early life stressors that shape the stress response in offspring have profound effects on mood and cognition in adulthood ( davidson & mcewen , 2012 ) .
chronic exposure to glucocorticoids contribute to the dysfunction of the inhibitory network and impairment of rhythmic oscillations , which are critical for the regulation of brain activity and complex cognitive processes ( hu & et  al , 2010 ) .
a dysfunctional gabaergic system is associated with the pathogenesis of neuropsychiatric diseases such as schizophrenia , anxiety and depression ( hines & et  al , 2012 ) . during brain development , gabaergic synapses
are formed prior to the formation of glutamatergic synapses and the activation of the gabaa receptor depolarizes immature neurons ( ben - ari and et  al , 2012 , ben - ari , 2002 , ben - ari and et  al , 2007 ) .
excitatory gaba transmission plays important roles in various neurodevelopmental processes including ; neuronal migration , cell proliferation , neurite outgrowth and generating synchronized network activity ( cherubini & et  al , 2011 ) .
cation chloride cotransporter ( ccc ) is the key controlling factor in controlling the switch of the gabaa receptor function .
cccs control the reversal potential of the gabaa receptor - mediated current ( egaba ) , which is important for the modulation of the gabaa receptor function .
there are two main types of cccs ; the outwardly directed potassium - chloride cotransporter 2 ( kcc2 ) , and the inwardly directed sodium  potassium - chloride cotransporter 1 ( nkcc1 ) . in immature neurons ,
kcc2 , on the other hand , reduces the chloride reversal potential thus it extrudes cl out of the cell and shifts the actions of the gaba from excitation to inhibition .
although the other chloride regulators channels and transporters also take part in this sequence ( blaesse and et  al , 2009 , medina and chudotvorova , 2006 ) , the expression of kcc2 is thought to initiate the developmental switch of the gabaa receptor function from excitatory to inhibitory transmission ( ben - ari , 2002 ) .
in addition to the expression of cation - chloride cotransporters , the gabaa receptor undergoes postnatal changes in its structure and function by the differential expression of different subunits ' composition ( jacob et  al . , 2008 ) . the presence of gabaa receptor 1 subunits mediates phasic inhibition by inducing a more rapid decay rate in gabaa - mediated synaptic currents ( dunning & et  al , 1999 ) .
in contrast , the gabaa receptor 5 subunits mediate tonic inhibition , which can be characterized by a slow decay rate of the synaptic current ( jacob et  al . , 2008 ) .
the 1 subunits are located at the synaptic sites and mostly found in mature neurons , while the 5 subunits are located at the extrasynaptic sites and found mostly in immature neurons prior to the formation of the inhibitory synapse ( jacob et  al . , 2008 ,
thus , the maturation of the gabaergic function requires the precise expression of specific subunits of the gabaa receptor during postnatal brain development .
the early expression of the gabaa receptor 5 subunit is required for the tonic inhibitory function of gaba , while the late expression of the 1 subunit is required for the phasic inhibition that indicates the maturation of the gabaa receptor function .
it is well documented that stress increases glucocorticoid hormones and thereby potentiates excitotoxic damage in hippocampal gabaergic neurons ( elliott and sapolsky , 1992 , stein - behrens and sapolsky , 1992 ) .
early life stress exerts an effect on the hippocampal neurons and predisposes individuals to psychosis ( stumpf and et  al , 1989 , tornello and et  al , 1982 , zhang and et  al , 1990 , scheller - gilkey and et  al , 2003 ) .
the hippocampus exhibits subtle alterations subsequent to neuropsychiatric diseases such as schizophrenia and mania depressive disorder ( benes , 1999 ) .
previous studies in postmortem brains from schizophrenia patients have shown a decrease in hippocampal gabaergic activity that could potentiate excitotoxic damage to hippocampal interneurons , consistent with abnormal oscillatory rhythms and increased basal metabolic activity ( benes , 1999 ) .
it is still unclear how prenatal stress affects the development of gabaergic synapses in the hippocampus of the offspring . in this study
we hypothesized that prenatal stress may affect the structural and functional maturation of the gabaa receptor in the hippocampus of rat pups .
