animals : female and male c57bl/6n mice were purchased from japan slc , inc . 

the animals used for the experiments were bred in a 
 laboratory at the department of animal science at kobe university , under a 12-hr light / dark 
 cycle at 2124c and 4060% humidity . a laboratory diet ( lab mr stock ;
nihon nosan co. , 
 yokohama , japan ) and filtered water were available ad libitum .
this 
 experiment was approved by the institutional animal care and used committee ( permission 
 number : 24 - 10 - 03 ) and carried out according to the kobe university animal experimentation 
 regulation .
, 
 osaka , japan ) was based on the dose associated with the lowest observed adverse effect level 
 ( loael ) [ 200 ng / kg body weight ( bw ) ] [ 11 , 12 ] .
the volumes for oral administration were adjusted to 
 25 l/30 g bw of sesame oil .
female mice in proestrus were mated 1:1 with 
 males overnight , and females that had a vaginal plug on the following morning were 
 designated as being at e0.5 .
vehicle or tcdd ( 20 , 200 , 2,000 or 5,000 ng / kg 
 bw ) was orally administered on e8.5 .
these groups were defined as the control , t20 , t200 , 
 t2000 and t5000 groups , respectively .
tissue preparation : at the time of necropsy , all dams were deeply 
 anesthetized with diethyl ether .
they were then fixed in 4% 
 paraformaldehyde in 0.1 m phosphate buffer overnight at 4c , dehydrated through a graded 
 series of ethanol followed by xylene and embedded in paraffin .
coronal serial sections of 10 
 m thickness were then cut and mounted on a glass slide ( platinum pro ; 
 matsunami glass ind . , ltd .
, kishiwada , japan ) , and a series of semi - serial sections was made 
 by collection at 100-m intervals ( one of every 10 tissue sections ) .
cresyl fast violet staining : after deparaffinization and rehydration , the 
 sections were stained with 0.1% cresyl fast violet solution for 5 min .
they were then placed 
 in distilled water and a graded series of ethanol for appropriate bleaching of dye , 
 dehydrated with ethanol and coverslipped with eukitt ( o. kindler , gmbh , freiburg , 
 germany ) .
immunohistochemistry and immunohistoplanimetry : the combination of 
 blocking agents and antibodies used for the detection of each protein by 
 immunohistochemistry is listed in table 
 1table 1.the combination of blocking agents and antibodies used for 
 immunohistochemistrydetectionblocking reagentprimary antibodysecondary antibodygfapblocking one histo(nacalai tesque , inc . ,
kyoto , 
 japan)mouse monoclonal antibody against gfap(ne1015 ; 
 1:1,000)(merck , kgaa , darmstadt , germany)histofine max - po ( m)(histofine simplestain system)(nichirei bioscience , inc . ,
tokyo , japan)pcnablocking reagent a and b(nichirei bioscience , 
 inc.)mouse monoclonal antibody against pcna(na03 ; 1:100)(merck , kgaa)histofine max - po ( m)(nichirei bioscience , inc.)dcxblocking one histo(nacalai tesque , inc.)goat polyclonal antibody against dcx(sc-8066 ; 1:100)(santa cruz biotechnology , inc . , dallas , tx , u.s.a.)horseradish peroxidase - conjugated antigoat igg mouse 
 igg ( ap186p ; 1:100)(merck , kgaa)gfap : glial fibrillary acidic protein , pcna : proliferating cell nuclear antigen , dcx : 
 doublecortin .. the sections were deparaffinized and rehydrated and were heated at 121c for 
 20 min in 10 mm sodium citrate buffer ( ph 6.0 ) for antigen retrieval .
after cooling , they 
 were immersed in absolute methanol and 0.5% h2o2 for 30 min each at 
 room temperature ( rt ) to quench the endogenous peroxidase activity .
the specimens were 
 incubated with each blocking agent for 1 hr at rt and then with each primary antibody for 18 
 hr at 4c ( table 1 ) . after washing with 

