Bauman Supplemental Material
Supplemental Material
Methods
Western Blots and Antibody Purification
All protocols were approved by the University of California, Davis Institutional Review Board. The IgG-ASD group was comprised of simplex mothers i.e., they had only one child on the autism spectrum. The mothers mean age at the time of the child’s birth was 31 years. At the time of the blood draw, none of the mothers had any known autoimmune disorders. All serum samples were screened for the presence of antibodies directed against fetal brain tissue using Western blots. 300µg of prepared fetal Rhesus macaque brain protein (FRB) was separated under reducing conditions in a prep well 4-12% gradient SDS-PAGE mini-gel (Invitrogen, Carlsbad, CA) and transferred electrophoretically to 0.2µm pore-size nitrocellulose. MagicMark molecular weight marker (Invitrogen) was loaded in the single marker lane allowing chemilluminescent visualization of marker bands. Blots were stained with Ponceau S (MP Biomedicals, Solon, Ohio) to verify uniform protein loading, migration and transfer. The nitrocellulose membrane was then cut into 3mm wide strips and probed with maternal plasma diluted 1:400. After washing, strips were incubated with 1:20,000 diluted horseradish peroxidase conjugated goat anti-human IgG (Invitrogen). The strips were then washed, incubated with SuperSignal West Chemilluminescent Substrate (Pierce) and aligned on a glass plate for imaging. Chemilluminescent images were acquired with a FluorChem 8900 imager using AlphaEaseFC software (Protein Simple). IgG was purified and pooled from 4 maternal samples derived from mothers of autistic children that demonstrated reactivity to the 37/73 kDa band pattern specific for autism. IgG from the 5 control samples did not show the reactivity to the fetal brain extracts and were also pooled. Each collection of pooled sera was diluted with Immunopure (G) IgG binding buffer (Pierce Biotechnology, Inc., Rockford, IL) and IgG antibodies were purified on Ultralink Affinity Pack immobilized protein G columns (Pierce Biotechnology, Inc, Rockford, IL).Purified IgG was then eluted from columns with Immunopure IgG elution buffer (Pierce Biotechnology, Inc., Rockford, IL). This process resulted in approximately 3.3 mg of purified IgG per 1 ml serum. The purified serum was screened for the presence of HIV and Hepatitis B and C and finally sterile filtered with a 0.2 µm filter prior to injection.
Subject Selection
Twenty-four pregnant rhesus macaques (Macaca mulatta) were selected from the colony timed-mating program, which provides accurate estimates of conception within +/- 2 days. Candidate females were between six and sixteen years of age (mean age = 11 years), had been reared in a naturalistic social group, demonstrated species-typical behaviors and had a successful history of raising offspring. Pregnancy was confirmed at approximately 20 days of gestation and was followed by blood assays to detect fetal DNA for sex determination (1). The dams were randomly assigned to experimental conditions (balancing for age and sex of the fetus). There were no group differences in weight and age of the gestating females. Due to limited availability of male fetuses, the IgG-CON group included more female fetuses (n=5) than males (n=3). One of the male IgG-CON pregnancies spontaneously aborted and was subsequently replaced with an untreated mother-infant pair. Pregnancies were monitored via ultrasound on gestational days 30, 40, 100 and 150. Twenty-one different sires were used, resulting in the three pair of offspring that were half-siblings. Each member of these sibling pairs was assigned to different treatment groups.
Offspring and Rearing Conditions
Behavioral data collected between 0-6 month of age include IgG-ASD37/73kDa (n = 8), IgG-Control (n = 7) and Untreated Controls (n = 9). Social group interactions took place in a large chain link enclosure (2.13 m W x 3.35 m D x 2.44 m H) equipped with perches and enrichment devices. The eight socialization groups were each composed of one mother/infant pair from each of the three experimental groups (IgG-ASD37/73kDa, IgG-CON and Untreated). The fourth mother/infant pair in each group was from a separate cohort of animals exposed to the heterogeneous autism-associated IgG and will be the focus of future publications. Thus each group was composed of two monkeys treated with autism-associated antibodies and two control offspring. The age range between the youngest and oldest infant within each group was approximately one month. At least one female and one male offspring were included in each social group. The dams demonstrated species-typical behavior during the formation of the rearing groups and quickly established a dominance hierarchy. Dominance assessments of the dams within each socialization cohort indicated that the mean ranking within each rearing group (1-4) groups did not significantly differ across the experimental groups (IgG-ASD37/73kDa = 3.0/4, IgG-CON = 2.14/4, Untreated control = 2.11/4). When the youngest subject within a rearing group reached six months of age, the infants were permanently separated from their mothers, but otherwise continued to experience the same housing and group socialization in the absence of their mothers. At this time, a new adult female was added to each socialization cohort to provide continued exemplars of adult female social behavior. After 6 months of age two untreated control animals were removed from the project due to poor weight gain/failure to thrive and were replaced with two age-matched colony animals. The replacement animals were accepted into their rearing groups; however, given that they had experienced a different rearing environment for the first six months of life (mother reared with limited peer access), we did not include these subjects in our focal observations. Behavioral data collected after 6 months of age include IgG-ASD37/73kDa (n = 8), IgG-Control (n = 7) and Untreated Controls (n = 7). The final group composition is shown in Table 1. The intermittent group socialization schedule continued until the monkeys reached approximately 18 months of age. At that time they became permanently socially housed (24 hours per day) with their original socialization cohort and three additional cohorts (16 juvenile animals) in a 2.13 m W x 3.35 m D x 2.44 m H chain link enclosure.
