2.1. Antibody acquisition
Human sera from 21 mothers of at least one child with autism and one or more additional children with autism spectrum disorders were purchased from the Autism Genetic Resource Exchange (Mothers of autistic children—MAC IgG). Human sera from 7 mothers of multiple typically developing children were collected locally (Mothers of typically developing children—MTDC IgG).
2.2. Western blots
All serum samples were screened for the presence of antibodies directed against fetal brain tissue using Western blots. Human fetal brain protein medley (300 μg/gel; Clontech Laboratories, Mountain View, CA) or neonatal monkey brain protein (acquired from the California National Primate Research Center) was prepared according to the protocol used for the human fetal brain as provided by Clontech Laboratories. The monkey brain protein extract was similarly used at 300 μg/gel. Protein samples were separated on a 4–15% gradient reducing gel using SDS–PAGE electrophoresis and transferred onto nitrocellulose paper. Antibody reactivity to the fetal brain extracts was then analyzed for all samples individually by Western blotting. The blots were incubated for 3 hin a 0.1Mphosphate buffered saline (PBS,pH7.4) solution containing serum from the above samples at a dilution of 1:400, with 5% milk and 0.3% Triton X-100. Following a series of rinses, the extracts were incubated for 1 h with a goat anti-human IgG peroxidase-conjugated antibody and visualized using chemiluminescence with a Fluorchem 8900 imager (Alpha Innotech, San Leandro, CA). IgG was purified and pooled from 12 maternal samples derived from mothers of autistic children that demonstrated one or more bands to human fetal brain proteins which were absent in serum from control mothers. IgG from the 7 control samples did not show the reactivity to the brain extracts and were also pooled.
2.3. Purification of IgG antibodies
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.
2.4. Subjects and living conditions
All procedures carried out on animal subjects were approved by the UC Davis Institutional Animal Care and Use Committee. The subjects for this study were 13 naturally born rhesus monkeys (Macaca mulatta). The mothers of the subjects were selected based on their proven birth record and on their high quality maternal behavior and randomly assigned to one of three conditions.
Pregnant rhesus monkeys (n = 4) were exposed to purified IgG (the only antibodies that cross the placental barrier) pooled from the serum of a subset of mothers of children with ASD that could be distinguished by the presence of reactivity to fetal brain proteins by Western blot (). A separate group of pregnant monkeys (n = 4) were exposed to purified IgG pooled from the serum of mothers of typically developing children. In all cases, 15–20 mg of purified IgG diluted in 5 ml of sterile saline was delivered intravenously on three separate occasions: days 27, 41, and 55 of gestation. Rhesus monkey gestation is approximately 165 days. Additional pregnant rhesus monkeys (n = 5) comprised an untreated control group.
Fig. 1 Western blot demonstrating reactivity of maternal serum against both human (HU) and monkey (MO) fetal brain proteins. Depicted are two representative samples from the mothers of multiple children with autism (AU) demonstrating the typical patterns of (more ...)
All infants were born and raised in standard home cages (61 × 66 × 81 cm). Each mother–infant pair was assigned to one of three socialization cohorts consisting of 6 mother–infant pairs and 1 adult male. There were 2 male and4 female infants in each cohort. Mother–infant pairs from each study group were distributed across the socialization cohorts so that there was at least 1 MAC IgG treated monkey, 1 MTDC IgG control monkey and 1 untreated control monkey in each cohort. In addition to the 13monkeys in this study, the socialization cohorts included 5 other mother–infant pairs that were not part of this study. Offspring were thus raised with their mothers and were socialized for 3 h daily with 5 other mother–infant pairs and 1 adult male in large group cages (2.13 × 3.35 × 2.44 m). Formal assessments of dominance within each socialization cohort indicated that the average dominance rankings of the mothers from each study group were roughly equivalent (MAC IgG treated = 4.25/6, MTDC IgG control = 3/6, Untreated control = 4/6). When the youngest subject within each socialization cohort reached ~6 months of age, all of the infants within that cohort were permanently separated from their mothers (weaned), a standard practice at the primate center, and permanently moved to large group cages. The adult males remained with each cohort and a novel adult female was added to each cohort for a period of 1 month following weaning to promote group stability.