phosphate buffered saline ( pbs ) , each secondary antibody was reacted for 1 hr at rt ( table 1 ) .
the excess antibodies were rinsed in pbs , 
 and the immunoreactivities were detected by incubation with 3,3-diaminobenzidine solution 
 ( envision+ kit / hrp [ dab ] ; dako , glostrup , denmark ) .
the sections were counterstained lightly 
 with hematoxylin . finally , they were dehydrated and cleared through an ethanol - xylene series 
 and coverslipped with eukitt ( o. kindler , gmbh ) .
gfap : glial fibrillary acidic protein , pcna : proliferating cell nuclear antigen , dcx : 
 doublecortin .
immunohistoplanimetry : coronal sections containing the hippocampus stained 
 immunohistochemically were observed under a light microscope ( nikon , corp . ,
the granular immunoreactivities of glial fibrillary acidic protein ( gfap ) and proliferating 
 cell nuclear antigen ( pcna)-positive cells were counted in 3 sections per 100 
 m . statistical analysis : statistical analysis was performed with excel 
 statistics 2012 for windows ( ssri , co. , ltd . ,
values were considered statistically significant if p<0.05 . 
 the tukey - kramer or steel - dwass test after one - way anova analysis was used to determine 
 differences between groups .
body and brain weights : no significant differences in bw or brain weight 
 were found between the control and tcdd - exposed groups ( data not shown ) .
the dg at e18.5 was immature , and the granular cell 
 layer could not be clearly distinguished from the molecular layer and polymorphic layer 
 based on its morphology ( fig .
( a  e ) the cell density in the dg was decreased in the t5000 compared with the 
 control group , whereas no obvious changes were observed in the shape or somatic size 
 of cells ( e ) .
( f  j ) the fibrous immunoreactivities of gfap were detected , but were 
 decreased in the t5000 group ( j ) .
( k  o ) the numbers of pcna - positive cells were 
 smaller in the t5000 ( o ) than the other groups ( k  n ) .
( p  t ) the intensity of dcx 
 staining did not differ between the control and tcdd - exposed groups .
mol : molecular 
 layer ; gcl : granule cell layer ; pia : pia mater ; bar=50 m . ) .
the cell density in the dg appeared to be decreased slightly in the t5000 group 
 compared with the control group , whereas no obvious changes were observed in the shape or 
 somatic size of cells .
however , no notable differences in the hippocampal fimbria were found 
 between the control and tcdd - exposed groups ( fig . 

2 . representative histology and immunohistochemistry in the hippocampal fimbria in the 
 hippocampus at e18.5 .
( a  e ) no notable differences were found between the control and 
 tcdd - exposed groups based on cresyl fast violet staining .
( f  j ) the fibrous and 
 granular immunoreactivities of gfap were observed , and the latter was reduced in the 
 t5000 group ( j ) .
lv : lateral ventricle ; bar=50 m ( inset : 20 
 m ) . ) .
( a  e ) the cell density in the dg was decreased in the t5000 compared with the 
 control group , whereas no obvious changes were observed in the shape or somatic size 
 of cells ( e ) .
( f  j ) the fibrous immunoreactivities of gfap were detected , but were 
 decreased in the t5000 group ( j ) .
( k  o ) the numbers of pcna - positive cells were 
 smaller in the t5000 ( o ) than the other groups ( k  n ) .
( p  t ) the intensity of dcx 
 staining did not differ between the control and tcdd - exposed groups .
mol : molecular 
 layer ; gcl : granule cell layer ; pia : pia mater ; bar=50 m . .
( a  e ) no notable differences were found between the control and 
 tcdd - exposed groups based on cresyl fast violet staining .
( f  j ) the fibrous and 
 granular immunoreactivities of gfap were observed , and the latter was reduced in the 
 t5000 group ( j ) .
immunohistological findings : the fibrous immunoreactivities of gfap were 
 detected in the cell body and process of neural stem cells ( nscs ) and astrocytes in the dg . 

gfap - immunoreactive nscs and astrocytes were localized in the molecular layer , granular cell 
 layer and next to the pia mater ( fig .
, the fibrous and granular gfap 
 immunoreactivities were observed in the cell body and process of nscs and astrocytes ( fig .
the granular immunoreactivities of gfap 
 appeared to be reduced in the t5000 group , and in fact , the number of them was significantly 
 decreased ( figs .
3.the results of immunohistoplanimetry for the number of pcna - positive cells in the dg 
 ( a ) and the granular immunoreactivities of gfap in the hippocampal fimbria ( b ) .
( a ) 
 the number of pcna - positive cells was decreased in all tcdd - exposed groups and 
 significantly reduced in the t20 , t200 and t5000 groups .
( b ) the number of the 
 granular immunoreactivities of gfap was significantly decreased in the t5000 group . 

