abnormal amounts of maternal circulating glucocorticoids ( gcs ) , which can be induced by 
 conditions such as chronic maternal stress , may produce protracted neurobehavioral 
 alterations in the offspring .
it has been shown that maternal prenatal ( but not postnatal ) 
 emotional stress is linked to abnormal infant affective reactivity at 4 mo of age ( 1 ) and reductions in gray matter density in various brain 
 regions , including the prefrontal , premotor , temporal , and cerebellar cortices , at 69 yr of 
 age ( 2 ) .
similar results have been reported in animal 
 models of prenatal stress , with offspring showing long - term anxiety - like behaviors and 
 delayed dendritic morphology in prefrontal ( 3 ) , 
 hippocampal ( 4 ) , and cerebellar purkinje cells ( 5 ) .
because stressful conditions produce elevated levels 
 of circulating gcs , neurobehavioral impairments have been attributed to the toxic effects of 
 excessive circulating prenatal gcs ( 6,7,8 ) . in our laboratory , we have systematically studied the effect of controlled prenatal 
 synthetic gc ( sgc ) administration ( betamethasone , bet ) to the mother on behavioral and 
 neuronal development in the offspring .
we have observed that administering bet to pregnant 
 rats at a therapeutic dose equivalent to that administered to women who are at risk of 
 preterm delivery produces a significant decrease in dendritic arborization in both dentate 
 granule and cerebellar purkinje cells ( 9 , 10 ) .
however , it is unknown whether similar neuronal 
 changes to pyramidal cortical cells occur during the early and late postnatal periods .
thus , 
 in the current study , we analyzed the impact of prenatal bet administration ( 0.17 mg / kg ) 
 ( 11,12,13 ) on the dendritic growth of layer ii / iii pyramidal 
 cells using the golgi - stained procedure along with histochemical staining of the dendritic 
 marker microtubule - associated protein 2 ( map2 ) in infant ( postnatal day 22 , p22 ) , adolescent 
 ( p52 ) , and young adult ( p82 ) rats .
eleven pregnant multiparous sprague - dawley rats were housed under controlled 
 environmental conditions ( temperature , 20  1 c ; 12h:12h light - dark cycle ) with food and 
 water available ad libitum .
pregnant animals were placed in individual 
 cages ( 45 cm  25 cm  20 cm ) , and gestational day 0 ( g0 ) was determined by the presence 
 of sperm detected in vaginal smears .
rats were randomly classified into the following two 
 groups : control - saline ( con , n = 6 ) and betamethasone - treated ( bet , n = 5 ) .
bet - treated 
 rats were given a single course of betamethasone phosphate subcutaneously ( 0.17 mg / kg of 
 body weight in the dorsal neck region ; cidoten , schering - plough , inc .
, santiago , chile ) 
 on gestational day 20 ( g20 ) , with the doses separated by an 8-hour interval .
importantly , 
 this rodent developmental stage corresponds approximately to a human fetus at 2836 
 gestational wk ( 14 ) .
furthermore , according to the 
 rat s bet pharmacokinetics and pharmacodynamics , the dose used in the current and previous 
 studies was equivalent to that administered to a woman who is at risk of preterm delivery 
 ( 11 , 13 ) . 

after the pre - weaning period ( postnatal day 22 , p22 ) , the con ( n : 23 ) and bet ( n : 28 ) 
 males were weaned and rearranged to house 23 animals per cage .
all procedures involving animals were approved by the local animal ethics committee and 
 were in accordance with the  guide for the care and use of laboratory animals 
( institute 
 for laboratory animal research , national research council , washington dc , 2011 ) .
all male animals were deeply anesthetized with pentobarbital ( 50 mg / kg of body weight ; 
 sigma - aldrich , co. , 3050 saint louis , misuri , usa ) , and intracardiac perfusion was 
 performed with 0.9% nacl followed by 4% paraformaldehyde ( sigma - aldrich ) .
each brain was 
 carefully dissected under a stereoscopic lens and freshly weighed ( g ) on a digital 
 analytical balance ( sartorious , entries 224 ) .
additionally , body weight ( g ) was assessed 
 using a classical balance ( radwag - wtb200 ) .
parietal slices were golgi - stained ( see 15 for further details ) , stabilized with collodion 
 solution ( fluka , sigma - aldrich ) , and sliced into 120-m thick sections .
