Animals and treatments
Outbred CrlFcen:CF1 female and male adult mice were obtained from the animal house at the Facultad de Ciencias Exactas y Naturales, University of Buenos Aires (Buenos Aires, Argentina). We chose this outbred strain because (1) it shows reliable intermediate levels of the behaviors analyzed, allowing the detection of increases and decreases in the behavioral parameters evaluated; (2) it has better breeding performance than inbred strains; and (3) it shows no VPA toxicity during pregnancy (neither litter size nor gestation time are affected). All animals were housed in the animal house on a 12:12 light to dark cycle and 18–22 °C temperature, with food and water ad libitum. All animal procedures were performed according to the regulations for the use of laboratory animals of the National Institute of Health (Washington, DC, USA) and approved by the institutional animal care and use committee of the Facultad de Ciencias Exactas y Naturales, University of Buenos Aires (CICUAL Protocol Nr. 6/2). Eight- to 10-week-old male mice were mated with nulliparous 8–10-week-old female mice. Females were controlled every morning, and the day when a vaginal plug was detected was considered the gestational day (GD) 0.5.
On GD12.5, pregnant female mice were injected subcutaneously with either 600 mg/kg of valproic acid sodium salt (VPA; Sigma, St. Louis, MO, USA) in saline solution or with saline solution (SAL), and housed individually. The parturition day was registered as postnatal day 0 (PD0), and the cage bedding was not changed during the first postnatal week to avoid nest and maternal care alterations.
On PD21, male pups prenatally exposed to VPA were weaned in cages containing four-five animals of the same treatment (VPA-VPA animals), or with control male pups (VPA-SAL animals) in cages containing two-three VPA-treated mice with three-two saline-treated mice (SAL-VPA animals). Control animals weaned with other control animals were denominated SAL-SAL animals (Fig. 2a). Mice interacted from PD21 to PD60 in their homecage. Littermates were assigned to different postnatal treatments (X-SAL and X-VPA), and offspring belonging to the same prenatal treatment group from different mothers were mixed at weaning. We performed all the studies on males because female offspring of VPA-injected dams does not show ASD-related behaviors (unpublished data; [17, 20]).
To evaluate sociability at weaning, an independent cohort of 16 VPA and 16 SAL animals was tested for juvenile play at PD21. We used an independent group to avoid interfering with the social enrichment process (postnatal treatment). Non-sibling mice from the same prenatal treatment were matched for similar body weights within 1-g difference. On PD20, one mouse in each pair was marked on its back with black marker to distinguish between animals during testing. On PD21, mice were isolated for 30 min and allowed to habituate to the testing room (10 lx). Each animal was then placed for a 10-min habituation period in the testing arena (floor: 30 cm × 30 cm of black PVC; walls: 30 cm high of black formic). Afterward, the pair of animals was placed again in the testing arena for 30 min, allowing them to interact freely while they were filmed. Testing was performed during the 2 h prior to the start of the dark phase (18:00 to 20:00 h) to maximize activity.
Behaviors were scored separately for each mouse in the pair. All behaviors were scored manually using keys and the video-tracking system ANY-maze (Stoelting, IL, USA) by an experimenter (M.C.) blind to treatment. We evaluated play solicitation (crawling and approaching events), investigative behaviors (events of anogenital sniffing, nose to nose sniffing or following), affiliative behaviors (time spent sitting side by side or in social grooming), and non-social behaviors (time spent exploring, self-grooming or sitting alone) as previously reported [21, 22].
Adult behavioral testing
All adult behavioral tests were performed during the light period (between 10:00 and 17:00 h), with the exception of the Y-maze test that was performed during the 2 h before the start of the dark phase (18:00 to 20:00 h) to maximize exploration. For adult behavior, data from three independent cohorts are presented. Each cohort consisted of five or six litters per prenatal treatment. Mice were 8 weeks old at the beginning of testing, and all tests were separated by 1-week intervals to reduce any inter-test effect. Tests were performed in the order listed below (Fig. 2b). Mice were tested in a room next to the holding room. Previous to the beginning of each test, all animals were habituated to the illumination in the testing room for 30 min. After testing, each mouse was identified and placed in a holding cage until all animals from a cage had been tested. All the behavioral testing and manual scoring were performed by two experimenters (M.C. and N.K.) blind to treatment groups.