therefore , the purpose of this study was to examine the effect of maternal restraint stress on the levels of nkcc1 and kcc2 , as well as gabaa receptors 1 and 5 subunits in the hippocampus of the offspring to provide insights about the involved mechanisms of maternal stress as a cause of dysregulation of gabaergic synapses that are known to be associated with the pathogenesis of psychiatric diseases at adulthood .
pregnant rats were obtained from the national laboratory animal centre , mahidol university , salaya , thailand .
they were housed in a single housing condition with a temperature and humidity controlled environment and maintained on a 12  h light / dark cycle with free access to food and water .
each pregnant female rat was weighed on gestation day ( gd ) 721 . in the morning of gd 21 ,
each pregnant rat received nesting material , and thereafter , the cage was checked twice daily for the appearance of a litter . the day
a litter gets discovered becomes designated as postnatal day 0 ( p0 ) , and the length of gestation was noted .
all experiments were conducted according to the guidelines for care and use of the laboratory animals and approved by the experimental animal ethics committee of the institute of molecular biosciences , mahidol university , thailand ( coa.mb-acuc 2015/003 ) .
pregnant rats were divided into two groups ; 1 ) control group , 2 ) maternal restraint stress group ( n  =  4/group ) . for the restraint stress ,
each pregnant rat was put into a small plexiglas cylindrical cage in which the length can be adjusted to accommodate the size of each animal .
the restraint stress was performed during gd14 - 20 , at four hours daily intervals during the light phase of the cycle as previously described ( surakul et  al . , 2011 ,
gestation days 1420 were selected because they represent the most sensitive period for the behavioral teratogenic effect of prenatal stress ( fride & weinstock , 1984 ) .
whole hippocampal tissues were collected from rat pups at different postnatal days ( p ) from p7 , p14 , p21 , p28 and p40 , with n  =  4/group .
brain tissues were then suspended in a lysis buffer composed of 50  mm tris ph 7.4 , 150  mm nacl , 1  mm edta , 0.5% sodium deoxycholate , 1% sds , 1  mm pmsf , 1% triton - x-100 and supplemented with a complete protease and phosphatase inhibitor cocktail set ( calbiochem , germany ) , then homogenized twice with a sonicator for 10  s each .
the homogenized samples were centrifuged at 14,000  rpm , 4  c for 15  min .
cell lysates were mixed with a sodium dodecyl sulfate ( sds ) sample buffer and boiled .
equal amounts ( 20  g ) of extracted protein samples were resolved in 10% sds - page and electrophoresis at 100  v for 150  min .
the protein bands were then transferred to the pvdf membrane ( amersham , usa ) at 100v for 135  min .
the membranes were then incubated in a blocking solution containing 3% skimmed milk for nkcc1 , kcc2 and gabaa receptor 1 and 5 subunits , and 5% skimmed milk for actin at room temperature for 60  min
. then the membranes were incubated with the following specific primary antibodies purchased from the available commercial sources .
polyclonal goat anti - nkcc1 ( sc-21545 ; 1:500 ) , polyclonal goat anti - kcc2 ( sc-19420 ; 1:500 ) , polyclonal goat anti - gabaa receptor 1 subunit ( sc-7348 ; 1:500 ) , polyclonal goat anti - gabaa receptor 5 subunit ( sc-7357 ; 1:500 ) , and monoclonal mouse anti - actin ( sc-69879 ; 1:5000 ) , all antibodies were purchased from santa cruz biotechnology , usa . the membranes were then thoroughly washed 3 times using 0.1% tween - tbs ( ttbs ) for 5  min each , and then incubated with an appropriate hrp - conjugated secondary antibody . after that , the membranes were washed 3 times using 0.1% ttbs for 5  min each , and then the signals were detected by an enhanced chemiluminescence system ( ecl prime , amersham biosciences , usa ) and film exposure .
the intensities of the band were quantified using densitometry software ( image j , national institutes of health , usa ) .
the immunoblot data were corrected for corresponding product of the -actin extracted from the same tissue which serve as an internal control .
quantitative results were expressed as mean    sem , calculated from the duplicate experiments .
the statistical significance of difference between means was evaluated using student 's t - test ( unpaired , unless otherwise stated ) .
changes produced by prenatal stress were analyzed at different postnatal ages using a two way anova with the prenatal stress and postnatal ages as independent variables and the protein levels as dependent variables ; followed by a tukey 's post hoc multiple comparison test .