MRI Acquisition
MRIs from the male IgG-ASD37/73kDa offspring (n=4) were compared to a combined control group consisting of male IgG-CON (n=2) and archived MRI data from an existing library of outdoor colony animals that were matched for age, sex and weight parameters (n=5). The indoor, untreated controls (n=4) were not scanned at the first 5 time points due to cost restrictions. However, to ensure that the differences in brain volume were not attributable to unanticipated environmental factors affecting the archived MRI data from field cage subjects, we also collected MRI data from available indoor, untreated male controls animals at the final MRI time point (2 years of age). The brain volume measures of the male indoor, untreated controls were consistent with the volumes of the controls (IgG-CON and MRI archives) used in the longitudinal data set. Significant findings were unaltered with the addition of the four indoor, untreated animals, therefore data in Figure 6 and Table 6 are presented using the same control group (n=7) that was used for the longitudinal model.
Infants were relocated from their respective home enclosures to temporary indoor housing the day prior to testing. On the day of MRI scanning, the mothers were sedated using ketamine (7 – 8 mg/kg, IM) and the infants were transferred to an incubator. For MRI scanning after weaning, infants were fasted a minimum of two hours prior to sedation. They were transported from the CNPRC to the Imaging Research Center (IRC). At the IRC, infants were sedated with ketamine (1mg/kg IM) in preparation for placement of an indwelling intravenous (IV) catheter (22 - 24 gauge) and intubation with an endotracheal tube (2.0 – 2.5 mm uncuffed, 3.0 – 3.5 mm cuffed). Infants were anesthetized with propofol (2 ml/kg/hr IV). Intravenous saline was administered throughout the scanning session. Heart rate and oxygen saturation were monitored in the control room of the scanner suite on an Invivo Omni-Trak 3150 MRI Vital Signs Monitor (Medco, Boise, Id). Based on this information, anesthetic levels could be increased or decreased remotely using a Continuum Wireless Two Pump configuration, MRI anesthesia pumps (MEDRAD, Indianola, PA). The infant was also monitored by video camera for the duration of the scan. The infant was positioned supine on the scanner bed and the head was centered in the head coil of the scanner. Body temperature was maintained by surrounding the animal with heated saline packs and blankets. Oxygen was delivered around the nose and mouth at a rate of 1.0 -2.0L/hour to maintain oxygen saturation. After completion of the scans, propofol delivery was stopped. The total time of sedation ranged from 60 to 90 minutes. During recovery from sedation, the infants were given subcutaneous saline with 5% dextrose in order to maintain hydration and normal blood sugar levels. Recovered infants were also provided access to glucose-enriched water in their incubators. Infants were transported back to the CNPRC following the scan. The younger animals were reunited with their mothers in their home enclosures; the older animals were returned directly to their home enclosures. All mothers immediately accepted their infants on each of the reunions.
Image Processing and Analysis
Images were acquired on a 1.5T GE Signa MRI system. The protocol includes a T1-weighted SPGR (TR 27 ms, TE 6 ms, Flip Angle 30, matrix 256 x 256, FOV 160mm, slice thickness .7mm). Total brain volume (TBV) was evaluated by a single rater who had achieved an intra-rater reliability (ICC) above 0.99 and was blind to the experimental groups. TBV was manually delineated on T1-weighted images using Analyze 8.1 software (Biomedical Imaging Resource, Rochester, Minnesota). Images were first resliced to yield cubic .625mm voxels and were then aligned along the AC-PC axis. Initial skull stripping of the brain was generated using Analyze's object extraction tool. The resulting segmentation was then overlaid onto the AC-PC-aligned scan and refined manually using the Region of Interest tool. The TBV protocol included the cerebrum, cerebellum, brainstem (subsected along a plane traversing the foramen magnum) and ventricles; the protocol excluded spinal cord, major nerves, blood vessels, and dura. TBV was analyzed at all ages in the same manner, i.e. manual tracing of the brain surface.