Social interaction test
The social interaction test was performed as previously described . Briefly, animals were exposed to a 40 cm × 15 cm black rectangular arena divided in three interconnected chambers and placed under dim light (10 lx). A clear Plexiglass cylinder (7.5 cm of diameter, with several holes to allow for auditory, visual, and olfactory investigation between a test and stimulus mouse) was placed in each side of the compartment at the beginning of the test. Prior to the start of each test, one of the end chambers was randomly designated as the “non-social side” and the other as the “social side”. Animals were placed in the central compartment and allowed to explore for 5 min (habituation). Then, an unfamiliar, young (3 weeks of age) CF1 male mouse (social stimulus) was placed in one of the cylinders (social side), and an object (white, plastic 3-cm-tall cylinder) was placed in the other cylinder (non-social side). Social interaction was evaluated during a 10-min period. The time the subject spent sniffing the social stimulus or the non-social stimulus (nose inside a hole of the cylinder) was recorded manually using a key in the video-tracking system ANY-maze (Stoelting, IL, USA). The entire apparatus, including the cylinders, was cleaned with a 20% ethanol solution between tests to eliminate odors. The apparatus floor was covered with bedding to reduce the stress and was replaced after each test.
Elevated plus maze test
The elevated plus maze test (EPM) was performed as previously described . The apparatus consisted of two open and two closed arms (open arms: 30 cm × 5 cm, 100 lx, surrounded by a 0.5-cm high border; closed arms: 30 cm × 5 cm, 43 lx, surrounded by 19 cm high walls), both elevated 50 cm above the floor. The walls and the floor were made of black PVC. Mice were placed into the central platform of the maze (5 cm × 5 cm, 100 lx) facing towards an open arm and allowed to explore the maze for 5 min. Locomotion data were collected by a video-tracking system (ANY-maze, Stoelting). Measured locomotor parameters were time spent in open arms, time in closed arms, time in the central platform, traveled distance in both open and closed arms, % distance in the open arms, and total traveled distance. Ethological parameters were scored manually during each session: number of rearings, grooming time, number of head dippings, and number of protected head dippings—which consisted in head dippings specifically performed in the center of the maze. The entire apparatus was cleaned with a 20% ethanol solution between tests to eliminate odors. For statistical analysis of this test, we selected uncorrelated variables (|r| < 0.7): the time spent in the open arms, the time spent in the closed arms, the time spent in the central platform, the distance traveled in closed arms, number of rearings, grooming time and number of both head dippings and protected head dippings.
Open field test
The open field test (OF) was performed as previously described . Mice were placed in the arena (floor: 45 cm × 45 cm of black PVC; walls: 30 cm high of black formic; 100 lx) for 30 min. Animals were initially placed along one side of the arena, and the center region was defined as the central 23 cm × 23 cm area. Locomotion data were collected by a video-tracking system (ANY-maze, Stoelting). Measured behavioral parameters were time spent in the center, time spent in the border, distance traveled in the center, distance traveled in the border, % distance in the center, and total distance traveled. Grooming time and the number of rearings were scored manually during each session. For statistical analysis of this test, we selected uncorrelated variables (|r| < 0.7): total distance traveled, time spent in the center, time grooming, and number of rearings.
The olfactory habituation/dishabituation test
Olfactory discrimination was investigated using a slightly modified habituation/dishabituation protocol . Each mouse was isolated in the testing cage (floor: 27 cm × 16 cm, height 12 cm) and habituated to a non-odorant cotton tip for 30 min. Then, animals were given three 2-min presentations of each odor: water, two non-social odors, and two social odors. Non-social odors were imitation vanilla and banana extracts, and social odors were male and female cage swipes. Odors were presented using a cotton tip with a 1-min inter-trial interval, which is the amount of time needed to change the cotton tip. The testing room was illuminated with 50 lx. The time spent sniffing the cotton tip was recorded manually using a key of the ANY-maze software (Stoelting). Animals that did not explore any of the presentations of an odor were excluded from the analysis (3 SAL-VPA, 2 VPA-SAL, 3 VPA-VPA).
