Animals

Female Wistar rats (Charles River, Arbresle, France), weighing 250±15 g, were mated overnight. The morning when spermatozoa were found was designated as gestational day 1. Pregnant rats were singly housed in Macrolon cages (40 (length) × 26 (width) × 20 (height) cm), under controlled conditions (temperature 20–21 °C, 55–65% relative humidity and 12/12 h light cycle with lights on at 0700 h). Food and water were available ad libitum. On gestational day 12.5, females received a single intraperitoneal injection of either sodium valproate (VPA) or saline (SAL). Newborn litters found up to 1700 h were considered to be born on that day (postnatal day (PND) 0). On PND 1, the litters were culled to eight animals (six males and two females), to reduce the litter size-induced variability in the growth and development of pups during the postnatal period. On PND 21, the pups were weaned and housed in groups of three. The experiments were carried out on the male offspring during infancy (PNDs 9 and 13), adolescence (PND 35) and adulthood (PND 90). One pup per litter from different litters per treatment group was used in each experiment. For every experiment, the exact sample size (n) for each experimental group/condition is indicated in the figure legends. The sample size was based on our previous experiments and power analysis.

The experiments were performed in Wistar rats because previous behavioral studies performed with the VPA preclinical model of autism used this rat strain.15 Furthermore, we focused on behavioral features, resembling the core and associated symptoms of ASD, that are well documented and can be easily studied in the Wistar rat strain, either from an ethological or pharmacological point of view.

The experiments were approved by the Italian Ministry of Health (Rome, Italy) and performed in agreement with the guidelines released by the Italian Ministry of Health (D.L. 26/14) and the European Community Directive 2010/63/EU.

Drugs

VPA (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in saline at a concentration of 250 mg ml−1 and administered at a dose (500 mg kg−1) and time (gestational day 12.5) that have been shown to induce autistic-like behavioral changes in the offspring.41

The anandamide hydrolysis inhibitor URB597 (Sigma-Aldrich, Milan, Italy) was dissolved in 5% Tween 80/5% polyethylene glycol/saline and administered intraperitoneally either 2 h (for the behavioral tests performed at adolescence and adulthood) or 30 min (for the behavioral tests performed at infancy) before testing. Drug dose and pre-treatment intervals were based on literature,29, 35, 37, 38, 42 and on pilot experiments. The solutions were administered in a volume of 2.5 ml kg−1 at infancy, 2 ml kg−1 at adolescence and 1 ml kg−1 at adulthood.

Western blot analysis of phosphorylated and total CB1 cannabinoid receptor

The rats were rapidly decapitated and their brains removed and cut into coronal slices on a cold plate. The prefrontal cortex, dorsal striatum, nucleus accumbens, hippocampus, amygdala and cerebellum were dissected by hand under microscopic control within 2 min. The tissues were stored at −80 °C until use.

Lysates from the tissues were homogenized in a homogenization buffer (0.01 m Tris-HCl, 0.001 m CaCl 2 , 0.15 m NaCl, 0.001 m PMSF (phenylmethylsulfonyl fluoride), pH 7.5) w/v 1:10 (ref. 43) by sonication. An aliquot of homogenate was resuspended in sample buffer (0.250 m Tris-HCl—pH 6.8—containing 20% SDS, 0.001 m PMSF). Lysate samples were boiled for 5 min before loading to the SDS-PAGE (polyacrylamide gel electrophoresis).

Proteins (20 μg) from lysate samples were resolved by 7% SDS-PAGE at 30 mA (constant current) for 120 min and blotted to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Immunoblots were incubated with primary antibodies anti P-CB1 and CB1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Catalog No. sc-17555 and sc-10068), followed by secondary peroxidase-conjugated antibodies (1:3000; Santa Cruz Biotechnology, Catalog No. sc-2020). Immunoreactivity was detected by enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK). The nitrocellulose membrane was stripped with Restore western blot stripping buffer (Pierce Chemical, Rockford, IL, USA) for 15 min at room temperature and re-probed with anti-tubulin (Sigma, Catalog No. T9026) antibody. Bound antibodies to proteins onto nitrocellulose were visualized by using enhanced chemoluminescence detection (GE Healthcare) and exposure to Amersham Hyperfilm ECL (GE Healthcare). The images were analyzed with ImageJ (National Institutes of Health).