the number of pcna - positive cells was decreased in all 
 tcdd - exposed groups and significantly reduced in the t20 , t200 and t5000 groups ( figs . 1k1o 
 and 3b ) .
doublecortin ( dcx ) immunoreactivity was 
 detected in axons and dendrites of the immature neurons .
the intensity of dcx staining did 
 not differ between the control and tcdd - exposed groups ( fig .
the results of immunohistoplanimetry for the number of pcna - positive cells in the dg 
 ( a ) and the granular immunoreactivities of gfap in the hippocampal fimbria ( b ) .
( a ) 
 the number of pcna - positive cells was decreased in all tcdd - exposed groups and 
 significantly reduced in the t20 , t200 and t5000 groups .
( b ) the number of the 
 granular immunoreactivities of gfap was significantly decreased in the t5000 group . 

this study demonstrated maternal tcdd exposure in c57bl/6n mice disrupted the fetal 
 hippocampal development at e18.5 . to our knowledge , this is the first in 
 vivo study reporting the effect of maternal exposure to tcdd on the dg and the 
 hippocampal fimbria in fetal mice in which the decreases of the gfap immunoreactivities and 
 the number of pcna positive cells in the dg , and the reduction of the number of granular 
 gfap immunoreactivities in the hippocampal fimbria were shown .
these results suggest that 
 the cumulative dioxins in fetus mediated via the placenta already affect the cells in the dg 
 and the hippocampal fimbria in the fetal period and result in an adverse effect on the 
 development of the hippocampus .
neurons and glial cells in the dg derive from a pseudostratified neuroepithelium of 
 ectodermal origin during early embryonic development . at around e910 in mice , cortical 
 neurogenesis begins , and neuroepithelial cells begin to differentiate into radial glia ( rg ) , 
 which are nscs and express gfap .
the rg 
 generates neurons directly or indirectly through neural precursor cells ( npcs ) . in the 
 present study ,
the gfap immunoreactivities were reduced in the dg of the t5000 group at 
 e18.5 , and the number of pcna - positive cells was significantly reduced in the dg of the t20 , 
 t200 and t5000 groups at e18.5 . moreover ,
the cell density in the dg was decreased in the 
 t5000 group compared with the control group .
these results suggest that tcdd exposure 
 impairs nscs , npcs or granular cells , resulting in a disruption of neuronal development in 
 the fetal dg .
tcdd exposure is 
 known to affect cell proliferation via the activation of mitogen - activated protein kinases 
 ( mapks ) , such as erk or jnk , and the induction of p21 or 
 p27 , which are involved in the cell cycle [ 3 , 22 , 33 , 38 ] .
moreover , tcdd also 
 inhibits dna synthesis in rat primary hepatocytes , and the proliferation of primary npcs is 
 arrested in the g1 phase of the cell cycle by tcdd exposure [ 18 , 25 ] . considering that the ahr is 
 present in proliferating nscs and npcs in vivo , tcdd exposure likely altered the proliferation of nscs or npcs in the 
 present study , resulting in a reduction in the number of pcna - positive cells .
another possibility is that the number of cells in the hippocampus was reduced through the 
 promotion of apoptosis . during the developmental period ,
a surplus of new neurons is 
 produced , and the inappropriate neurons are excluded by apoptosis [ 7 , 31 ] . as a result , the net number 
 of neurons is properly retained .
it has been reported that ahr - mediated oxidative 
 stress generated by the induction of cytochrome p450 enzymes may be an upstream event in the 
 apoptosis cascade .
in addition , tcdd exposure 
 has been shown to increase apoptosis of granule neuron precursor cells in the developing 
 cerebellum . in this study , it is possible that the 
 mechanism for regulating the number of cells in the hippocampus was disrupted via 
 tcdd - induced promotion of nsc , npc or granular cell apoptosis . in the hippocampus , astrocytes and nscs express an astroglial intermediate filament , gfap 
 [ 4 , 49 ] . 

