efforts toward the identification of such factors contributing to the female specific normalization or protection against perturbed post-synaptic development may widen our understanding on the manifestation of ASD endophenotypes as well as general mechanism governing synapse maturation.

Imbalanced excitatory/inhibitory (E/I) signaling in the brain may underlie the manifestation of autistic symptoms (Rubenstein and Merzenich 2003; Dani et al. 2005). Especially, attenuated GABAergic signal (Oblak et al. 2011; Blatt et al. 2001) and excess glutamatergic signal (Shinohe et al. 2006) have been reported, which might be responsible for the autistic behaviors including impaired social interaction (Mehta et al. 2011; Wei et al. 2012; Chez et al. 2007; Rinaldi et al. 2007). Expression of GABAA receptor (Samaco et al. 2005; Fatemi et al. 2009) and glutamic acid decarboxylase (GAD) (Fatemi et al. 2002) was decreased in the brain of autistic patients. Disrupted inhibitory architectures in the brain of ASD patients was also reported (Casanova et al. 2002). Moreover, it is well-known that GABAergic defects in the brain underlay epileptic symptoms (Lloyd et al. 1986; Sherwin and van Gelder 1986), which showed substantial incidence in ASD patients from 5% (Bryson et al. 1988) to 46% (Hughes and Melyn 2005).

Prenatally VPA-exposed autism animal models also showed E/I imbalance, similar to human ASD patients. Prenatal VPA exposure elevated expression of NR2A, NR2B, and α-CaMKII, with enhanced post-synaptic long-term potentiation (Rinaldi et al. 2007). Abnormal expression of GABAA receptor subunit and dysfunction of benzodiazepine binding site have been reported in VPA-exposed rats (Derry et al. 2004; Fukuchi et al. 2009), and VPA exposure reduced GAD expression in young neurons (Fukuchi et al. 2009). Prenatally VPA-exposed mice also exhibited a reduction of parvalbumin (PV)-positive inhibitory neuron, which may underlie disruption of E/I balance in the brain (Gogolla et al. 2009). Recent study suggests that VPA induces synaptic E/I imbalance by increased post-synaptic proteins (neuroligin-1, neuregulin-1, PSD-95) in culture systems (Wang et al. 2012). The significant sensitivity to electroshock of prenatally VPA-exposed rats also suggested aberrations on E/I imbalance with diminished GABAergic function (Kim et al. 2011a).

Developing from E/I imbalance, functional and structural synaptic dysregulation is a critical pathophysiological feature of ASD and many researchers recently acknowledged the excessive synapse formation in ASD (Hutsler and Zhang 2010; Geschwind and Levitt 2007; Toro et al. 2010). Especially, hyperconnectivity in microcircuitry in mPFC along with excessive glutamatergic differentiation has been reported in several different model systems. In this study, we reported the up-regulation of PSD95 and α-CAMKII in the brain of prenatally VPA-exposed rat offspring consistent with the up-regulation of NR2A and NR2B expression along with altered NMDA receptor dependent electrophysiological responses (Rinaldi et al. 2007). Surprisingly, we found that the statistically significant up-regulation of PSD95 and α-CAMKII is observed only in male offspring. These results suggest that the increase in post-synaptic density in VPA-exposed animals is a main determinant underlying the gender-specific autistic behaviors in this model system. Interestingly, vGluT1 expression was elevated and GAD67 expression was decreased in both male and female offspring, which may imply the increased glutamatergic neuronal differentiation in female brain as well. Whether these results suggest the existence of factors down-regulating the ectopic maturation of post-synaptic density even with overt glutamatergic neuronal number in female brain remains to be determined as well as the existence of such finding in human brain. Interestingly, GAD67 expression in VPA-exposed female hippocampus is higher compared with male offspring suggesting that the mechanism governing gender specificity of ASD is different in other brain areas and inhibitory neuronal differentiation in hippocampus is another determinant of gender specificity in VPA model of ASD.

Although all these results point to the distorted excitatory/inhibitory balance toward hyperexcitation, various domains of ASD phenotype governed by specific brain regions may be differentially regulated by slightly different neurobiological/neurodevelopmental mechanisms, which mandates domain specific treatment strategies. Determining whether other brain regions such as striatum and amygdala show similar changes in neuronal differentiation and synaptic changes may provide additional level of proof for this assumption. For example, anatomical abnormalities of cerebellum were frequently observed in the human ASD patients (Bauman and Kemper 1985; Ritvo et al. 1986; Williams et al. 1980). In spite of controversial reports, hypoplasia of cerebellar vermal lobules VI, VII was commonly observed (Courchesne et al. 1988, 1994) and this loss was negatively correlated to the rate of repetitive behavior in the ASD children (Pierce and Courchesne 2001). A recent volumetric analysis of MRI data showed the reduction of vermis volume in high functioning autism group (Scott et al. 2009). Loss of Purkinje cells was also reported in human ASD subjects (Allen and Courchesne 2003). Previously, pre-natal VPA exposure (600 mg/kg, i.p.) at E12.5 resulted in the reduction of cerebellar volume and the number of Purkinje cells in rat offspring (Ingram et al. 2000; Sandhya et al. 2012), which might be related to the toxic effect of VPA on cerebellar granule cells in vivo. Although, we did not examine cerebellar defects in this study, above mentioned studies as well as a recent study reporting decreased spine density in cerebellum of VPA-exposed rat offspring (Mychasiuk et al. 2012) suggest a possible aberration in cerebellar synaptic structure and function in VPA animal model of ASD. We are now actively investigating this possibility.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

All authors declare no conflict of interest. This study was supported by Mid-career Researcher Program (CY Shin, No. 2011-0014258) and the Framework of International Cooperation Program (CY Shin, No. 2012K2A1A2032549) through the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST).

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12147-sup-0001-FigureS1-S2.pdfapplication/PDF235K

Figure S1. Effect of prenatal VPA exposure on tail formation and motor coordination.

Figure S2. Increased locomotor activity in VPA-exposed male rats.

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