animals : all experiments were approved by the animal care and use committee of the faculty of agriculture at the university of tokyo , according 
 to guidelines adapted from the consensus recommendations on effective institutional animal care and use committees by the scientists center for 
 animal welfare .
experimentally nave male wistar rats ( aged 8 weeks ) were purchased from charles river laboratories japan ( yokohama , japan ) .
they were housed with three animals 
 per cage in a room with an ambient temperature of 24  1 c and a humidity of 45  5% .
the room had a 12-hr light /12-hr dark cycle ( lights were switched on at 
 8:00 ) .
all rats were housed separately and were handled for 3 min twice daily , commencing 3 days before 
 the conditioning day .
fear conditioning : fear conditioning was performed in an illuminated room between 9:00 and 18:00 , as described in our previous studies [ 11 , 23 ] . a subject from the conditioned group
was placed in an acrylic conditioning box 
 ( 28  20  27 cm ) for 20 min , where it received 7 repetitions of a 3-sec tone ( cs , 8 khz , 70 db ) that terminated concurrently with a foot shock ( 0.5 sec , 0.75 
 ma ) .
we also prepared the non - conditioned group by presenting the tone and foot shock separately during a 20-min period .
fear - expression test : we performed a fear - expression test as described in our previous studies [ 11 , 14 ] .
a test box ( 25  25  35 cm ) was placed in a 
 dark room illuminated with dim red light .
the box had three acrylic walls , one wire mesh wall and a wire mesh ceiling .
the wire mesh wall was constructed with 
 1-cm gauge mesh in the lower section ( 20 cm ) and vertical bars spaced 1 cm apart in the upper section ( 15 cm ) , which prevented the rats from 
 climbing up to the ceiling .
the subject was placed in the box and kept undisturbed during a 3-min acclimation period .
then , a cs was presented five times for 3 sec each at 1-min intervals 
 during the first half of the 10-min experimental period .
the behavior of the subjects during the acclimation and experimental periods was recorded with a video 
 camera ( hdr - hc9 ; sony , tokyo , japan ) and an hdd - bd recorder ( dmr - bw770 ; panasonic , osaka , japan ) .
sixty minutes after the beginning of the acclimation period , each subject was deeply anesthetized with sodium pentobarbital and intracardially perfused with 
 saline , followed by 4% paraformaldehyde in 0.1 m phosphate buffer .
the brain was removed and immersed overnight in the same fixative , and then placed in 30% 
 sucrose / phosphate buffer for cryoprotection .
we used the avidin - biotin - peroxidase immunohistochemistry method to detect fos expression , as described previously 
 [ 15 , 16 , 19 ] .
briefly , we collected 
 successive 30-m sections from bregma 2.16 mm through bregma 3.24 mm .
half of the sections were stained with cresyl violet to confirm the 
 location of the nucleus .
the remaining sections were incubated with a primary antibody to c - fos protein ( 1:7,500 ; pc38 , merck millipore , billerica , ma , u.s.a . ) 
 for 65 hr and a biotinylated anti - rabbit secondary antibody ( ba-1000 , vector laboratories , burlingame , ca , u.s.a . ) for 2 hr .
the sections were then processed 
 using the abc kit ( vector laboratories ) , and staining was developed by incubating the tissue in a diaminobenzidine solution with nickel intensification .
data analyses and statistical procedures : the data are expressed as means  standard error of the means ( sem ) .
a researcher who was blind to the experimental conditions recorded the duration of freezing ( immobile 
 posture , with cessation of skeletal and vibrissae movement , except for that associated with respiration ) and the frequency of walking ( number of steps taken with 
 the hind paws ) using microsoft excel - based visual basic software to record the duration and number of key presses , as in our previous studies [ 17 , 19 , 24 ] .
the behavioral data for the 
 conditioned and non - conditioned subjects during the acclimation and experimental periods were compared using a manova followed by fisher s plsd post hoc test . for immunohistochemical analyses of the bla and cea
, we counted the number of fos - immunoreactive cells in each bilateral nucleus in all sections .
we then 
 compared the total number of immunoreactive cells between the conditioned and non - conditioned groups using a student s t test .
when we detected 
 activation of the nucleus , we compared the total number of immunoreactive cells between the right and left sides in each group using a paired t 
 test . in order to assess the relationship between fear and neural responses
, the correlation between duration of freezing and number of fos - immunoreactive cells in the 
 bla and cea was analyzed using pearson s correlation analysis .