the probability level of p    0.05 was considered to have a statistically significant difference between the two sets of data .
we examined the effects of maternal restraint stress , during the gestation day ( gd ) 1420 , on the levels of nkcc1 and kcc2 in the hippocampus of rat pups and compared between the groups at different postnatal ages .
the results showed that maternal restraint stress caused a transient but significant increase in the level of nkcc1 in the hippocampus at p14 ( p  <  0.05 ) but no significant difference when observed at the other periods ( fig .  1 ) . for kcc2 ,
the results show that maternal restraint stress caused a transient but significant increase in the kcc2 level in the hippocampus of rat pups during the weaning period ( p21 ) ( p  <  0.01 ) and this was followed by a transient but significant decrease during the preadolescence period ( p28 ) ( p  <  0.05 ) ( fig .  2 ) . however , there was no difference in the level of kcc2 when compare between groups during the adolescence period ( p40 ) .
we found that maternal restraint stress significantly increases the nkcc1/kcc2 ratio in the pup 's hippocampus at p14 and p28 ( p  <  0.01 ) . during this period , the nkcc1/kcc2 ratios in the hippocampus of prenatally stress pups exhibited more fluctuations than those observed in the control group ( fig . 
the developmental expressions of gabaa receptor 5 and 1 subunits in the hippocampus appear in the opposite way . the 5 subunit was highly expressed during p7p14 , and then declined during p21p40 ( fig .  4 , white bar )
while the 1 subunit was expressed at a very low level during p7p14 , then continually increased during p21p28 ( fig . 
maternal restraint stress caused a transient but significant decrease in the level of the gabaa receptor 5 subunit at p14 ( p  <  0.05 ) and followed by a long term increase at p21 ( p  <  0.01 ) , p28 ( p  <  0.05 ) and p40 ( p  < 
0.05 ) as compared to the control ( fig .  4 ) . in contrast , maternal restraint stress caused a transient but significant increase in the level of the gabaa 1 subunit at p14 ( p  <  0.05 ) followed by a significant decrease at p21 ( p  <  0.01 ) and p28 ( p  <  0.01 ) ( fig . 
we found no significant difference in the level of the 1 subunit when comparing between the groups at p40 .
when the ratios of the 5/1 subunit of the gabaa receptor were calculated and compared across the different postnatal ages , we found that maternal restraint stress causes a significant decrease in the ratio of the 5/1 subunits during p7p14 ( p  <  0.01 ) , but a significant increase in the ratios of the 5/1 subunits during p21p28 ( p  <  0.05 ) ( fig .  6 ) .
in fact , we found small increase in the 5/1 ratios at p40 ; however , there was no statistically significant difference when compared with the control group .
gabaa receptor depolarization maintained by nkcc1 is important for proper brain development since it is a key factor in the control of several ca2-dependent developmental phenomena , including neuronal proliferation , migration and targeting ( rivera & et  al , 1999 ) .
kcc2 , in contrast , shifts the gabaa receptor activity from depolarization to hyperpolarization in mature neurons . in a developing hippocampus
, the level of nkcc1 continuously increases starting from p21 to adulthood ( yan et  al . , 2001 ) while the hyperpolarizing gaba is completed by the second postnatal week due to the progressive reduction of nkcc1 activity in parallel with the enhanced activity of kcc2 ( rivera and et  al , 1999 , emri and et  al , 2001 , gulyas and et  al , 2001 , stein and et  al , 2004 ) .
previous studies reported that kcc2 expression significantly increases during the second postnatal week , which is the co - incidence time point when the developmental switch of gabaa receptor activity is observed ( rivera and et  al , 1999 , clayton and et  al , 1998 ) and continually increased until p28 ( lu et  al . , 1999 ) . in the hippocampus ,
nkcc1 and kcc2 expressions show relatively similar developmental patterns indicating that both are required for the reciprocal regulation of cl homeostasis , which is important for the functional maturation of the gabaa receptor . in this study ,
maternal restraint stress induced a transient increase in the level of nkcc1 in the hippocampus of rat pups only at p14 , while there was a kcc2 increase at p21 and then a decrease at p28 .
previous studies reported that the alteration in nkcc1 under a stress response has no effect on the gabaa receptor function .
in contrast , the stress - altered kcc2 level has a profound effect on the modulation of intracellular cl concentrations ( hewitt & et  al , 2009 ) .