immunoreactivities of gfap were observed fibrously and granularly in the hippocampal fimbria 
 at e18.5 .
the former is considered as the cell processes of astrocytes and nscs and the 
 latter as the branching point of the cell process .
the number of granular gfap 
 immunoreactivities was significantly decreased in the t5000 group , which may indicate the 
 delay of astrogenesis . during embryonic development , rgs give rise to neurons first and 
 glial cells later . at the end of embryonic 
 development ,
the basic helix - loop - helix genes hes1 and 
 hes5 are known notch signaling genes , and hes1 and hes5 keep nscs 
 undifferentiated during early development and promote differentiation into glial cells 
 .
these data 
 suggest that the toxicity of tcdd mediated via the ahr disrupts the maintenance of nscs at 
 the point of their differentiation into astrocytes , resulting in the induction of 
 developmental delay of astrogenesis in the hippocampal fimbria .
the hippocampal fimbria is 
 mainly composed of a band of efferent axons that forms part of the cerebral fornix .
ca3 
 subcortical efferents in the hippocampal fimbria support the acquisition of contextual fear 
 .
it is known that astrocytes surrounding the 
 synapses participate in neurotransmission and that astrocytes are also an important 
 component in glutamate uptake and metabolism . 

therefore , astrocytes are also considered to support neural activity in the hippocampal 
 fimbria .
these facts suggest that the developmental delay of astrocytes by tcdd exposure 
 disrupts neuronal activity in the hippocampal fimbria . in adult mice ,
new neurons are produced in the subgranular zone ( sgz ) of the dg . unlike in fetal mice , radial astrocytes in the adult 
 sgz
play a role as the primary precursors of new neurons in the adult hippocampus   and increased in sgz differentiation into granular 
 cells at 14 weeks .
neurogenesis in the dg 
 becomes persistent throughout postnatal life and is regulated by behavior and the 
 environment .
the hippocampus is functionally 
 engaged in learning and memory , and newborn cells are increased by hippocampal - dependent 
 learning tasks [ 41 , 47 ] .
therefore , it is suggested that neurogenesis exerts an influence on learning 
 and memory .
it has been reported that in utero and lactational exposure to 
 dioxin deficit the fear memory of male offspring in adulthood , and latency in the active 
 avoidance learning was longer in the tcdd exposed male offspring in the 4144 postnatal day 
 [ 13 , 30 ] . 

considering these reports , the effects of tcdd observed in the fetal period , such as the 
 decrease in the numbers of gfap- and pcna - positive cells , may persist into adulthood , or 
 neurogenesis in the dg may be negatively affected by tcdd exposure - mediated lactation .
based on the present data , we conclude that maternal tcdd exposure has adverse impacts on 
 nscs , npcs , granular cells or astrocytes in the dg or hippocampal fimbria , resulting in a 
 disturbance of neuronal development in future generations of mice .
it is possible that these 
 effects remain until adulthood , and so , whether the influences of tcdd toxicity on the 
 developing hippocampus persist and impair learning and memory in adulthood is needed to be 
 examined as a further study .
 dioxins are widespread persistent environmental contaminants with adverse 
 impacts on humans and experimental animals . 
 behavioral and cognitive functions are 
 impaired by 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) exposure . 
 tcdd 
 exerts its toxicity via the aryl hydrocarbon receptor ( ahr ) , a ligand - activated 
 transcription factor . 
 the hippocampus , which plays important roles in episodic memory and 
 spatial function , is considered vulnerable to tcdd - induced neurotoxicity , because it 
 contains the ahr . 
 we herein investigated the effects of tcdd toxicity on hippocampal 
 development in embryonic mice . 
 tcdd was administered to dams at 8.5 days postcoitum with a 
 single dose of 20 , 200 , 2,000 and 5,000 ng / kg body weight ( groups t20 , 
 t200 , t2000 and t5000 , respectively ) , and the brains were dissected from their pups at 
 embryonic day 18.5 . 
 immunohistochemical analysis demonstrated that the glial fibrillary 
 acidic protein ( gfap ) immunoreactivities in the dentate gyrus ( dg ) were reduced in the 
 t5000 group . 
 granular gfap immunoreactivity was observed in the hippocampal fimbria , and 
 the number of immunoreactive fimbria was significantly decreased in the t5000 group . 
 the 
 number of proliferating cell nuclear antigen ( pcna)-positive cells was decreased in all 
 tcdd - exposed groups and significantly reduced in the t20 , t200 and t5000 groups . together , 
 these results demonstrate that maternal tcdd exposure has adverse impacts on neural stem 
 cells ( nscs ) , neural precursor cells ( npcs ) and granular cells in the dg and disrupts the 
 nsc maintenance and timing of differentiation in the hippocampal fimbria , which in turn 
 interrupt neuronal development in future generations of mice . 
MATERIALS AND METHODS
RESULTS
DISCUSSION