Novel object recognition test
The novel object recognition test (NOR) was modified from a previous study . Animals were first habituated to the experimental box (floor: 30 cm × 30 cm of black PVC; walls: 30-cm high of black formic; 10 lx) during 5 min and then placed individually in a holding cage during 5 min. After that, animals were placed again in the experimental box containing two identical objects, allowing them to explore the objects during a 5-min trial (training session). Animals were placed again in their cage for a 5-min period, to generate new short-term memories. Finally, animals were placed again in the experimental box, allowing them to explore two different objects (testing session) for 5 min. One of the objects was identical to those explored during the training session (familiar object), and the other was a different object (novel object). The objects were presented in the same locations as in the training session. The location of the novel object was randomly assigned (left or right) for each animal to avoid place preference during the testing session. The objects used were a small transparent glass Erlenmeyer or a 1.5-ml Eppendorf tube of similar size. The time spent sniffing each object was scored manually using keys in the ANY-maze software (Stoelting). The objects and the experimental box were cleaned with 20% ethanol solution between animals to eliminate olfactory cues. One VPA-SAL animal was removed from the analysis because it did not explore any of the objects during the test.
Mice were scored for spontaneous grooming behaviors as described earlier [4, 21]. Each mouse was placed individually into a clear Plexiglass cylinder (20 cm high, 5.5 cm wide), illuminated at 10 lx. Before testing, animals were habituated to the test cylinder in the testing room, for 1 h in two consecutive days. On the testing day, each mouse was scored during 10 min for cumulative time spent grooming all body regions, using a key of the ANY-maze software (Stoelting).
Light-dark box test
The light-dark (LD) test was performed as previously described . A 45 cm × 45 cm arena was divided in half with an inverted black box (lit side: 100 lx; dark side: 1 lx). Animals were able to cross from one compartment to the other through a small hole in the wall (12-cm height, 8-cm width). Each mouse was placed under the hole facing the illuminated side of the box and tracked for 5 min using the ANY-maze software (Stoelting). Behavioral parameters analyzed were time spent in the lit compartment and distance traveled in the lit compartment.
Tail suspension test
The tail suspension test (TST) was performed as previously described . Animals were suspended in the air using adhesive tape wrapped around the subject’s tail (about 4/5 from the base) and fixed to a wire at 25 cm of height from a wooden surface. This test was performed under 50 lx. The immobility time was scored during 5 min, using a key of the ANY-maze software (Stoelting). One animal was removed from this test because it learned to climb its tail.
Forced swimming test
The forced swimming test (FST) was performed as previously described . Animals were gently placed in a beaker glass (15 cm in diameter and 25 cm in height), filled with 14 cm of water at 25 °C and illuminated with 50 lx. Immobility time was scored during 6 min using a key in the ANY-maze software (Stoelting). At the end of the test, animals were dried with a paper towel and placed into a holding cage.
Y maze test
The Y-maze was constructed of Plexiglas with three identical arms (42-cm long, 12-cm tall walls, 30 lx), and visual cues were located on the walls outside the maze. The maze floor was made of black PVC. One of the three arms was arbitrarily designated as the start and exploration was recorded using the ANY-maze software (Stoelting). Total locomotion and percentage of alternation are reported. Percentage of alternation was calculated as (alternations × 100)/(Total arm visits− 2), where an alternation was considered as consecutively visiting the three arms. One VPA-SAL animal was excluded because it performed less than fifteen alternations.
Pre-clinical PET imaging
Images were acquired using a preclinical PET TriFoil LabPET 4 with LYSO and GYSO crystals and 1536 APD detectors groups. Approximated spatial resolution FWHM = 1.2 mm (full width at half maximum), 3.7 cm axial and 11 cm trans-axial FOV (field of view).
Pre-clinical PET imaging was performed on 15–17 animals for each experimental group, from two independent cohorts. Animals were starved during 18 h and then injected with 25 μCi/gr of [18F]-FDG i.p. and left undisturbed in an individual temperature-controlled (29 °C) cage for 30 min during radiopharmaceutical incorporation. Mice were then anesthetized using a mixture of isoflurane and O2 (inhalation, 4.5% induction and 1.5% maintenance dose) and maintained in a warm table (35 °C) during the acquisition.
Acquisition and reconstruction setup
Each subject was acquired for 12 min using list-mode acquisition. Images were reconstructed using an OSEM 3D algorithm with 30 iterations, to maximize SNR (signal-to-noise ratio). If motion was detected during acquisition, a dynamic reconstruction was performed in order to correct it using SPM5 on MATLAB® realign algorithm.
Image spatial processing
The images were confined to a bounding-box that only includes the brain. A normal subject-based template was created in order to have an anatomic reference for realignment and normalization. All subjects were smoothed using an isotropic Gaussian kernel with 1 mm FWHM. All images were co-registered and normalized to this template using SPM5 on MATLAB®, according to these parameters: Normalized mutual information as objective function and 7-mm smoothing histogram for rigid co-registration; and affine regularization to the averaged template size, no-smooth and 2–0.1 mm of separation for the non-rigid normalization. Previous to intensity normalization and statistical analysis, a brain masking avoiding Harderiand glands was applied to all subjects since the uptake in these glands can significantly modify the intensity normalization values.