pyramidal cells 
 were selected according to the following criteria : ( 1 ) having a well - defined pyramidal shape , ( 2 ) having a single apical dendrite oriented perpendicularly to the pial surface , 
 ( 3 ) demonstrating an adequate staining of the soma 
 and dendrites , ( 4 ) having no extensive processes 
 overlapping neighboring neurons , and ( 5 ) located in 
 a cortical strip at 350600 m under the pial surface ( cortical layers ii / iii were 
 delimited by the stereotaxic coordinates as described in paxinos and watson , 1998 ) ( 16 ) .
selected neurons were then photographed ( 400  ) 
 and digitized , and the basilar dendritic length ( m ) per neuron was quantified .
the number 
 of pyramidal cells assessed at each stage was as follows : con - p22 : 178 ; bet - p22 : 135 ; 
 con - p52 : 120 ; bet - p52 : 160 ; con - p82 : 160 ; and bet - p82 : 160 . the contralateral hemisphere of each animal was post - fixed for one hour and stored in 30% 
 sucrose at 4 c for 7 d ( cryoprotection ) . for the immunohistochemical procedure ,
the 
 parietal zone was sliced into 20-m - thick sections with a cryostat thermo scientific 
 microm hm525 ( walldorf baden - wurtemberg , germany ) , 46 sections per rat .
sections that had 
 been previously attached to a coded slide were washed twice in phosphate - buffered saline 
 ( pbs ) for 10 min per wash at 90 rpm and then incubated with 0.5% 
 h2o2 ( merck ) for 30 min at room temperature .
after two additional 
 washes in pbs , the sections were blocked for 1 h with 3% bovine serum albumin ( bsa ; 
 sigma - aldrich ) and 0.4% triton x-100 ( sigma - aldrich ) .
the primary antibody used was 
 monoclonal anti - map2 ( m4403 , sigma - aldrich ) .
the sections were incubated with the primary 
 antibody in blocking solution overnight at room temperature and under agitation ( 40 rpm ) . 
 the tissue was then washed three times with pbs and incubated in 1.5% bsa and 0.2% triton 
 x-100 for 2 h at room temperature and under agitation ( 40 rpm ) .
the secondary antibody used was conjugated goat anti - affine 
 pure rabbit igg ( h + l ) for map2 incubation ( 1:500 ) diluted in 1.5% bsa and triton x-100 
 at 0.2% for 2 h at room temperature , without agitation . to visualize the labeled parietal 
 map2 expression , an avidin - biotin peroxidase complex ( vectastain elite abc kit ; vector 
 laboratories )
was prepared in 1.5% bsa and triton x-100 and incubated for one hour prior 
 to addition to the substrate coupled with diaminobenzidine ( dab ) for 20 min without 
 stirring ( immpact dab peroxidase substrate ; vector laboratories ) .
the sections were 
 finally washed in distilled water for 10 sec , attached to coded slides , air - dried , 
 enclosed with entellan ( merck ) , and coverslipped .
images from the parietal cortex coronal 
 sections were captured with a bioblue model , bb.1153.pli , euromex microscope , considering 
 the same cortical region used in the golgi - stained cortical tissue ( located at 350600 m 
 under the pial surface , corresponding to approximately layers ii / iii ) .
map2 
 immunoreactivity was evaluated with imagej software ( nih , bethesda , md , usa ) using 
 grayscale images ( % of controls , arbitrary values ) .
the number of brain sections assessed 
 per animal was as follows : con - p22 , n : 4 ; bet - p22 , n : 3 ; con - p52 , n : 4 ; bet - p52 , n : 4 ; 
 con - p82 , n : 3 ; and bet - p82 , n : 3 .
a one - way analysis of variance ( anova ) and scheff post - hoc test were used to analyze the 
 basal dendritic length per neuron . for immunohistochemical data
, we used the 
 kruskal - wallis ( kw ) non - parametric test .
eleven pregnant multiparous sprague - dawley rats were housed under controlled 
 environmental conditions ( temperature , 20  1 c ; 12h:12h light - dark cycle ) with food and 
 water available ad libitum .
pregnant animals were placed in individual 
 cages ( 45 cm  25 cm  20 cm ) , and gestational day 0 ( g0 ) was determined by the presence 
 of sperm detected in vaginal smears .
rats were randomly classified into the following two 
 groups : control - saline ( con , n = 6 ) and betamethasone - treated ( bet , n = 5 ) .
bet - treated 
 rats were given a single course of betamethasone phosphate subcutaneously ( 0.17 mg / kg of 
 body weight in the dorsal neck region ; cidoten , schering - plough , inc .
