Sex-specific Behavioral Characteristics of Rats with Fetal Valproate Syndrome

Both male and female Wistar rats with an experimental model of autism spectrum disorders induced by prenatal administration of valproate sodium at a dose of 500 mg/kg have behavioral abnormalities similar to people with these disorders, including the abnormalities of social interaction; locomotor, orientation, and exploratory behavior; coordination of movement; spatial exploration behavior; and hyperactivity in previously explored “familiar” environments. The severity of behavioral abnormalities in rats with fetal valproate syndrome exhibits sex differences, which must be taken into account when conducting experimental studies in this model.

Autism spectrum disorders (ASD) is a group of neurological and developmental disorders of heterogeneous etiology characterized by impaired social interaction and communication as well as by restricted interests and repetitive patterns of behavior. Starting with the first reports on this disorder by Leo Kanner in 1943 and then by Hans Asperger in 1944, ASD was shown to prevail in boys (4-5 : 1), which had various consequences for both scientific research and clinical practice [1,2]. Recently, large-scale epidemiological studies have shown that the prevalence of ASD in boys and in girls can vary within a range of 2-5 : 1 and at even lower ratios; thus, there is a tendency towards detection of previously omitted girls with ASD with high-level functioning [3]. On suspicion of ASD of any subtype in adults, the disease was diagnosed in 72% of men and 66% of women; the adults with highly functional ASD (IQ > 70; N = 827) did not exhibit any marked gender differences in the severity of symptoms in the area of social communication and in the prevalence of comorbid psychopathologies in case of different ASD subtypes [4].
The girls and boys with ASD have different clinical and neurobiological characteristics determining different behavioral disorders. In girls, the typical symptoms of ASD, especially in the absence of mental deficiency, are less pronounced or different; they can better disguise their social difficulties so that disease manifestations can be less aggressive and destructive; at the same time, they more often suffer from anxiety and depression [4,5]. Hence, ASD in girls, especially in milder forms, is more difficult to diagnose. The knowledge of gender differences in ASD manifestations can be useful for recognizing and treating this disease in both sexes.
The experimental model suitable for the study of gender differences in ASD is a model of fetal valproate syndrome (FVS) [6] induced by administration of valproic acid (VPA) salts at high doses to rodents in the critical period of neural tube occlusion, which corresponds to the first trimester of pregnancy in humans. The FVS model in rodents is recognized as one of the most adequate models: the changes in the state of CNS in animals are similar to those in humans, as is manifested at the morphological, neurotransmitter, and behavioral levels [7][8][9]. Most of the studies with the FVS model are conducted in male rodents; researchers took account of the sex of animals and the related differences in behavioral and molecular changes in the brain in only a few works [6]. It has been shown that male rats with FVS are characterized by more marked social disabilities compared to females, enhanced convulsive readiness (especially in the maximal electroshock seizure test) and anxiety, as well as reduced pain sensitivity [10][11][12]. Male mice with FVS exhibited more marked social deficits compared to females, which was manifested in the shorter duration of sniffing the social object in the social interaction test, with similar behavior in the open field maze and elevated plus maze tests [13]. In other studies, the rats of both sexes with FVS demonstrated stereotyped behavior [12], attention and sensorimotor impair-ments, with the minimum difference between the specific parameters of ASD [14]. It has been shown clinically that ASD is twofold more common in boys among the children prenatally exposed to VPA [8]; however, another study has shown almost the same incidence of ASD in subjects of both sexes as a result of prenatal exposure to VPA [9].
The presence of contradictory data requires further detailed study of sex differences in ASD manifestations, which later on will probably contribute to elucidation of phenotypic peculiarities and mechanisms of development of this pathology in individuals of both sexes.
In the present work, we have studied the differences in ASD manifestations in male and female Wistar rats with FVS induced by prenatal exposure to VPA.