Image statistical analysis
All subject groups were analyzed as a full-factorial ANOVA test using SPM5 on MATLAB®. Intensity normalization was considered as a regressor variable for each factor using grand mean scaling (ANCOVA). Global calculation of individual means was calculated over each masked brain. Parametric statistical images were calculated for all possible experimental group contrasts. In order to correct for multiple comparisons, false discovery rate (FDR) approach was applied using SPM5 (p value FDR: 0.05). In order to have an accurate anatomical reference, all results of statistical differences where co-registered with an MRI atlas . Spatial transformation was applied to the MRI atlas to correct for the differences between mice strains and methodological animal handling.
An independent group of animals (N = 4–5 animals per group) was used for catecholamine determination as previously described . Mice were sacrificed via cervical dislocation and punches of piriform cortex were taken and quickly frozen and kept at − 80 °C. Tissue was homogenized in 1 ml of 0.3 M perchloric acid, centrifuged for 15 min at 3000 g at 4 °C and then frozen at 80 °C. Levels of DA, DOPAC, 5-HT, and 5-HIAA were measured by high pressure liquid chromatography coupled to electrochemical detection (HPLC-EC) using a Phenomenex Luna 5 μm, C18, 250 mm × 4.60 mm column (Phenomenex, Torrance, CA, USA) and LC-4C electrochemical detector with glassy carbon electrode (BAS). The working electrode was set at + 0.65 V versus an Ag/AgCl reference electrode. The mobile phase contained 0.76 M NaH2PO4·H2O, 0.5 mM EDTA, 1.2 mM 1-octane sulfonic acid, and 5% acetonitrile; pH was adjusted to 3.0. Catecholamine quantification was referred to total protein content, measured using the NanoDrop 1000 Spectrophotometer (Thermo Scientific).
Animals from the second cohort of behavioral testing were randomly selected (7 SAL-SAL, 6 SAL-VPA, 5 VPA-SAL, and 6 VPA-VPA). Two weeks after the last behavioral test, mice were deeply anesthetized with ketamine/xilacine and transcardially perfused with 4% paraformaldehide (PFA). Brains were post-fixed for 4 h in 4% PFA and criopreserved in 30% sucrose. 0.035-mm thick coronal sections were prepared on a cryostat (Leica, Wetzlar, Germany), and cFos immunohistochemistry was performed as previously described . We used the primary antibody rabbit anti-c-Fos (1:1000 in blocking solution; EMD Millipore, Burlington, MA, USA), the secondary antibody biotin-SP-conjugated donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA) and the ABC kit (Vector Laboratories, Burlingame, CA, USA). Sections were then stained with cresyl violet (5 mg/ml in 0.6% acetic acid). Positive cells in the layer 2 of the piriform cortex were counted on × 400 magnification in a light-field microscope (CX31: Olympus, Buenos Aires, Argentina) by a researcher (N.S.) blinded to treatments. Total c-Fos-positive nuclei are presented normalized by the volume of the layer 2. To this end, each counted section was photographed under × 40 magnification using a digital camera (Infinity 1; Lumera Corporation, Ottawa, ON, Canada) attached to the microscope. The volume of layer 2 in each image was determined using ImageJ : the area in pixels was transformed to square millimeters and multiplied by the thickness of the section (0.035 mm). The limit between the anterior and posterior parts of the piriform cortex was determined with the aid of the mouse brain atlas  and set to the bregma position when the posterior branch of the anterior commissure is visible (AP − 0.26 mm).
Sample sizes were estimated based on similar, previously conducted studies. No statistical methods were used to predetermine sample size. Group comparisons were done using unpaired Student’s t test, or two- or three-way ANOVAs for normally distributed data (confirmed by the D’Agostino & Pearson omnibus normality test), with litter as a nested factor. The statistical designs and outcomes are outlined in Additional file 1: Tables S1-S4. Whenever appropriate, Tukey’s multiple comparisons test was used for post hoc comparisons. All tests were performed with the Statistica software (version 8, StatSoft Inc., Tulsa, OK, USA), and statistical significance was assumed where p < 0.05. We only used statistical methods to correct for multiple testing in the PET studies. Animal exclusions in behavioral tests are specified in each behavioral test description.