stress is defined as any condition that seriously disrupts 
 physiological and psychological homeostasis ranging from anxiety to 
 posttraumatic stress disorder , and affects cognitive functions both in 
 animal models and in humans [ 24 ] .
the hippocampus is of special significance 
 in this respect because it has been shown to play a major role in regulating 
 stress [ 5 , 6 ] , and to be involved in some aspects of learning and memory [ 713 ] . at present , long - term potentiation ( ltp ) of synaptic 
 transmission in the hippocampus
is the most studied neurophysiological model 
 for learning and memory processes in the mammalian nervous system .
depending on the type of stress and 
 the procedures used , stress has been shown to have different effects on 
 different measures of synaptic plasticity .
there is a general agreement that 
 ltp in area ca1 of the hippocampus is impaired following stress [ 4 , 1418 ] .
some studies have also shown that stress impairs ltp in the dentate 
 gyrus ( dg ) of the hippocampus [ 16 , 19 , 20 ] , while others reported intact ltp 
 in the dg following stress [ 14 , 21 ] .
thus , dg ltp is considered to be less 
 sensitive to stress compared to ltp in ca1 .
although ltp is a widely accepted model of learning and 
 memory , debates continue over its validity , and controversial results regarding 
 its behavioral correlates are reported ( for review , see ) .
a different 
 level of processing that is likely to be relevant to memory formation is local 
 circuit activity . when examining this level of processing , the focus is on 
 interactions between local , mostly inhibitory gabaergic neurons and pyramidal 
 or granular principle cells in the hippocampus and cortex [ 24 , 25 ] .
this is 
 in contrast to the focus on ltp of input excitatory synapses onto principle 
 cells , which is responsible for transmitting information from one region to 
 another .
inhibitory interneurons exert a powerful control over local circuit 
 activity through feedforward and feedback inhibition .
modification of local 
 circuits can affect the computational properties of the region , and therefore 
 affect its involvement in behavior .
in the current study , local circuit activity and plasticity 
 were measured by using frequency - dependent inhibition ( fdi ) and commissural 
 modulation protocols , following exposure to behavioral stress .
fdi is suggested to reflect gaba - mediated inhibition by 
 perforant path- ( pp- ) activated interneurons onto granule cells . 

increasing stimulus frequency from 0.1  hz to 1.0  hz results in the reduction of 
 the population spike ( ps ) of the field potential response to stimulation of the 
 pp . our lab has previously shown that fdi in the dg is nmda - dependent 
 , gaba - mediated , and that delivering theta - burst stimulation ( tbs ) to the 
 pp of the hippocampus induced a lasting reduction in fdi .
the dg commissural pathway is activated by stimulating the 
 contralateral dg at different intervals prior to pp stimulation .
stimulation of 
 the commissural pathway induces a biphasic , inhibitory / excitatory effect on 
 granule cell responsiveness to pp stimulation .
although the effect of behavioral stress on induction of 
 hippocampal ltp has been studied extensively , to our knowledge no research has 
 established the relationship between stress and local circuit activity and 
 plasticity .
the current study addresses this issue in order to further explore 
 the potential relevance of local circuit activity to learning and memory .
our 
 aim in this study was to characterize local circuit activity and plasticity in 
 the dg of the hippocampus following exposure to behavioral stress .
adult , male sprague dawley rats , weighing 240330  g , from harlan 
 ( jerusalem , israel ) maintained five per cage on a 12-hour light / dark cycle 
 with water and laboratory rodent chow ad libitum .
trunk blood was collected following decapitation and 
 samples were centrifuged at 3000 r.m.p . for 20 minutes at 4c .
corticosterone was measured using a radioimmunoassay kit 
 ( coat - a - count , diagnostic products corporation , los angeles , calif , usa ) .
rats were anaesthetized ( 6% chloral hydrate in 100  ml 
 saline ; 0.5  ml/100  g. ip ) and prepared for acute stimulation of the 
 perforant path and for recording of field potentials in the dentate gyrus as 
 described before .
rats were placed in a head holder in a 
 stereotaxic frame and small holes were drilled in the skull to allow the 
 insertion of electrodes in the brain .
a recording microelectrode ( glass , tip 
 diameter 25  m filled with 2  m nacl , resistance 14  m ) was placed in the dentate gyrus ( coordinates : 4  mm posterior to bregma , 2.5  mm lateral to midline ) . 

