Behavioral Tasks

We assessed the nest-seeking (NS) response mediated by olfactory discrimination as described previously (Schneider and Przewłocki, 2005) at the postnatal day 10 (P10). All litter (males and females) was evaluated, since sex is very difficult to determine during behavioral tests before P10 and one would have to manipulate the litter, which could introduce a stress component. The apparatus used was a plastic container (30 × 20 × 13 cm) that was divided in thirds and had the lateral sections covered with wood shavings, but leaving a clear uncovered center. One side is filled with the home-cage bedding (nest shavings) and in the other side with sterilized shavings. The pup was placed in the center of the apparatus and the latency to reach the nest shavings and the time to make any choice was registered. The total time of the test was limited to 60 s. In order to maintain the smell of the litter/mother, we did not change the shavings in the home-cage in the 2 days preceding the test.

During this test, the animal behavioral response to direct vibrissae stimulation was observed in P30 animals. All tests and analyses were performed blindly. Since this is a sensory test, prior to testing, animals were familiarized with handling of the experimenter and habituated with the empty housing (57.1 × 39.4 × 15.2 cm) coated with an absorbent pad. To perform the test, the vibrissae are stimulated with a wooden toothpick for three consecutive periods of 5 min (15 min in total) with a 30 s interval between stimulation (Figures 1E,F). Animals were scored according to a scale developed by McNamara et al. (2010), in which freezing, stance and body position, breathing, whisker position, whisking response, evading stimulation, response to stick presentation and grooming are classified from 0 to 2 according to the response (0 = absent/typical, 1 = present/light response and 2 = profound/accentuated response; Supplementary Table S1). The sum of all scores is then calculated. Low scores (0–4) indicate normal responses, in which the animal is calm or indifferent to stimulation. High scores (8–16) indicate abnormal responses to stimulation, in which the animal freezes, shakes, or is aggressive (McNamara et al., 2010).

Animals from the valproic acid (VPA) model of autism demonstrate impaired sensorial behavior, which is counteracted by resveratrol (RSV). (A) Representative illustration from the Nest Seeking (NS) test. RSV administration could not prevent all behavioral alterations in VPA groups, which presented an increasing time to reach the nest (B) and increased latency to make any choice (C). Whilst VPA induced significant decrease in the accuracy of the choice, prenatal RSV treatment prevented this feature (D). (E,F) Representative illustration of the stimulation in the Whisker Nuisance Task (WNT; E) and possible position of whiskers: protracted (curiosity), retracted (avoidance) or not showing any particular response (F). (G) Behavioral analysis of WNT, demonstrating the hyper-responsiveness of VPA animals, a behavior prevent by RSV treatment. Different letters indicate statistically significant differences between groups. Values plotted are Mean ± standard deviation (SD). Statistical analysis: ordinary one-way analysis of variance (ANOVA) followed by Bonferroni.