, santiago , chile ) 
 on gestational day 20 ( g20 ) , with the doses separated by an 8-hour interval .
importantly , 
 this rodent developmental stage corresponds approximately to a human fetus at 2836 
 gestational wk ( 14 ) .
furthermore , according to the 
 rat s bet pharmacokinetics and pharmacodynamics , the dose used in the current and previous 
 studies was equivalent to that administered to a woman who is at risk of preterm delivery 
 ( 11 , 13 ) . 

after the pre - weaning period ( postnatal day 22 , p22 ) , the con ( n : 23 ) and bet ( n : 28 ) 
 males were weaned and rearranged to house 23 animals per cage .
all procedures involving animals were approved by the local animal ethics committee and 
 were in accordance with the  guide for the care and use of laboratory animals 
( institute 
 for laboratory animal research , national research council , washington dc , 2011 ) .
all male animals were deeply anesthetized with pentobarbital ( 50 mg / kg of body weight ; 
 sigma - aldrich , co. , 3050 saint louis , misuri , usa ) , and intracardiac perfusion was 
 performed with 0.9% nacl followed by 4% paraformaldehyde ( sigma - aldrich ) .
each brain was 
 carefully dissected under a stereoscopic lens and freshly weighed ( g ) on a digital 
 analytical balance ( sartorious , entries 224 ) .
additionally , body weight ( g ) was assessed 
 using a classical balance ( radwag - wtb200 ) .
parietal slices were golgi - stained ( see 15 for further details ) , stabilized with collodion 
 solution ( fluka , sigma - aldrich ) , and sliced into 120-m thick sections .
pyramidal cells 
 were selected according to the following criteria : ( 1 ) having a well - defined pyramidal shape , ( 2 ) having a single apical dendrite oriented perpendicularly to the pial surface , 
 ( 3 ) demonstrating an adequate staining of the soma 
 and dendrites , ( 4 ) having no extensive processes 
 overlapping neighboring neurons , and ( 5 ) located in 
 a cortical strip at 350600 m under the pial surface ( cortical layers ii / iii were 
 delimited by the stereotaxic coordinates as described in paxinos and watson , 1998 ) ( 16 ) .
selected neurons were then photographed ( 400  ) 
 and digitized , and the basilar dendritic length ( m ) per neuron was quantified .
the number 
 of pyramidal cells assessed at each stage was as follows : con - p22 : 178 ; bet - p22 : 135 ; 
 con - p52 : 120 ; bet - p52 : 160 ; con - p82 : 160 ; and bet - p82 : 160 .
the contralateral hemisphere of each animal was post - fixed for one hour and stored in 30% 
 sucrose at 4 c for 7 d ( cryoprotection ) . for the immunohistochemical procedure ,
the 
 parietal zone was sliced into 20-m - thick sections with a cryostat thermo scientific 
 microm hm525 ( walldorf baden - wurtemberg , germany ) , 46 sections per rat .
sections that had 
 been previously attached to a coded slide were washed twice in phosphate - buffered saline 
 ( pbs ) for 10 min per wash at 90 rpm and then incubated with 0.5% 
 h2o2 ( merck ) for 30 min at room temperature .
after two additional 
 washes in pbs , the sections were blocked for 1 h with 3% bovine serum albumin ( bsa ; 
 sigma - aldrich ) and 0.4% triton x-100 ( sigma - aldrich ) .
the primary antibody used was 
 monoclonal anti - map2 ( m4403 , sigma - aldrich ) .
the sections were incubated with the primary 
 antibody in blocking solution overnight at room temperature and under agitation ( 40 rpm ) . 
 the tissue was then washed three times with pbs and incubated in 1.5% bsa and 0.2% triton 
 x-100 for 2 h at room temperature and under agitation ( 40 rpm ) .
the secondary antibody used was conjugated goat anti - affine 
 pure rabbit igg ( h + l ) for map2 incubation ( 1:500 ) diluted in 1.5% bsa and triton x-100 
 at 0.2% for 2 h at room temperature , without agitation . to visualize the labeled parietal 
 map2 expression , an avidin - biotin peroxidase complex ( vectastain elite abc kit ; vector 
 laboratories )
was prepared in 1.5% bsa and triton x-100 and incubated for one hour prior 
 to addition to the substrate coupled with diaminobenzidine ( dab ) for 20 min without 
 stirring ( immpact dab peroxidase substrate ; vector laboratories ) .