a bipolar 125  m stimulating electrode was implanted in the ipsilateral 
 angular bundle to stimulate the perforant path ( coordinates : 8  mm posterior to bregma , 4  mm lateral to midline ) .
the 
 depth of the electrodes was adjusted to maximize the size of the evoked 
 positive - going excitatory postsynaptic potential ( epsp ) recorded in the hilus 
 of the dentate gyrus .
evoked responses were digitized 
 ( 10  khz ) and analyzed using the cambridge electronic design 1401 + and its 
 spike2 software .
offline measurements were made of the amplitude of the ps and 
 the slope of the epsp using averages of 5 successive responses to a given stimulation 
 intensity applied at 0.1  hz .
test stimuli ( monopolar pulses , 100-microsecond duration , 
 intensity adjusted to yield a ps of 3050% of the maximal pretetanus value ) 
 were delivered at 0.1  hz .
after positioning the electrodes , the rat was left 
 for 20 minutes before commencing the experiment . during recording the rats were 
 maintained at 37  1c with a homeothermic blanket system ( harvard ) .
ltp was induced by a tbs ( 3 sets of 10 trains , 
 each consisting of 10 pulses at 100  hz .
intertrain interval : 200 milliseconds , 
 and the interval between each set : 1 minute , trains are delivered at 2x test 
 stimulus intensity ) .
ltp was measured as the difference in epsp 
 slope before and 60 minutes after tbs . 

we defined ltp as an increase of the least 20% in the epsp slope of the 
 evoked potentials 60 minutes after application of tbs .
to determine fdi , 10 
 pulses were delivered at 0.1  hz followed by 10 pulses at 1.0  hz , as described 
 before .
the ps or epsp slope of the 10 responses at 0.1  hz were averaged and compared to the 10 
 responses at 1.0  hz in each set .
inhibition is expressed as an fdi index which was assessed by dividing the 
 averaged response at 1.0  hz by the averaged response at 0.1  hz . the dg commissural 
 pathway was activated by stimulating the contralateral dg at different 
 intervals prior to pp stimulation ( 15 , 30 , 80 , and 150 milliseconds ) as 
 described before .
stimulation of 
 the commissural pathway induces a biphasic inhibitory / excitatory effect on 
 granule cell responsiveness to pp stimulation [ 31 , 32 ] .
commissural - induced modulation is expressed by commissural 
 index which was evaluated as the ratio of the size of the response to pp stimulation 
 after commissural stimulation divided by that of the response to pp stimulation 
 with no priming stimulation . in order to measure tbs - induced alterations on frequency - dependent 
 inhibition or on commissural - induced inhibition and facilitation
, the 
 stimulation intensity following the induction of ltp was adjusted to yield a ps 
 size comparable to pre - tbs level . 

apparatusthe maze employed is a four - armed 
 black opaque plexiglas platform , elevated 50  cm above ground .
two opposite arms are 
 enclosed by 40  cm high plexiglas walls on both sides and on the outer edges of the platform , that 
 is ,  closed ,  while the two remaining opposite arms are  open ,  and are 
 surrounded only by a 1  cm high plexiglas rim , which serves as a tactile guide to animals in the open 
 areas .
individual rats were placed 
 in the central platform , faced towards different arms in randomized order . 
 
the maze employed is a four - armed 
 black opaque plexiglas platform , elevated 50  cm above ground .
two opposite arms are 
 enclosed by 40  cm high plexiglas walls on both sides and on the outer edges of the platform , that 
 is ,  closed ,  while the two remaining opposite arms are  open ,  and are 
 surrounded only by a 1  cm high plexiglas rim , which serves as a tactile guide to animals in the open 
 areas .
individual rats were placed 
 in the central platform , faced towards different arms in randomized order .
each 
 five - minute session was recorded using an overhead video camera connected to a 
 monitor / recorder in an adjacent observation room .
animals were placed in the 
 central platform and allowed to explore for five minutes .
animals were then 
 subjected to the acute swim stress procedure , and then allowed to rest for 90 
 minutes .
after the resting period animals were placed once again in the maze 
 for a five - minute poststress test .
animals were 
 scored as being in an open or closed arm only when all four paws passed over the 
 open / closed dividing line .
the water container used for the forced 
 swim procedure consists of a circular container of water ( 50  cm in diameter with a rim 75  cm high ) .
individual rats were subjected 
 to a single 15-minute swim session in the water container .
after this single 
 swim session , rats were allowed to dry in a resting cage for 30 minutes , and 
 then anesthetized and taken to electrophysiology .
in order to validate that 
 animals subjected to the acute swim stress procedure were indeed behaviorally 
 affected , we used the elevated plus maze test . a paired sample t - test revealed that in the poststress 
 session , animals spent less time in the open arms ( see figure 
 1(a ) , t(7 ) = 4.25 , p < .005 ) .
an independent t - test 
 revealed that stress was associated with a significant increase in 
 corticosterone levels ( see figure 1(b ) , t(13 ) = 4.29 , p <
the stimulation intensity used to elicit a baseline 
 response was not different between the control and stress groups ( t(25 ) = 0.93 , n.s . ) . 