Quantitative PCR analysis of fatty acid amide hydrolase and N-acylphosphatidylethanolamines-phospholipid D expression

Two micrograms of the total RNA extracted from the whole rat brain were reverse-transcribed using an oligo-dT primer to prime the reverse transcription and the SuperScript II Reverse Transcriptase system (Life Technologies). The quantitative PCR (qPCR) of fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamines-phospholipid D (NAPE-PLD) was performed using a Rotor-Gene 6000 thermal cycling system (Corbett Research, Mortlake, NSW, Australia) and the Hydra SYBR qPCR Master Mix (BioLab, Rome, Italy) as detection system. The thermal cycling conditions for all the transcripts analyzed were: 95 °C for 2 min and 40 cycles at 95 °C for 10 s and 60 °C for 30 s. PCR primers were the following: FAAH_FW: 5’-IndexTermTGCTGAAGCCTCTGTTTCCT-3’; FAAH_RV: 5’-IndexTermTCTCATGCTGCAGTTTCCAC; NAPE_FW: 5’-IndexTermTCGCTGGGGATACTGGTTAC-3’; NAPE_RV: 5’-IndexTermAAGCTCCAATGGGAATAGCC; β-ACTIN_FW: 5’-IndexTermAGGCCATGTACGTAGCCATCCA-3’; β-ACTIN_RV: 5’-IndexTermTCTCCGGAGTCCATCACAATG-3’. The fold changes of FAAH and NAPE transcripts expression in VPA- vs SAL-treated samples were calculated using the 2–ΔΔCT method.

Reproduction data

Body weights of the dams were taken daily throughout pregnancy and the length of pregnancy was determined. Litter size, male/female ratio, weight gain of pups and postnatal vitality were also measured.

Isolation-induced ultrasonic vocalizations

On PND 9, the pups were individually placed into a Plexiglas arena (30 (length) × 30 (width) × 30 (height) cm), located inside a sound-attenuating and temperature-controlled chamber, with a camera positioned above the arena. The ultrasonic vocalizations (USVs) emitted by the pup were detected for 3 min by an ultrasound microphone (Avisoft Bioacoustics, Glienicke, Germany) sensitive to frequencies between 10 and 200 kHz. Pup axillary temperature was measured before and after the test by a digital thermometer. The USVs were analyzed, both quantitatively and qualitatively, using Avisoft Recorder software (Version 5.1), and classified in six different groups according to number of syllables, frequency modulation and duration:44, 45, 46 flat calls (calls with constant frequency with a maximum variation of ±5 kHz); up/downward (single syllable calls emitted at ±5 kHz with a single frequency modulation); flat high frequency calls (calls with similar constant frequency, but emitted at >75 kHz); syllable calls (calls composed by two or more syllables); complex calls (calls displaying complex frequency modulation); short calls (calls with durations shorter than 5 ms).

Homing behavior

On PND 13, the litter was separated from the dam and kept for 30 min in a temperature-controlled holding cage. Then, each pup was placed into a Plexiglas box whose floor was one-third covered with bedding from the pup’s home cage and two-third with clean bedding. The pup was located at the side of the box covered by clean bedding, and its behavior was videorecorded for 4 min for subsequent analysis. The following parameters were scored using the Observer 3.0 software (Noldus Information Technology, Wageningen, The Netherlands): latency (seconds) to reach the home-cage bedding area; total time (seconds) spent by the pup in the nest bedding area; total number of entries into the nest bedding area.45