the sections were 
 finally washed in distilled water for 10 sec , attached to coded slides , air - dried , 
 enclosed with entellan ( merck ) , and coverslipped .
images from the parietal cortex coronal 
 sections were captured with a bioblue model , bb.1153.pli , euromex microscope , considering 
 the same cortical region used in the golgi - stained cortical tissue ( located at 350600 m 
 under the pial surface , corresponding to approximately layers ii / iii ) .
map2 
 immunoreactivity was evaluated with imagej software ( nih , bethesda , md , usa ) using 
 grayscale images ( % of controls , arbitrary values ) .
the number of brain sections assessed 
 per animal was as follows : con - p22 , n : 4 ; bet - p22 , n : 3 ; con - p52 , n : 4 ; bet - p52 , n : 4 ; 
 con - p82 , n : 3 ; and bet - p82 , n : 3 .
a one - way analysis of variance ( anova ) and scheff post - hoc test were used to analyze the 
 basal dendritic length per neuron . for immunohistochemical data
, we used the 
 kruskal - wallis ( kw ) non - parametric test .
golgi - stained layer ii / iii pyramidal cell basal dendrites located in the parietal cortex of 
 animals treated prenatally with bet showed a significant reduction in length compared with 
 the age - matched con animals at both p52 and p82 ( fig . 

1.(a ) mean basilar dendritic length per neuron in 
 golgi - stained superficial ( layer ii / iii ) pyramidal cells .
p22 , p52 , p82 : postnatal days 22 , 52 , and 82 , respectively .
the 
 data are presented as the mean  sem ( * * p<0.01 , * * * p<0.001 , one - way analysis of 
 variance ) .
( b ) representative photomicrographs of golgi - stained pyramidal cells 
 ( layers ii / iii ) from con and bet animals .
bar : 40 m . ; * * p<0.01 , * * * p<0.001 , anova ) .
1b shows representative layer ii / iii pyramidal 
 cells from each condition and age .
( a ) mean basilar dendritic length per neuron in 
 golgi - stained superficial ( layer ii / iii ) pyramidal cells .
p22 , p52 , p82 : postnatal days 22 , 52 , and 82 , respectively .
the 
 data are presented as the mean  sem ( * * p<0.01 , * * * p<0.001 , one - way analysis of 
 variance ) .
( b ) representative photomicrographs of golgi - stained pyramidal cells 
 ( layers ii / iii ) from con and bet animals .
however , animals exposed to prenatal bet showed a significant reduction in map2 
 immunohistochemical expression at both p22 and p52 but not at later ages ( p82 ) ( fig . 2afig .
con : control group ; bet : betamethasone group ; p22 , 
 p52 , p82 : postnatal days 22 , 52 , and 82 , respectively .
the data are shown as mean  
 sem and are presented as a percentage of the control value ( * p<0.05 , 
 kruskal - wallis ) .
( b ) representative photomicrographs of the parietal cortical tissue 
 stained with anti - map2 antibodies from con and bet animals .
2b shows representative neocortical 
 ( parietal ) micrographs from con and bet offspring with the immunohistochemical map2 labeling 
 of superficial ( layers ii / iii ) pyramidal cells at p22 , p52 , and p82 .
con : control group ; bet : betamethasone group ; p22 , 
 p52 , p82 : postnatal days 22 , 52 , and 82 , respectively .
the data are shown as mean  
 sem and are presented as a percentage of the control value ( * p<0.05 , 
 kruskal - wallis ) .
( b ) representative photomicrographs of the parietal cortical tissue 
 stained with anti - map2 antibodies from con and bet animals .
in the current study , we showed that a single course of prenatal bet causes a reduction in 
 basilar dendritic length per neuron in layer ii / iii neocortical pyramidal cells and a 
 transient reduction in neocortical histochemical map2 immunoreactivity .
consistent with our previous studies in the rat cerebellar cortex , adolescent ( p52 ) and 
 young adult ( p82 ) animals exposed prenatally to a single course of bet showed a significant 
 reduction in basal dendritic trees in the superficial neocortical pyramidal neurons ( layers 
 ii / iii ) .
the long - term morphological alteration of dendritic development is the most 
 consistent result observed in our studies .