MATERIALS AND METHODS
The study was conducted on Wistar rats; the parental generation was obtained from the Stolbovaya Branch  FVS was induced by a single intraperitoneal prenatal administration of VPA (Sigma-Aldrich, United States) at a dose of 500 mg/kg on day 12.5 of pregnancy to female Wistar rats [15,16]. Normal saline solution in an equivalent volume (0.2 mL per 100 g of animal weight) was administered to female rats of the control group. Newborn rats (43 individuals: 25 control, 18 FVS rats) were weaned on day 21 of life and placed by 4-5 animals per T/3C cage (43.5 × 27.5 × 15.5 cm) with respect to sex. Behavioral testing of the rats was performed from day 35 to 43 of life, which corresponded to the average period of puberty characterized by enhanced anxiety and increased number of social gaming contacts [17,18].
Social disorders were assessed on day 35 of postnatal development of the rats by the social interaction test [15,16,19] in a plexiglas chamber (40 × 72 × 40 cm) divided into equal compartments by partitions with guillotine doors. The tested animal was placed into the central compartment and reticulated metal cylinders were put into extreme compartments; in one of them there was an unfamiliar animal ("social object") of the same age and sex as the tested animal and the other cylinder was empty ("unsocial object"). The preference for the social over the unsocial object was assessed by the time of staying in the compartments, the number of entries, as well as the number of contact approaches to the cylinder with the social object (at a distance of less than 2 cm) and the time of staying nearby during a 10-min observation. The findings were used to calculate the coefficient of preference for the social object (the ratio of the time of staying in the compartment with the social object to the total time spent in both compartments).
The orientation and exploratory behavior and anxiety of the rats with FVS were analyzed on day 36 of their postnatal development in the open field (OF) test (TS0501-R, Open Science Research and Production Company, Russia) by 3-min recording under normal lighting conditions of the indices of horizontal (movement in the periphery, precentral, and central parts of the field) and vertical motor activity, the number of entries into holes, and grooming acts [20]. The ratio of the total index of horizontal activity in central and precentral sectors of the field to the total index of horizontal motor activity was used to calculate the index of anxiety in rats.
The spatial working memory of rats with FVS was assessed on day 38 of postnatal development in a Y-maze (TS1301-R, Open Science Research and Production Complex, Russia). The entry into one of the maze branches was closed 24 h before the experiment; the rat was placed into one of the branches ("home") and allowed to explore this and the other ("alternative") open branch freely for 5 min. In 24 h, the rat was placed into the center of the maze with three branches available for exploration; the number of animal's entries to each branch ("new," "home," and "alternative") and the time of staying there were recorded for 5 min [21].
The anxiety of rats with FVS was tested on day 40 of their postnatal development in a light-dark box (LDB) test (TS0702-R, Open Science Research and Production Company, Russia) simulating a conflict between the orientation and exploratory motivation aimed at familiarization with the entire chamber and the hole exploratory behavior (hole reflex)-running into the preferred dark compartment [20]. The time of observation was 5 min.
The locomotor behavior and the coordination of movement of rats with FVS were assessed on days 35-39 and 43 of their postnatal development in the rotating rod test in a Rat Rota-Rod device (Ugo Basile, Italy): a cylinder (6 cm in diameter) divided into four equal parts by five discs and rotating with an acceleration increasing from 5 to 54 rpm.
Statistical data were processed using Statistica 10.0. Normal distribution was checked by the Shapiro-Wilk test followed by the Leven's test of equality of variances. Since experimental groups were characterized either by the absence of normal distribution or by nonobservance of the intergroup equality of variances, the nonparametric Mann-Whitney U test was used for further processing. The statistical significance of differences between the repeated measurements in the group was assessed by the pairwise Wilcoxon rank sum test. The results in the tables are presented as the mean ± error of the mean. The differences between the groups were considered significant at p < 0.05.