As anticipated, behavioral data from the control IgG monkeys and the untreated control monkeys were very similar and did not approach significance. These two groups were therefore pooled to form a single control group (n = 9) for comparisons with the MAC IgG treated monkeys.
2.5. Behavioral observations
Behavioral data were collected with The Observer software (Noldus, Sterling, VA; (Noldus, 1991) by three trained observers, demonstrating an inter-observer reliability of at least 85% (agreements/[agreements + disagreements] × 100). All observers had previous experience using Noldus software for the collection of rhesus macaque behavioral data (9 months, 2 years and 3 years, respectively). Reliability was evaluated using the most experienced behavioral observer as the reliability standard. Inter-rater reliability was attained for each behavioral task, with observers meeting at least 85% reliability over two consecutive days. All observers were blind to the experimental status of all subjects.
2.6. Preweaning social group and dyad observations
Beginning at approximately one month of age, the infants were observed in their socialization cohorts. Each subject was observed for 5 min twice per week during weeks in which no other testing took place. One month prior to weaning, two mother–infant pairs were placed together in the large testing enclosures for a 20 min tetradic social interaction (Bauman et al., 2004a
). Each mother–infant pair was observed in pairings with every other mother–infant pair in its own socialization cohort. For both the social group and mother–infant pairings, trained observers blind to the condition of the animals used the Observer software program on laptop computers to score the behavior of each subject in real time during 5 min focused observations (ethograms of behaviors recorded in the social groups and the familiar dyads have been provided as supplementary material
2.6.1. Mother preference test
On the first 4 days immediately following weaning, each infant was observed in a test designed to evaluate one aspect of mother–infant attachment, the infant’s preference for its mother over another familiar adult female (Bauman et al., 2004a
). Five daily 2 min trials were conducted, with each trial consisting of a choice between the infant’s mother and one of the five other adult females from the infant’s socialization group (the stimulus female). A different stimulus female was used for each trial in a predetermined pseudo-random order. Before each trial, the test subject was hand-caught by a technician and placed in a plastic release box in the center of an unfamiliar chain link testing enclosure (5.56 × 1.91 × 2.13 m). The front of the subject’s release box was transparent and the remaining three sides were opaque allowing the test subject to view only the observers until released. The subject’s mother was placed in one of two holding cages, located at either end of the testing enclosure, and the other female was placed in the opposite holding cage (holding cage assignments were balanced across trials). For the safety of the infant, transparent plastic panels prevented physical contact between the test subject and the adult females. At the onset of the trial, the subject’s release box and the opaque panels in front of the holding cages were raised simultaneously, allowing the test subject to freely move around the testing enclosure and see both its mother and the other female. During each 2 min trial, trained observers recorded the behaviors exhibited by the test subject, including which adult was first approached (scored when the subject moved within a 1 m half-circle painted on the floor in front of each holding cage. An ethogram of behaviors for the maternal preference testing has been provided as supplementary material
2.6.2. Solo and familiar dyad observations
One month following weaning (when the animals were on average 8.5 months old), each subject was observed in a test setting designed to study the behavior of the subject alone and during interactions with familiar peers (Bauman et al 2004b
; Emery et al 2001
). Subjects were removed from their socialization cohorts and placed alone in a large testing enclosure similar to their home environment and observed for two consecutive 5 min sessions. Immediately following these initial solo observations, the first day of familiar social dyad observations began. Subjects were placed into the testing enclosures in pairs to form social dyads. Each social dyad consisted of two subjects from the same socialization cohort. Social dyad sessions were 20 min in duration with the focal subject (the subject for whom data were collected) alternating every 5 min. Each subject met with each other subject in their socialization cohort on two occasions separated by at least one day. Social dyads were spread out over 5 consecutive testing days with each subject participating in 2 separate 20 min dyads each day in a predetermined pseudo-random order. On the final day of familiar social dyad testing, each subject was again observed alone for two consecutive 5 min sessions. Trained observers blind to the condition of the animals used the Observer software program on laptop computers to score the behavior of each focal subject in real time from a behavioral ethogram of approximately 50 normal and abnormal rhesus monkey behaviors (ethogram of behaviors has been provided as supplementary material
). Behaviors included whole-body stereotypies such as pacing, back-flipping, twirling, and swinging. When a subject was engaged in a particular whole-body stereotypy for longer than 6 s, an extended stereotypy was scored.
