Sex-Specific Behavioral Features of the Prenatal Valproic Acid Rat Model of Autism Spectrum Disorder

Abstract

Background/Objectives: Autism is a complex neurodevelopmental disorder characterized by restricted behaviors and impaired social and communication skills. The exact cause of autism remains unknown. One promising animal model for studying autism is the valproic acid rat model. Due to a 1 to 4 bias for males in autism occurrence, most animal model studies investigate only males and neglect females. However, female autism often appears different from that observed in males. Females are said to be less regularly diagnosed because they can "mask" their symptoms. Female autism is as necessary to investigate as male autism. Methods: Fertile adult female Sprague-Dawley rats were impregnated and injected with valproic acid on gestational day 13. Male and female offspring were subjected to behavioral tests to investigate autistic symptoms. Tests included novel object recognition, balance-beam, Y-maze, hole-board, three-chamber, marble burying, olfactory, light/dark and hot plate tests. Results: The tests revealed that VPA-exposed rats had increased anxiety-like behaviors, hyperactivity, and impaired non-verbal communication. However, they did not display repetitive behaviors or cognitive impairments. Notably, male and female rats showed different autism-like traits, with both showing hyperactivity, and males (but not females) additionally showing impaired sociability and increased anxiety. Conclusions: The findings suggest that prenatal exposure to VPA induces autism-like behaviors in both male and female Sprague-Dawley rat offspring. However, males appear more impacted by VPA exposure as evinced by their display of more autism-like symptoms relative to females. This study provides support for including both sexes in all studies modelling autism, as outcomes are seemingly impacted by the sex being observed.

Keywords: anxiety; autism model; repetitive behavior; sociability; valproic acid.

Figure 1

Illustrating the schedule of animal impregnation and treatment to induce prenatal valproic acid exposure in pups; and subsequent sequence and timeline of behavioral testing. SD—Sprague-Dawley rats, VPA—valproic acid, SAL—0.9% saline solution, GD—gestational day, PND—postnatal day.

Figure 2

Sociability. Graphs showing outcomes from the three-chamber test: (a) total time spent in the social chamber (SC), center chamber (CC), and non-social chamber (NSC), indicating significant difference between time spent in SC vs. NSC amongst all groups. (b) Total number of entries into the social and non-social chambers among all groups, indicating significant difference among VPA males and VPA females. (c) Sociability index, indicating no significant difference between the males and females of both the VPA-exposed and the control groups. All data are presented as mean ± standard deviation. VPA: valproic acid. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Non-verbal communication. Graphs showing outcomes from the cotton-tip test, total time spent sniffing (habituation): Time spent sniffing the water scent for males (A) and females (B). The time spent sniffing the lemon scent for males (C) and females (D). The time spent sniffing the urine scent by males (E) and by females (F). Outcomes for dishabituation: (G) shows the time spent sniffing familiar water scent (trial 3) followed by novel lemon scent (trial 1); (H) shows the time spent sniffing novel urine scent (trial 1) following familiar lemon scent (trial 3). Summarized habituation and dishabituation patterns for all groups (I). All data are presented as mean ± standard deviation. VPA: valproic acid, SAL: 0.9% saline solution. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 4

Repetitive/restrictive behavior, lack of interest and anxiety. Outcomes from the hole board test: total number of investigations (head dips into holes) in the hole board test, indicating no significant differences among the groups (a). Total distance travelled while undertaking the hole board test, indicating the VPA-exposed females travelling longer than their male counterparts, and significantly longer than their control female counterparts (b). (c) indicates the average speed of travel, where a significant difference between the females in the VPA-exposed and control groups was seen, along with a significant difference between the males and females of the VPA-exposed group. Graph showing the outcome from the marble-burying test: (d) number of marbles buried by each group, where no significant difference was found. Graphs showing the outcomes of the light/dark test: (e) shows the anxiety index, where a significant difference was found between the male groups. The total time spent immobile in the light zone indicated a significant difference within the male groups (f). (g) indicates the number of entries made into the light zone, indicating no significant difference between any groups. The average speed in the light zone revealed a significant difference between the males of the VPA-exposed and the control males (h), along with a significant difference between males and females within the control group. All data are presented as mean ± standard deviation. VPA—valproic acid, LZ—light zone. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 5

Motor and sensory stimuli. Graphs showing outcomes for the balance beam test: The average speed across the balance beam indicated non-significant differences between all groups (a). The number of foot slips was not significantly different between the groups (b). Graphs showing the outcomes for the hot plate thermal test: (c) shows the time taken to react to thermal stimuli, which indicated no significant difference between the groups. All data was presented as mean ± standard deviation. VPA—valproic acid.

Figure 6

Cognition. Graph showing the outcomes for the novel-object recognition (NOR) test: (a) shows the discrimination index which revealed no significant difference between the four groups. Graph showing the outcome of the y-maze test: (b) shows the percentage of spontaneous alternations, indicating no significant difference between all groups. All data are presented as mean ± standard deviation. VPA—valproic acid.