balb / c female mice ( 68 week old ) were obtained from jackson laboratory .
animal use conformed to institutional guidelines of weill cornell medical college and the feinstein institute for medical research .
mice were immunized with multi - antigenic peptide coupled to dweysvwlsn ( mp , 100 g ) , control polylysine core ( mc , 100 g ) , scrambled wsdyevwlsn ( ms , 100 g ) , or aaaaavwlsn ( ma , 100 g , anaspec ) as previously described15 .
we used mouse monoclonal aab r4a or control igg2b ( 200 g , southern biotech ) , human monoclonal aab g11 , human control aab b1 or human anti - dna ab d9 ( 150 g ) , obtained by isolating peptide - reactive or non - reactive b cells and expressing the ig heavy and light chain genes in 293 cells39,40 .
the d form of dweysvwlsn peptide ( 200 g ) or irrelevant peptide nemqssrlre was given i.p .
to mice at the time of intravenous r4a administration , and again 24 h and 48 h later .
eu - labeled monoclonal abs ( perkin elmer ) were intravenously administered ( 100 g ) to gestating dams at e14 .
fetal brains exposed to eu - r4a were stained with ab to cd-31 to identify blood vessels . for tunel assays , coronal sections ( 20 m thick ) of e15 brains were processed according to the manufacturer ( r&d systems ) and counterstained with methyl green . for ph3 assays of mitotic cells , frozen brain sections ( 20 m ) were blocked with 10% normal goat serum and incubated overnight at 4c with ph3 ab ( 1:1000 ; chemicon , millipore , billerica , ma ) followed by red x goat anti - rabbit igg ( jackson immuno research laboratories )
. measurements of the cp and the cerebral width were performed on nestin ( 1:200 ; chemicon ) and dapi ( 1:1000 ; invitrogen , carlsbad , ca ) stained e15 coronal sections , with openlab software ( improvision , perkinelmer ) .
quantification of ph3 + and tunel+ cells was performed using automated image analysis programs on an axio - image microscope ( zeiss , thornwood , ny ) .
anatomic regions were identified using within section coordinates and positive cells were counted in comparable areas of interest .
negative geotaxis was measured23 in offspring born from mph ( n = 19 ) , mpl ( n = 14 ) and mc ( n = 15 ) dams at ages p11 , p15 or p16 and p20 .
mice were placed with their heads facing downward on an incline ( 45 ) and the time to turn 180 and climb the incline was measured ( maximum 30 s , if the animal did not reach the top of the incline in the allotted time , a score of 30 s was assigned ) .
each mouse was given three trials and the mean latency was calculated . adult male offspring from mph ( n = 10 ) , mpl ( n = 8) and mc ( n = 10 ) groups underwent a behavioral battery15,16 ( described in supplementary fig .
we used a blinded format and randomized the order in which the cohorts of animals were tested in different tasks .
the novel object recognition task27 consisted of three phases . in the first ( sample ) , mice explored two identical objects for 5 min . in the second ( delay ) , mice stayed in their home cage for 10 min .
test objects replaced sample objects , so that one was identical to those in the sample phase ( familiar object ) and the other was different ( novel object ) . for the final phase ( choice ) , mice inspected the test objects for 5 min .
object exploration was scored automatically with the pc - based anymaze software ( stoelting ) .
we defined a3 as the exploration time for the familiar object , and b1 as the exploration time for the novel object , and measured the discrimination ratio , ( b1a3 ) / ( b1+a3 ) .
the topological task28 consisted of five sessions ( 5 min ) with varying intersession intervals ( 3 min , 10 min , 3 min and 10 min ; sequentially ) .
four different objects were arranged in a square . in sessions 1 and 2 , mice were familiarized to this arrangement . before session 3 , two objects were transposed , creating a novel configuration ii .
animals were expected to explore displaced objects more avidly during session 3 and 4 . before session 5
, the two objects that had remained unchanged were switched , thus creating configuration iii .
we defined d1 as the sum of the times exploring the displaced objects in configurations ii and iii ( sessions 3 and 5 ) and d2 as the sum of the times exploring the non - displaced objects ( sessions 3 and 5 ) .