2.6.3. Unfamiliar dyad observations
One month following the solo and familiar dyad observations (when the animals were on average 9.5 months old), each subject was observed in a test setting designed to study interactions with unfamiliar peers. Four age-appropriate monkeys (2 males and 2 females) served as unfamiliar peers (stimulus monkeys). These stimulus monkeys were maternally reared and socialized in group housing prior to their temporary assignment to this study. Subjects were again removed from their socialization cohorts and placed in individual holding cages. Subjects were then paired with one of the four stimulus monkeys in the same testing enclosures used for solo and familiar dyad observations. These unfamiliar dyad sessions were also 20 min in duration with the focal subject alternating every 5 min. Each subject met with each of the stimulus monkeys on two occasions separated by at least one day. The unfamiliar social dyads were therefore spread out over 4 consecutive testing days, with each subject participating in 2 separate 20 min dyads each day, again in a predetermined pseudo-random order.
2.6.4. Social group observations
In addition to the acquisition of behavioral data in novel testing environments, each subject was also observed within their home cage socialization cohorts. Each subject was observed for 5 min twice per week during weeks in which no other testing took place. A total of 30 observations were conducted on each subject, with all testing taking place in the weeks following the unfamiliar dyads.
2.6.5. Activity monitoring
When the monkeys were approximately 1 year old, activity was measured with an actimeter (Actiwatch-64; MiniMitter, Bend, OR) housed in a metal casing (40 × 32 × 13 mm) attached to a nylon primate collar (Primate Products, Woodside, CA). The Actiwatch-64 is a small device (17 g) capable of detecting the degree and speed of omnidirectional motion with an accelerometer. Changes in the degree and speed of motion produce changes in voltage that are stored as activity counts. For this study, each actimeter was programmed to sample activity at a frequency of 32 Hz and record activity counts in 30 s intervals. Each monkey was fitted with the primate collars with the attached actimeters and allowed to acclimate to the collars for 1 week before monitoring began.
The activity of each monkey was monitored in two separate conditions. In the first, the activity of each monkey was monitored for 7 consecutive days while the monkeys remained in their routine social housing situation. In the second condition, the monkeys were removed from their social groups and their activity was monitored while they were individually housed in standard home cages (61 × 66 × 81 cm) over two separate 24 h periods. During each 24 h period, each monkey was observed over four separate 10 min sessions, two between 8 a.m. and 12 p.m. and two between 3 p.m. and 6 p.m. Behaviors were scored in real time using the same ethogram as the solo and dyadic observations. In between each individual housing period, the monkeys were returned to their social groups for 24 h. During both monitoring conditions, special precautions were made to minimize disturbances to the monkeys.
2.7. Statistical analyses
Due to the large number of zero values scored for some of the behavioral observations, the data were not normally distributed and the variance was not homogenous. We therefore used nonparametric Mann– Whitney tests with a .05 alpha level to determine between-groups differences in these data. For all data in which parametric test assumptions of normality and homogeneity of variance were not violated, Independent t-tests with a .05 alpha level were used. All statistical procedures were carried out using SPSS 14.0 statistical software.