our finding , that maternal restraint stress increases the kcc2 level in the pup 's hippocampus at p21 , indicates the protective mechanism that counteracts the higher level of nkcc1 at p14 .
maternal restraint stress , which increases kcc2 level at p21 and then decreases it at p28 , indicates the dysregulation of intracellular cl concentrations and the gabaa receptor mediated current during the preadolescence period . in the mature pyramidal neurons , kcc2 inhibition positively shifts the gabaa receptor reversal potential , thus
, gabaa receptor activation causes depolarization ( rivera & et  al , 1999 ) .
our results indicate that maternal restraint stress may induce prolonged depolarizing of gaba in the hippocampus of rat pups until the pre - adolescence period , while in the control pups , hyperpolarizing gaba was completed by the early postnatal week .
different brain regions may exhibit differential vulnerability to the effects of stress on the level of kcc2 and its activity . for examples , in the rat hypothalamic paraventricular nuclei
, acute restraint stress has no effect on the level of kcc2 , but attenuates the kcc2 activity ( hewitt & et  al , 2009 ) .
maternal restraint stress has no effect on the level of kcc2 in the amygdala of male pups at p14 and p22 ( laloux & et  al , 2012 ) .
in contrast , prenatal stress causes a significant decrease in the kcc2 level and its activity in the hippocampus , as measured by phosphorylation of kcc2 on ser 940 residue ( sarkar & et  al , 2011 ) .
these findings , together with our results , indicate that maternal restraint stress might alter gabaa transmission in the hippocampus of prenatal stress pups during the post - weaning ( p21 ) to pre - adolescence period ( p28 ) and this mechanism might be due to the alteration in the levels of kcc2 . the underlying mechanism by which prenatal stress alters kcc2 levels remains unclear .
studies have shown that brain a derived neurotrophic factor ( bdnf ) regulates the expression of kcc2 in both the young and adult brains ( aguado and et  al , 2003 , rivera and et  al , 2002 ) .
bdnf is seen to down - regulate kcc2 expression in the adult hippocampal slices via the activation of tyrosine receptor kinase b ( trkb ) ( rivera & et  al , 2002 ) .
in addition , bdnf promotes kcc2 expression in the developing mice forebrain ( aguado & et  al , 2003 ) .
therefore , bdnf regulation of kcc2 expression varies depending on the developmental stages and brain regions .
prenatal stress has been reported to decrease bdnf levels in the rat hippocampus at p21 ( van den hove & et  al , 2006 ) .
taken together , the results suggest that an alteration in bdnf levels caused by prenatal stress might affect the kcc2 level in the pup 's hippocampus .
although it has been noted that bdnf - induced alteration in kcc2 expression was not caused by neuronal excitability and network activity ( aguado & et  al , 2003 ) , the endogenous action that is regulating the changes is still elusive .
it is assumed that maternal restraint stress altering kcc2 level in the rat pups hippocampus at preadolescence period could affect the excitatory glutamatergic synapses as well .
it has been reported that kcc2 has an important roles in the modulation of the dendritic spines and ampa receptor diffusion by interacting with sub - membranous actin cytoskeleton ( gauvain & et  al , 2011 ) , therefore indicating kcc2 is also require for the production of long - term potentiation ( ltp ) in the hippocampus of young animals .
prenatal stress has been linked to an increased risk of psychiatric disorders such as schizophrenia and depression ( charil & et  al , 2010 ) .
recent studies show that the kcc2 level is significantly decreased in the hippocampus of schizophrenia patients , while there was no change in the nkcc1 level ( hyde & et  al , 2011 ) .
thus , an increase in the nkcc1/kcc2 ratio indicates the delayed maturation of cation - chloride cotransporters in the patient 's brain , which may underlie the pathology of neuropsychiatric diseases .
recently , it was demonstrated the significant increase in the level of oxsr1 ( oxidative stress response kinase1 ) and wnk3 ( with no k [ lysine ] protein kinase3 ) in the post - mortem brain of schizophrenia patients ( arion & lewis , 2011 ) .