there was no significant difference between control and stressed rats in the 
 amplitude of the baseline ps ( t(25 ) = 1.44 , n.s . ) ) and fepsp slope ( t(25 ) = 0.26 , n.s . ) . upon altering the frequency of stimulation from 0.1  hz to 
 1.0  hz
, a marked reduction of the ps was observed in both control and stressed 
 rats as indicated by the fdi index ( see figure 2(a ) ) .
this 
 reduction was apparent prior to tbs application and 60 following it ( see figure 
 2(b ) , f(1,14 ) = 101.16 , p < .05 ) .
no significant differences in fdi index were 
 found between the two groups on both tests ( f(1,14 ) = 0.18 , n.s . )
although no main effect was observed for group under the 
 different conditions , the analysis of variance indicated a significant group x 
 test interaction ( f(1,14 ) = 6.53 , p < .05 ) .
the initial pre - tbs fdi 
 index of stressed rats was lower than that of controls whereas the post - tbs fdi 
 index was higher . altering stimulation frequency from 0.1  hz to 1.0 
hz resulted 
 in a slight but significant reduction in the epsp slope in both groups . in 
 control rats ,
stimulation at 1.0  hz reduced the slope of the epsp to 0.850.05 of its size during stimulation at 0.1  hz ( t(7 ) = 11.49 , p < .
hz reduced the slope of the epsp to 0.880.02 of its size during stimulation at 0.1  hz [ t(7 ) = 3.66 , p < 0.05 ] . 

there was no significant difference in epsp fdi between the two groups and , in 
 contrast to the ps , the application of tbs did not affect epsp fdi in either 
 group . in both control and
stressed rats , the response to the pp 
 stimulation was inhibited by priming stimulation of the commissural path at 
 interpulse intervals of 15 milliseconds and 30 milliseconds ( see figure 3(a ) ) . 
 when using the 15-millisecond interpulse interval , exposure to stress induced 

significantly higher inhibition , as expressed by the small commissural index of 
 stressed rats compared to controls .
this increase in inhibition was apparent 
 before and after tbs application ( f(1,12 ) = 64.36 , p < .005 ) .
the 
 application of tbs did not cause any significant changes in commissural - induced 
 inhibition in either group ( f(1,12 ) = 0.126 , n.s ) ( see figures 3(b ) and 3(c ) ) . when priming stimulation of the commissural path at interpulse 
 interval of 30 milliseconds , stressed rats exhibited significantly higher 
 commissural - induced inhibition than control rats before and after tbs 
 application ( f(1,12 ) = 90.42 , p <
.001 ) , as seen in the 15-millisecond interpulse 
 interval . interestingly , both groups have shown a significant decrease in commissural - induced 
 inhibition following tbs application ( f(1,12 ) = 13.68 , p < .005 ) .
( see figures 
 3(b ) and 3(c ) ) . when using the 80-millisecond and 150-millisecond interpulse 
 intervals ,
the response to the pp stimulation was facilitated in both control 
 and stressed rats , as expressed by the 
 large commissural indexes . in both groups ,
commissural - induced facilitation was 
 significantly increased after tbs application ( 80 milliseconds : f(1,12 ) = 17.55 , p < 
 .005 ; 150 milliseconds : f(1,12 ) = 7.87 , p < .05 ) .
no differences were 
 found between the two groups before or after tbs application ( 80 milliseconds : 
 f(1,12 ) = 0.68 , n.s ; 150 milliseconds : f(1,12 ) = 0.057 , n.s ) .
sixty minutes following the application of tbs , there was a 
 clear potentiation of the slope of the epsp in control rats ( 38%  5.9 , t(13 ) = 
 6.38 , p < .001 ) and stressed rats ( 35%  9.3 , t(10 ) = 3.54 , p < .05 ) , compared 
 to pre - tbs levels .
the application of 
 tbs also resulted in an increase in ps amplitude in both groups ( control : 128% 
  32 , t(13 ) = 4.02 , p
< .005 ) ; 
 stress : 127%  38 , t(10 ) = 4.6 , p < 
 .05 ) .
no significant difference in ltp was found between the two 
 groups ( epsp - ltp : t(23 ) = 0.33 ; ps - ltp : t(23 ) = 0.93 , n.s ) ] ( see figure 4 ) .
in the present study , we have examined the effects of behavioral 
 stress on local circuit activity and plasticity in the dg .
we report that when 
 using fdi , this form of local circuit activity was reduced following the 
 application of tbs in both control and stressed rats . however , this reduction 
 in fdi plasticity was greater in the stressed rats compared to controls . when using commissural - induced modulation , inhibition was 
 significantly higher in stressed rats both before and after tbs at 15- and 30-millisecond 
 intervals . fdi results from direct afferent excitation of inhibitory 
 interneurons or of other cells that excite inhibitory cells [ 26 , 35 ] , and thus 
 provides a simple method for measuring local circuit activity mediated by 
 dentate interneurons .
in the present study , an attempt was made to find out 
 whether behavioral stress would have an effect on fdi activity and plasticity . 