RESULTS AND DISCUSSION
The VPA salt administered to female Wistar rats at a dose of 500 mg/kg disturbed the development of the vertebral column in offspring manifested by 100% occurrence of a curved tail in both male and female rats.
In the social interaction test, the control male rats preferred the compartment with a social object, spending 2.3-fold (p < 0.05) more time here than in the compartment with an unsocial object. Yet, the time of staying in the compartments of different social significance was the same for male rats with FVS (Fig. 1). In addition, male rats with FVS spent equal periods of time exploring social and unsocial objects, while the control male rats spent 2.13-fold (p < 0.05) more time on the social object (Fig. 1).
The behavior of control female Wistar rats in the social interaction test did not differ from the behavior of control males. The control females also preferred the social object, spending 3.0-fold (p < 0.05) more time in this compartment and 3.9-fold (p < 0.05) more time on its exploration compared to the analogous parameters recorded in the compartment with the unsocial object.
The female rats with FVS demonstrated a marked preference for the unsocial object. The time of staying in the compartment with the social object and the time of its exploration in the female rats with FVS was 2.4-(p < 0.05) and 3.3-fold (p < 0.05) less, respectively, than in the control female rats (Fig. 1).
The number of entries into the compartments with significant and insignificant social objects, as well as the total locomotor behavior, showed no statistical differences between animals from all groups.
The data on the apparent lack of preference for the social object in male and female rats with FVS in the social interaction test demonstrate the loss of sociability typical of ASD [15,22,23]. The observed disorders were more marked in the female rats with FVS as indicated by a 60.0% decrease in the coefficient of preference for the social object (p < 0.05) relative to the respective control (Fig. 2).
The orientation exploratory and locomotor behavior and the level of anxiety in the rats with FVS were analyzed in the Open Field maze. On day 36 of postnatal development, the male rats with FVS showed a  Table 1). The index of anxiety in the male rats with FVS was 1.6 times (p < 0.05) below the index in the control, indicating a higher level of anxiety in animals ( Table 1). The findings are confirmed by the data on reduced locomotor activity in the rats with FVS in the central part of the open field maze in the absence of marked changes in the total number of locomotions [16]. The recorded decrease in the locomotor and orientation exploratory behavior of VPA-exposed male rats in the OF test was even more marked in females. Thus, for example, the horizontal activity of female rats with experimental ASD in the periphery of the maze showed a statistically significant decrease by 34.6%; the number of passages in the central and precentral zones of the maze decreased by 75.0 and 68.2%, respectively, while the number of upright postures decreased by 62.6% compared to the respective values in the control female rats ( Table 1). The index of anxiety in the female rats with FVS was 2.0 times (p < 0.05) lower and the number of grooming acts was by 52.3% (p < 0.05) higher compared to the respective values in the control ( Table 1).
The results of the OF test show that the locomotor and orientation exploratory activity in young rats with FVS decreases with the simultaneous increase in anxiety, being more marked in females.
Prenatal administration of VPA disturbed the typical behavioral patterns of rodents based on the exploratory motivation and the ability to choose previously unexplored areas for visitation. In the Y-maze test, the male rats with FVS demonstrated a marked preference for the previously explored "home" and "alternative" branches. The proportion of time spent in the "new" branch by the male rats with FVS showed a statistically significant decrease by 25.7% compared to the value in the control (Table 2). Yet, the time spent by the female rats with FVS in the "new" branch was not different from the time recorded for the control female rats. In addition, the animals of both sexes with FVS demonstrated hyperactive behavior, which was recorded by the average number of passages increasing 1.62-fold (p < 0.05) in male rats and 1.95-fold (p < 0.05) in female rats compared to the analogous values in the respective control (Table 2).
Thus, the rats with FVS demonstrated the impaired spatial exploratory behavior (more typical for males) and hyperactivity, which is in agreement with the data on stress-induced stereotypy characteristic of animals with ASD as well as reduced locomotor activity in the new, unfamiliar situation and enhanced locomotor activity in the familiar, previously explored environment [19,24].
In the LDB test, the control animals of different sexes demonstrated different behaviors. For example, female Wistar rats showed lower anxiety and the higher orientation exploratory activity compared to male rats: the time of their staying in the light compartment was 2.1-fold longer (p < 0.05) ( Table 3).
The male rats with FVS were shown to have marked behavioral deviations in the LDB test (Table 3). For example, the male rats with FVS spent 1.7-fold (p < 0.05) more time in the light compartment and statistically significantly less time in the dark compartment compared to the control group of males (Table 3).  Such behavior was also observed in the male rats with FVS in other studies [19], wherein the enhanced activity in LDB was recorded in animals from day 33 to day 40 of life. The enhanced anxiety of female rats with FVS demonstrated in the LDB test was manifested in a 2.8fold shorter (p < 0.05) time of staying in the light compartment and significant preference for the dark compartment compared to the respective control group ( Table 3).
The locomotor activity and coordination of movement in the rotating rod test analyzed from day 35 to 43 of postnatal development showed no differences between the control groups of male and female Wistar rats (Fig. 3). The rats of both sexes with FVS demonstrated a statistically significant decrease in locomotor activity and coordination of movement compared to the respective control groups; however, this disorder was more marked in the group of female rats with FVS (Fig. 3).
The present study confirmed the presence of disorders typical of ASD in both male and female rats with FVS, as confirmed by preference for the unsocial object over the social one in the social interaction test, impaired spatial exploratory behavior and hyperactivity of animals in the previously explored "familiar" environment of the Y-maze, reduced locomotor and orientation exploratory activity with simultaneous enhancement of anxiety in the open field test, and Time of remaining on the rod, s * * * * * impaired locomotor coordination in the rotating rod test. The observed impairments were more marked in the female rats with FVS. The behavioral differences between male and female rats with FVS were also observed in the LDB test. The male rats with FVS demonstrated distortion in the behavior typical of healthy animals (avoidance of brightly lit unfamiliar places, preference for the dark closed "safe" zone), staying for a long time in the light compartment, whereas the female rats with FVS displayed enhanced anxiety with preference for the dark compartment. Thus, the results of this work show that the male and female Wistar rats are equally susceptible to the toxic effect of prenatally administered VPA, which simulates the state similar to ASD; however, pathological manifestations may be different in severity or even opposite depending on sex assignment, which should be taken into account in the experimental study of these disorders as well as in their therapy.

FUNDING
The work was performed within the scope of State Assignment no. 0521-2019-0007.