The rostral extent of the frontal lobe was marked by the frontal pole. The caudal boundary was delimited by the central sulcus on the lateral, dorsal, and dorsomedial brain surface and the cingulate sulcus on the medial and ventral surface. The insula, internal capsule, and striatum were excluded. The rostral, caudal, and dorsal boundaries of the cingulate cortex were marked by the cingulate sulcus; the ventral boundary was defined by the corpus callosum and the calcarine sulcus. The rostral boundary of the parietal lobe comprised the caudal boundary of the frontal lobe. The caudal parietal boundary was defined as the lunate sulcus, dorsolaterally, the inferior occipital sulcus, ventrolaterally, and the parietoccipital sulcus, medially. The ventral border was marked by the lateral sulcus, laterally, and the cingulate sulcus, medially. The rostral extent of the temporal lobe was marked by the temporal pole. The caudal boundary on the lateral surface was marked by the junction of the superior temporal sulcus and lateral sulcus, superiorly, and by the first appearance of the inferior occipital sulcus (ios), inferiorly. Cortex posterior to the first appearance of the ios was designated as occipital. The caudal boundary on the ventral temporal surface began at the termination of the hippocampus, ventromedially, and progressed posteriorly and laterally along the occipitotemporal sulcus until the first appearance of the ios on the lateral surface. The dorsal boundary was defined by the lateral sulcus. The amygdala, hippocampus, and entorhinal cortex (cortex medial to the rhinal sulcus) were excluded from the temporal lobe. The rostral boundary of the occipital lobe comprised the caudal boundary of the parietal and temporal lobes. The caudal limit of the occipital lobe was the occipital pole. Lobe specific white matter volumes were obtained by first segmenting cortical white matter for each subject using the high-dimensional warping procedure and then masking this segmentation with each total lobe segmentation. Grey matter volumes were obtained by subtracting white matter from total lobe volumes.
Behavioral Data Collection
All data were collected using the Observer software (Noldus, Sterling, VA) by trained observers demonstrating an inter-observer reliability > 85% (agreements/ [agreements + disagreements] X 100). Each infant received a dye mark, allowing the observers to score behaviors while remaining blind to their experimental condition. Our behavioral assays are designed to detect changes in macaque social and communication development and screen for the presence of repetitive behaviors and restricted interests (2-5). We also include a broad array of additional biobehavioral assays to quantify other aspects of development (i.e., neonatal maturation, endocrine responses, etc.) that may contribute to behavioral pathologies.
1 Week Neonatal motor/reflex development. Neuromotor reflexes, behavioral maturation and attention processes were evaluated at 1 week of age with a standardized test battery modified from published macaque neonatal evaluations (6, 7). All animals were rated on a three point scale (2 = reflex well developed, 1 = reflex partially developed, 0 = reflex absent) for measures of: 1. Visual orientation (i.e., focusing on and following a pen light), 2. Motor maturity (i.e., head posture in prone and supine positions, coordination, maturity spontaneous locomotor pattern), 3. Reflexes (i.e., placing, moro, righting and rooting reflexes), and 4. State control (i.e., agitation, consolability).
3 Month Biobehavioral Assessment (BBA). Infants were temporarily separated from their mothers for a 25 hour biobehavioral assessment between the ages of 90 and 115 days. The BBA is a colony wide assessment of infant macaques at the CNPRC that includes assays of health, behavior, temperament, adrenal regulation, responses to threatening (i.e., human intruder), social (i.e. videos and pictures of conspecifics) and nonsocial (i.e., novel objects) stimuli. Detailed descriptions of the complete BBA assessment battery are available in previous publications (8-11). Following relocation to a temporary holding cage, blood samples were drawn via femoral venipuncture at four time points during the testing period, in order to assess the infants' response to relocation and separation from peers and to evaluate HPA axis regulation. Infants were awake and not sedated during venipuncture. Sample 1 (1.0ml) was drawn at approximately 11:00am (2 hours after removal from the mother). Sample 2 (0.5ml) was drawn after completion of the Human Intruder test (see below) at 16:00hr. Immediately after the Sample 2 venipuncture, each monkey was given an injection of 500µg/kg dexamethasone intramuscularly. Sample 3 (0.5ml) was taken following completion of Holding Cage observations on Day 2 at 08:30hr, immediately after which subjects were administered 2.5IU ACTH. The final sample (0.5ml) was drawn 30min after ACTH was given. Blood was drawn into unheparinized syringes and was immediately transferred to EDTA tubes, which were subsequently centrifuged at 1,000g for 10min at 4°C. Plasma was decanted into microtubes for storage at −80°C until assayed for cortisol concentration by RIA (Diagnostic Products Corp., Los Angeles, CA).
Human Intruder Paradigm. At 1, 3 and 6 months of age response to threat was evaluated using a modified version of the human intruder paradigm (12). Infants were tested in a room in which they could not hear or see conspecifics. Testing took place in a standard macaque indoor housing cage. Infants were left alone in the test cage for one-minute before testing commenced. After the one-minute baseline period had expired, two experimenters (behavioral observer and the human intruder) entered the room. The same human intruder (an adult female) was used for each infant at each time point. The human intruder stood in each presentation condition for one minute. For the first trial, the human intruder positioned herself three feet in front of the test cage, and presented her left profile to the infant (profile-far). At the end of the first minute, the human intruder took one step sideways toward the test cage and positioned herself approximately one foot from the front of the test cage, while still holding the profile position (profile-near). Next, the human intruder returned to the position three feet away and turned to face the infant and established direct eye contact with the infant (stare-far). Finally, the human intruder took one step forward while maintaining eye contact (stare-near). Behaviors commonly elicited by rhesus monkeys during this paradigm were assessed (Table S1). Data was collected live during each one-minute posture presentation via one-zero sampling at 10-second intervals. The maximum value per behavior per trial was six and the minimum value zero.