our results reveal that both control and stressed rats have shown a decrease in 
 fdi following tbs , and that differences between pre- and posttetanic levels of 
 fdi were not significantly different between the two groups . when examining the 
 differences between pre- and post - tbs levels of fdi within each group ,
a 
 greater difference was observed in the stressed group , which may suggest that 
 undergoing behavioral stress has caused an increase in fdi plasticity .
this increased plasticity is somewhat surprising since 
 exposure to stress is typically shown to suppress plasticity and to impair 
 learning [ 3640 ] .
it should be noted though that most studies 
 related to stress effects on plasticity focused mainly on ltp and on ca1 area 
 of the hippocampus .
the effects of stress on ltp in other brain regions are 
 less consistent [ 14 , 21 , 41 ] . 

furthermore , even in ca1 , while stress may suppress ltp , it is reported 
 to enhance other forms of plasticity , such as ltd [ 17 , 42 ] .
stressed rats have shown a marked increase in inhibition 
 both before and after the application of tbs , indicating a lasting modification 
 in this form of local circuit activity .
this result is in agreement with other 
 studies that have shown that stress levels of corticosterone produce a profound 
 and long - lasting inhibitory influence on hippocampal cell activity [ 4347 ] .
although stress has caused a reduction in local circuit activity in 
 this case , no alteration in commissural local circuit plasticity was observed 
 after tbs application .
the fact that fdi activity was not affected by the stress 
 while commissural - induced modulation was significantly affected suggests that 
 stress may affect only a subpopulation of inhibitory interneurons .
indeed , it 
 has been previously suggested that interneurons in the hippocampus may be 
 divided into those showing no activity - dependent plasticity and those that do . 

it was further suggested that activity - dependnet plasticity of this sort may 
 contribute to mood and anxiety disorders .
the fact that an increase in inhibitory activity was 
 observed in the hippocampus may imply that 
 deficits in cognitive functioning and flexibility might take place , as has been 
 suggested before for aged rats .
this does not necessarily mean that rats 
 that have undergone stress would show impaired learning in a specific task , but 
 it is possible that they would be less adaptive if required to shift to a new 
 coping strategy during task acquisition .
for example , stress has been shown to 
 cause impairment in reversal learning and induced perseveratory behavior in the 
 morris water maze without having a significant effect on task acquisition . in the present study ,
as indicated above , the effects of stress on dentate gyrus 
 ltp may depend on the exact nature of the stress experience [ 14 , 21 , 41 ] . 

interestingly , although stress seemed to have caused alterations in local 
 circuit plasticity ( as observed in the fdi findings ) and in local circuit 
 activity ( as observed by the commissural modulation findings ) , it had no effect 
 on ltp induction .
this further supports the notion that local circuit activity 
 and plasticity is independent of synaptic plasticity such as ltp [ 18 , 28 ] .
the study of the effects of stress on gabaergic 
 neurotransmission is of special interest because it has been suggested that 
 gaba plays a role in the pathophysiology of mood and anxiety disorders [ 5054 ] .
the results presented here may further support the potential role 
 of gaba - impaired modulation of neural activity and plasticity in stress - related 
 disorders .
our results suggest that stressful experience may lead to 
 alterations in local circuit activity and plasticity . understanding the 
 alterations that take place in these local interneurons
may contribute to a 
 better understanding of their involvement in memory formation and regulation 
 under normal and psychopathological conditions .
 studies have shown that , depending on its severity and context , stress can affect neural plasticity . 
 most related studies focused on synaptic plasticity and long - term potentiation ( ltp ) of principle cells 
 . however , evidence suggests   that following high - frequency stimulation , which induces ltp in principal cells , modifications also take place at the level of complex interactions with interneurons within the dentate gyrus , that is , at the local circuit level . 
 so far , the possible effects of stress on local circuit activity and plasticity were not   studied . 
 therefore , we set out to examine the possible alterations in local circuit activity and plasticity following exposure to stress . 
 local circuit activity and plasticity were measured by using frequency dependant inhibition ( fdi ) and commissural modulation protocols following exposure to a 15 minute - forced swim trial . 
 exposure to stress did not alter fdi . 
 the application of theta - burst stimulation ( tbs ) reduced fdi in both control and stressed rats , but this type of plasticity was greater in stressed rats . 
 commissural - induced inhibition was significantly higher in stressed rats both before and after applying theta - burst stimulation . 
 these findings indicate that the exposure to acute stress affects aspects of local circuit activity and plasticity in the dentate gyrus . 
 it is possible that these alterations underlie some of the behavioral consequences of the stress experience . 
1. INTRODUCTION
2. METHODS
3. RESULTS
4. DISCUSSION