Three-chamber test

The apparatus was a rectangular three-chamber box, with two lateral chambers (30 (length) × 35 (width) × 35 (height) cm) connected to a central chamber (15 (length) × 35 (width) × 35 (height) cm). Each lateral chamber contained a small Plexiglas cylindrical cage. The test was performed as previously described.47 Each experimental rat was individually allowed to explore the apparatus for 10 min, and then confined in the central compartment. An unfamiliar stimulus animal was placed into the Plexiglas cage in one chamber of the apparatus, whereas the cage in the other chamber was left empty. Both doors to the side chambers were then opened, allowing the experimental animal to freely explore the apparatus for 10 min. The percent of time spent in social approach (sniffing the stimulus and the cage confining it) was scored using the Observer 3.0 software (Noldus Information Technology).

Social play behavior

Rats were individually habituated to the test cage for 10 min on each of the 2 days before testing, as previously described.37, 42, 48 On the test day, the animals were isolated for 3 h before testing.49, 50, 51 The test consisted of placing VPA- or SAL-exposed rats together with an untreated animal for 15 min. The behavior was assessed for each individual animal of a pair separately using the Observer 3.0 software (Noldus Information Technology). The behaviors of the animals were recorded using a camera with zoom lens, video tape recorder and television monitor.

In rats, a bout of social play behavior starts with one rat soliciting (‘pouncing’) another animal, by attempting to nose or rub the nape of its neck. The animal that is pounced upon can respond in different ways: if the animal fully rotates to its dorsal surface, ‘pinning’ is the result (one animal lying with its dorsal surface on the floor with the other animal standing over it), which is considered the most characteristic posture on social play behavior in rats.52

We determined ‘play responsiveness’ (that is, the percentage of response to play solicitation) as the probability of an animal of being pinned in response to play solicitation (pouncing) by the stimulus partner.42

Elevated plus maze

The apparatus comprised two open and two closed arms (50 (length) × 10 (width) × 40 (height) cm) that extended from a common central platform (10 (length) × 10 (width) cm). The rats were individually placed on the central platform of the maze for 5 min. Each session was recorded with a camera positioned above the apparatus for subsequent behavioral analysis performed using the Observer 3.0 software (Noldus Information Technology). The following parameters were analyzed:53

1 % time spent in the open arms (% TO): (seconds spent on the open arms of the maze/seconds spent on the open+closed arms) × 100; 2 % open-arm entries (% OE): (the number of entries into the open arms of the maze/number of entries into open+closed arms) × 100; 3 Number of closed-arm entries (number of CE).

Hole-board test

The apparatus was a gray square metal table (40 (length) × 40 (width) × 10 (height) cm) with 16 evenly spaced holes (4 cm in diameter), inserted in a Plexiglas arena (40 (length) × 40 (width) × 60 (height) cm). The rats were individually placed in the apparatus and their behavior was observed for 5 min. Dipping behavior was scored by the number of times an animal inserted its head into a hole at least up to the eye level. Each session was recorded with a camera positioned above the apparatus for subsequent behavioral analysis performed using the Observer 3.0 software (Noldus Information Technology).

Statistical analysis

The data are expressed as mean±s.e.m. To assess the effects of the prenatal treatment (VPA or SAL) on the offspring, data were analyzed with Student’s t-tests. Two-way analysis of variance (ANOVA) was used to assess the effects of prenatal and postnatal treatments, using prenatal (VPA or SAL) and postnatal (URB597 or vehicle) treatments as between-subjects factor. Two-way ANOVA was followed by Student's–Newman–Keuls post hoc test where appropriate.

The western blot and qPCR experiments were performed in duplicate and the results were comparable.

Behavioral experiments were scored and analyzed by a trained observer who was unaware of the treatment conditions (Noldus Information Technology). Similarly, biochemical experiments were performed and analyzed in blinded conditions.

Supplementary Figure 1 shows three different timeline diagrams for: (1) the behavioral characterization of the VPA- and SAL-exposed offspring; (2) the behavioral effects of URB597 in the VPA- and SAL-exposed offspring; (3) the western blot and quantitative PCR analyses of the brain of VPA- and SAL-exposed animals.