topological processing was measured with the ratio , ( d1d2 ) / ( d1+d2 ) .
the fear extinction task26 initially required animals to associate the conditioned stimulus ( cs , 20-s tone , 5 khz , 80 db ) with the unconditioned stimulus ( us , foot - shock , 1 s , 0.6 ma ) , as previously described16 .
extinction learning was tested 24 h later by exposing mice to 20 trials of cs alone in a novel chamber , with an intertrial interval of 120 s. to determine fear extinction , we measured the amount of freezing during each tone , using freezeframe software ( actimetrics ) .
eight months after birth , mice that had been subjected to behavioral studies were anesthetized and perfused .
the anterior striatum , and the anterior and posterior commissure were the internal landmarks to block and section the tissue in order to determine the volume of neocortical areas , hippocampus and amygdala .
tissue was stained with cresyl violet and mouse anti - neun ( 1:1000 ; chemicon ) .
internal cortical coordinates were the interhemispheric fissure and the anterior corpus callosum ; for hippocampus and amygdala ( basolateral and lateral nuclei ) , the coronal perimeters were captured on video screen by ibas software ( zeiss ) from the dorsal third ventricle to the posterior commissure . for neuron size determinations , video capture of neurons was performed using an optical dissector randomly sampling each region of interest .
statistical comparisons were performed with kruskal - wallis and fisher 's analysis of variance , student 's t test ( unpaired , two - sided ) , mann - whitney u test , and kolmogorov - smirnov non - parametric test .
we used the software packages spss ( spss inc . ) , statistica ( statsoft ) and origin ( originlab ) for our analyses .
balb / c female mice ( 68 week old ) were obtained from jackson laboratory .
animal use conformed to institutional guidelines of weill cornell medical college and the feinstein institute for medical research .
mice were immunized with multi - antigenic peptide coupled to dweysvwlsn ( mp , 100 g ) , control polylysine core ( mc , 100 g ) , scrambled wsdyevwlsn ( ms , 100 g ) , or aaaaavwlsn ( ma , 100 g , anaspec ) as previously described15 .
we used mouse monoclonal aab r4a or control igg2b ( 200 g , southern biotech ) , human monoclonal aab g11 , human control aab b1 or human anti - dna ab d9 ( 150 g ) , obtained by isolating peptide - reactive or non - reactive b cells and expressing the ig heavy and light chain genes in 293 cells39,40 .
the d form of dweysvwlsn peptide ( 200 g ) or irrelevant peptide nemqssrlre was given i.p .
to mice at the time of intravenous r4a administration , and again 24 h and 48 h later .
eu - labeled monoclonal abs ( perkin elmer ) were intravenously administered ( 100 g ) to gestating dams at e14 .
fetal brains exposed to eu - r4a were stained with ab to cd-31 to identify blood vessels .
for tunel assays , coronal sections ( 20 m thick ) of e15 brains were processed according to the manufacturer ( r&d systems ) and counterstained with methyl green . for ph3 assays of mitotic cells , frozen brain sections ( 20 m ) were blocked with 10% normal goat serum and incubated overnight at 4c with ph3 ab ( 1:1000 ; chemicon , millipore , billerica , ma ) followed by
measurements of the cp and the cerebral width were performed on nestin ( 1:200 ; chemicon ) and dapi ( 1:1000 ; invitrogen , carlsbad , ca ) stained e15 coronal sections , with openlab software ( improvision , perkinelmer ) .
quantification of ph3 + and tunel+ cells was performed using automated image analysis programs on an axio - image microscope ( zeiss , thornwood , ny ) .
anatomic regions were identified using within section coordinates and positive cells were counted in comparable areas of interest .
negative geotaxis was measured23 in offspring born from mph ( n = 19 ) , mpl ( n = 14 ) and mc ( n = 15 ) dams at ages p11 , p15 or p16 and p20 .
mice were placed with their heads facing downward on an incline ( 45 ) and the time to turn 180 and climb the incline was measured ( maximum 30 s , if the animal did not reach the top of the incline in the allotted time , a score of 30 s was assigned ) . each mouse was given three trials and the mean latency was calculated .