for example , dentate granule cells of adolescent 
 rats ( p52 ) treated with prenatal bet ( g20 ; 0.17 mg / kg ) showed a significant reduction ( 35% ) 
 in total dendritic length compared with age - matched controls ( 9 ) .
a similar reduction in dendritic domain ( 39% ) has been observed in cerebellar 
 purkinje cells at p52 in offspring treated antenatally with bet ( 10 ) . in both studies ,
the neuronal changes were related to significant 
 impairments in spatial memory tasks along with anxiety - like behaviors .
although we did not 
 evaluate functional variables , it is possible that the reduced dendritic arborization could 
 alter pyramidal cell function because the majority of synaptic connections , transmission , 
 and signal integration significantly depend on the number , length , and complexity of the 
 cell s dendritic branching ( 17 ) .
moreover , since the 
 glucocorticoid receptor ( gr ) is highly expressed in layers ii / iii cerebrocortical 
 neocortical neurons ( 18 ) , the morphological changes 
 in pyramidal cells reported here are probably due to long - term effects of prenatal sgc 
 administration on cortical neuronal maturation .
the results of the current study in 
 conjunction with previous studies on dendritic morphometry indicate that prenatal sgc 
 administration could produce subtle microstructural changes that may be related to the 
 long - term behavioral and cortical findings described in children whose mothers experienced 
 stressful prenatal conditions ( 1 , 2 ) .
moreover , map2 is the most predominant cytoskeletal protein isoform present in dendritic 
 branches ; it contributes to dendritic growth and plasticity ( 19 , 20 ) .
regarding the results obtained in 
 the present study , although we observed that the reduction of map2 in the neocortex of 
 animals exposed prenatally to bet is protracted ( infant and adolescent rats ) , we did not 
 observe significant differences when the animals reached young adulthood ( p82 ) .
this 
 transient reduction in map2 is consistent with our observation in previous studies carried 
 out in cerebellar neurons at the same ontogenetic age ( p52 , 56% ) ( 12 ) .
it should be noted that the mere transience of the reduction in 
 immunohistochemical expression of map2 does not rule out the possibility that map2 
 underexpression changes the course of neuronal dendritic maturation and causes permanent 
 structural dendritic developmental impairments , as observed in the current golgi - stained 
 layer ii / iii pyramidal cells .
however , because immunohistochemical map2 staining is an 
 indirect measure of protein expression , it is difficult to determine whether the transient 
 map2 reduction observed in bet - treated animals clearly reflects quantitative changes in map2 
 protein expression . to make this differentiation , it is necessary to complement the 
 immunohistochemical approach used in the current work with other quantitative methods such 
 as western blot analysis .
in addition , since our previous studies carried out in the 
 cerebellar cortex indicated significant changes in the immunohistochemical expression of 
 bdnf and its trkb receptor , along with map2 , in the purkinje cells of animals exposed to 
 prenatal bet , it is not possible to rule out that the changes observed in the present study 
 also involve changes in these neurotrophic variables . in conclusion , the present data indicate that administration of prenatal bet to pregnant 
 dams at g20 is associated with a long - term reduction in the basilar dendritic length of 
 cortical pyramidal cells and a transient reduction of the immunohistochemical expression of 
 map2 in superficial ii / iii neocortical cells of the offspring .
 abstract.previous animal studies have indicated that excessive prenatal circulating glucocorticoid 
 ( gc ) levels induced by the antenatal administration of synthetic gc ( sgc ) significantly 
 alter neuronal development in the cerebellar and hippocampal neurons of the offspring . 
 however , it is unknown whether antenatal sgc administration results in long - term 
 neocortical pyramidal cell impairment . in the current study 
 , we examined whether an 
 equivalent therapeutic dose of antenatal betamethasone phosphate ( bet ) in pregnant rats 
 alters the golgi - stained basilar dendritic length and histochemical expression of 
 dendritic microtubule - associated protein 2 ( map2 ) of neocortical pyramidal cells in 
 infant , adolescent , and young adult offspring . 
 the results obtained showed that in 
 utero bet exposure resulted in a significant reduction in the basilar dendritic 
 length per neuron and a transient reduction in histochemical map2 immunoreactivity . 
 
 consistent with previous hippocampal and cerebellar data , the present findings suggest 
 that prenatal bet administration alters the dendritic growth of cerebrocortical pyramidal 
 cells . 