consequently , changes in the level of oxsr1 and wnk3 can shift the balance of chloride transport and leading to an abnormal gabaergic transmission in the prefrontal cortex , thereby contributing to the impaired neural network synchrony and cognitive dysfunction in affected individuals ( arion & lewis , 2011 ) . for the development of gabaa receptor subunits , our results are consistent with those that have been previously reported ( ramos and et  al , 2004 , laurie et  al . , 1992 ) .
we found the 5 subunit is highly expressed during the early postnatal period and declined to the adult level around the 3rd postnatal week , while the 1 subunit was initially expressed at a small level during the 1st and the 2nd postnatal week and gradually increased until reaching its peak around the 3rd postnatal week .
prenatal stress delays the developmental shift of the gabaa receptor 1 and 5 subunits that normally occur around p21 in the control pups .
this was clearly observed in the control pups that were seen to have manifested developmental increments in the 1 subunit at p21 , but not in the prenatal stress pups , at least until p40 . on the contrary , the control pups show a developmental decrease in the 5 subunit at p21 , but not in the prenatal stress pups that were seen to maintain the expression of the 5 subunit at least until p40 . as a result ,
the ratio of the 5/1 subunits in the prenatal stress pups exhibits a significant increase during p7 and p14 , but shows a significant decrease when observed at p21 and p28 , as compared to the control group .
the underlying mechanism of prenatal stress that induces a prolonged increase of 5 subunits in the rat pup 's hippocampus at preadolescence is still elusive .
a prolonged increase in the 5 subunit in the pup 's brain may create an unpredictable effect on gaba inhibitory transmission ; especially during puberty ( shen & et  al , 2007 ) . in the hippocampal ca1 and neocortical pyramidal neurons , the extrasynaptic 5 subunit of the gabaa receptor mediates tonic inhibition and plays an important role in memory and learning ( rudolph & mohler , 2014 ) . an increased expression of the gabaa receptors ' 5 subunit
is associated with memory loss ( wang & et  al , 2012 ) while the antagonist of the receptor can enhance learning and memory processes ( rudolph and mohler , 2014 , ling and et  al , 2015 ) .
thus , our results suggest that prenatal stress induces an increase in the gabaa receptor 's 5 subunit expression and the 5/1 ratios in the hippocampus at the preadolescence period may underlie the long term effects of prenatal stress on learning and memory impairment in the offspring at adulthood . for the gabaa receptors 1
subunit , our findings are consistent with previous report that reveal exposure to stress in juvenile rats can induce biphasic changes in their behavior , including hyperactivity at juveniles which in adulthood becomes hypoactivity accompanied by behavioral anxiety that are associated with the decrease of 1 subunits in the hippocampus and amygdala ( jacobson - pick & richter - levin , 2012 ) . taken together
the results are in agreement , the juvenile period is a sensitive time and is more vulnerable to stress than other periods and this supports the hypothesis that prenatal stress is a predisposing factor for various neuropsychiatric diseases and memory impairment at later life . in this study , we added further information that the developmental expression of the gabaa receptor 1 subunit is similar to the developmental pattern of the kcc2 . both the gabaa 1 subunit and
kcc2 reach their peak around p21p28 and this indicates that the gabaa 1 subunit and kcc2 might coordinate in enhancing the gabaa receptor mediated synaptic inhibition that occurs around this period .
interestingly , we found that prenatal stress induces changes in the levels of the gabaa 1 subunit and the kcc2 in a similar way .
our results correspond to what has been previously documented in that kcc2 could modulate the expression level of the gabaa receptor 1 subunit via an alteration in intracellular [ cl ] and the decay rate of gaba - mediated inhibitory transmission ( houston & et  al , 2009 ) .
indeed , lower intracellular [ cl ] resulted in a faster decay rate of the gaba transmission ( moroni & et  al , 2011 ) .
it has been shown that kcc2 can manipulate the expression of gabaa receptor subunits , i.e. , overexpression of kcc2 results in the reduction of intracellular [ cl ] and leads to an increase in the level of 1 and  subunits ( succol & et  al , 2012 ) .
taken together , these results are in accordance with the hypothesis that prenatal stress reduces the kcc2 levels , which might lower the intracellular [ cl ] , that acts as the intracellular signal and induces a faster decay rate of the gabaa receptor gating and , thus , decreases the expression of the 1 subunit of the gabaa receptor ( succol & et  al , 2012 ) .