adult male offspring from mph ( n = 10 ) , mpl ( n = 8) and mc ( n = 10 ) groups underwent a behavioral battery15,16 ( described in supplementary fig .
we used a blinded format and randomized the order in which the cohorts of animals were tested in different tasks .
the novel object recognition task27 consisted of three phases . in the first ( sample ) , mice explored two identical objects for 5 min . in the second ( delay ) , mice stayed in their home cage for 10 min .
test objects replaced sample objects , so that one was identical to those in the sample phase ( familiar object ) and the other was different ( novel object ) . for the final phase ( choice ) , mice inspected the test objects for 5 min .
object exploration was scored automatically with the pc - based anymaze software ( stoelting ) .
we defined a3 as the exploration time for the familiar object , and b1 as the exploration time for the novel object , and measured the discrimination ratio , ( b1a3 ) / ( b1+a3 ) .
the topological task28 consisted of five sessions ( 5 min ) with varying intersession intervals ( 3 min , 10 min , 3 min and 10 min ; sequentially ) .
four different objects were arranged in a square . in sessions 1 and 2 , mice were familiarized to this arrangement . before session 3 , two objects were transposed , creating a novel configuration ii .
animals were expected to explore displaced objects more avidly during session 3 and 4 . before session 5 , the two objects that had remained unchanged were switched , thus creating configuration iii .
we defined d1 as the sum of the times exploring the displaced objects in configurations ii and iii ( sessions 3 and 5 ) and d2 as the sum of the times exploring the non - displaced objects ( sessions 3 and 5 ) .
topological processing was measured with the ratio , ( d1d2 ) / ( d1+d2 ) .
the fear extinction task26 initially required animals to associate the conditioned stimulus ( cs , 20-s tone , 5 khz , 80 db ) with the unconditioned stimulus ( us , foot - shock , 1 s , 0.6 ma ) , as previously described16 .
extinction learning was tested 24 h later by exposing mice to 20 trials of cs alone in a novel chamber , with an intertrial interval of 120 s. to determine fear extinction , we measured the amount of freezing during each tone , using freezeframe software ( actimetrics ) .
eight months after birth , mice that had been subjected to behavioral studies were anesthetized and perfused .
the anterior striatum , and the anterior and posterior commissure were the internal landmarks to block and section the tissue in order to determine the volume of neocortical areas , hippocampus and amygdala .
tissue was stained with cresyl violet and mouse anti - neun ( 1:1000 ; chemicon ) .
internal cortical coordinates were the interhemispheric fissure and the anterior corpus callosum ; for hippocampus and amygdala ( basolateral and lateral nuclei ) , the coronal perimeters were captured on video screen by ibas software ( zeiss ) from the dorsal third ventricle to the posterior commissure . for neuron size determinations , video capture of neurons
statistical comparisons were performed with kruskal - wallis and fisher 's analysis of variance , student 's t test ( unpaired , two - sided ) , mann - whitney u test , and kolmogorov - smirnov non - parametric test .
we used the software packages spss ( spss inc . ) , statistica ( statsoft ) and origin ( originlab ) for our analyses .
 systemic lupus erythematosus ( sle ) is an autoimmune disease mediated by autoantibodies ( aabs ) and preferentially affecting women of childbearing age . 
 since the offspring of mothers with sle exhibit a high frequency of learning disorders1 - 5 , we hypothesized that maternally transferred aabs that bind dna and the n - methyl - d - aspartate receptor ( nmdar)6 - 12 could play a pathogenic role during fetal brain development . here we describe a maternal sle murine model wherein pregnant dams harbored dna - specific , nmdar - specific aabs throughout gestation . 
 high titers of these aabs in maternal circulation led to histological abnormalities in fetal brain and subsequent cognitive impairments in adult offspring . 
 these data support a paradigm in which in utero exposure to neurotoxic aabs causes abnormal brain development with long - term consequences . 
 this paradigm may apply to multiple congenital neuropsychiatric disorders . 
METHODS
Animals and immunization
Immuno-histology of fetal brains
Behavioral assessment
Adult Histology
Statistical analysis
Supplementary Material