these changes indicate a delayed maturation of the gabaergic function in the hippocampus of prenatal stress pups , especially during the preadolescence period .
additionally , it was reported that stress disrupts the gabaergic function in the brain in many ways .
stress induces dysfunction of the inhibitory network and impairs rhythmic oscillations leading to cognitive deficits commonly found in psychiatric disorders ( hu & et  al , 2010 ) .
prenatal stress disturbs the distribution of gabaergic interneurons in the cortical plate , reflecting the changes occurring in tangential migration and radial integration in the developing cortex ( stevens & et  al , 2013 ) . prenatal stress causes a significant decrease in the frequency of spontaneous ipscs in the immature hippocampal neurons ( grigoryan & segal , 2013 ) and increasing the vulnerability to stressful situations in the offspring during adulthood accompanied by a reduction of benzodiazepine binding in the hippocampus ( fride & et  al , 1985 ) . in summary
, this study has shown that maternal restraint stress has the ability to differentially alter the levels of nkcc1 , kcc2 , and gabaa receptor 1 and 5 subunits in the hippocampus of rat pups .
consequently , these changes can lead to an imbalance of inhibitory transmission that may delineate the linkage between prenatal stress and neuropsychiatric disorders in later life .
our findings reveal that there is a strong connection between early life stress exposures with an increased risk of developing psychiatric disorders at adulthood .
furthermore , a reduction of kcc2 levels has been linked to the cause of epilepsy , which is considered as a risk factor for schizophrenia and autism . moreover ,
a prolonged increase in the 5 subunits in the hippocampus of rat pups during adolescence indicates a prolongation of the slow decay rate of inhibitory transmission in the pup 's hippocampus and predisposes it for neuropsychiatric diseases and memory impairment in adulthood .
experiencing adverse events during pregnancy has a negative impact on brain development and may increase vulnerability to developing neurological and psychiatric disorders later in life .
as we demonstrated , fetal exposure to maternal stress hormones delays structural and functional development of gaba transmission in the rat pup 's hippocampus during the preadolescence period .
these changes may lead to the dis - regulation of gaba inhibitory transmission in the developing hippocampus .
similar patterns of changes in the kcc2 and gabaa receptor 1 subunits were observed in response to early life stress , accompanied by supporting evidence that indicates changes in kcc2 levels may underlie the effect of maternal stress on the alterations in the 1 subunit of the gabaa receptors .
moreover , prenatal stress also increases gabaa receptor 5 subunit expression throughout the preadolescence period , which may underlie the learning and memory impairments in the offspring at adulthood . in summary , we have provided an explanation of certain prenatal factors mediating structural and functional development of the gabaergic synapse that may be the link between prenatal stress and the emergence of neuro - psychiatric disorders at adulthood .
 the gabaergic synapse undergoes structural and functional maturation during early brain development . 
 maternal stress alters gabaergic synapses in the pup 's brain that are associated with the pathophysiology of neuropsychiatric disorders in adults ; however , the mechanism for this is still unclear . in this study 
 , we examined the effects of maternal restraint stress on the development of cation - chloride cotransporters ( cccs ) and the gabaa receptor 1 and 5 subunits in the hippocampus of rat pups at different postnatal ages . 
 our results demonstrate that maternal restraint stress induces a transient but significant increase in the level of nkcc1 ( sodium  potassium chloride cotransporter 1 ) only at p14 , followed by a brief , yet significant increase in the level of kcc2 ( potassium - chloride cotransporter 2 ) at p21 , which then decreases from p28 until p40 . 
 thus , maternal stress alters nkcc1 and kcc2 ratio in the hippocampus of rat pups , especially during p14 to p28 . 
 maternal restraint stress also caused biphasic changes in the level of gabaa receptor subunits in the pup 's hippocampus . 
 gabaa receptor 1 subunit gradually increased at p14 then decreased thereafter . on the contrary , 
 gabaa receptor 5 subunit showed a transient decrease followed by a long - term increase from p21 until p40 . 
 altogether , our study suggested that the maternal restraint stress might delay maturation of the gabaergic system by altering the expression of nkcc1 , kcc2 and gabaa receptor 1 and 5 subunits in the hippocampus of rat pups . 
 these changes demonstrate the dysregulation of inhibitory neurotransmission during early life , which may underlie the pathogenesis of psychiatric diseases at adolescence . 