rats were either fear - conditioned ( conditioned group : n = 10 ) or non - conditioned ( non - conditioned group : n = 11 ) to an auditory 
 cs on the conditioning day .
then , 24 hr after the conditioning procedure , each subject was placed in the test box and kept undisturbed for 3 min as an acclimation 
 period .
then , during a subsequent 10-min experimental period , five cs tones were presented . as summarized in table 1table 1.behavioral responses during the acclimation periodgroupfreezing ( sec)walking ( steps)non - conditioned10.9  5.545.9  5.4conditioned3.1  1.254.6
 6.0data are expressed as means  standard error of the mean . , we found no difference between the conditioned and non - conditioned groups during the initial acclimation period 
 ( f(2,18)=0.964 , p=0.400 ) .
in contrast , behavioral responses during the experimental period were significantly 
 affected by the conditioning procedure ( f(2,18)=14.3 , p<0.01 ) .
a post hoc test revealed that the conditioned group 
 showed increased freezing ( p<0.01 ) and decreased walking ( p<0.01 ) compared with the non - conditioned group ( fig .
( a ) duration of freezing and frequency of walking ( mean + sem ) and ( b ) the number of 
 fos - immunoreactive cells ( mean + sem ) in the basolateral complex of the amygdala ( bla ) and central amygdala ( cea ) of fear - conditioned and non - conditioned 
 subjects . * p<0.05 according to a manova followed by fisher s plsd post hoc test for behavioral results , and according to a student s 
 t test for fos expression in the bla and cea . ) . along with these behavioral responses ,
the conditioned group showed increased fos expression in the bla ( t19= 3.29 , 
 p<0.01 ) and cea ( t19= 2.30 , p<0.05 ) compared with the non - conditioned group ( fig .
( a ) duration of freezing and frequency of walking ( mean + sem ) and ( b ) the number of 
 fos - immunoreactive cells ( mean + sem ) in the basolateral complex of the amygdala ( bla ) and central amygdala ( cea ) of fear - conditioned and non - conditioned 
 subjects . *
p<0.05 according to a manova followed by fisher s plsd post hoc test for behavioral results , and according to a student s 
 t test for fos expression in the bla and cea . in the bla ( fig .
2.representative photomicrograph showing fos immunoreactive cells in the left ( a ) and right ( b ) basolateral complex of the amygdala and in the left ( c ) and 
 right ( d ) central amygdala of fear - conditioned subjects . )
, fos expression was similar between the right and left hemispheres , both in the conditioned ( t9= 1.25 , 
 p=0.244 ) and non - conditioned groups ( t10= 0.698 , p=0.501 ) ( fig . 

3.number of fos - immunoreactive cells ( mean + sem ) in the right and left basolateral complex of the amygdala ( bla ) and central amygdala ( cea ) of 
 fear - conditioned and non - conditioned subjects . *
p<0.05 according to a paired t test . ) . in the cea ( fig .
2c and 2d ) , we found that fos expression in the conditioned group was greater in the right 
 hemisphere compared with that in the left hemisphere ( t9= 2.75 , p<0.05 ) ( fig . 

fos expression in the non - conditioned group was similar between the right and left hemispheres ( t10= 1.25 , 
 p=0.241 ) ( fig .
representative photomicrograph showing fos immunoreactive cells in the left ( a ) and right ( b ) basolateral complex of the amygdala and in the left ( c ) and 
 right ( d ) central amygdala of fear - conditioned subjects .
number of fos - immunoreactive cells ( mean + sem ) in the right and left basolateral complex of the amygdala ( bla ) and central amygdala ( cea ) of 
 fear - conditioned and non - conditioned subjects . * p<0.05 according to a paired t test . in the fear - conditioned group , we further assessed the correlation between the duration of freezing during the experimental period and fos expression in the 
 bilateral bla ( p=0.265 ) , bilateral cea ( p=0.683 ) , right bla ( p=0.445 ) , left bla ( p=0.160 ) , 
 right cea ( p=0.557 ) and left cea ( p=0.979 ) .
when fear - conditioned subjects were re - exposed to the auditory cs , they showed fear responses , such as increased freezing and decreased walking . along with these 
 behavioral responses , fear - conditioned subjects exhibited activation of the bla and cea , as assessed by fos expression .
these results confirmed that the 
 fear - conditioning was successful in our subject group . when we compared fos expression between the hemispheres , fos expression in the cea , but not in the bla , was 
 greater in the right hemisphere compared with the left hemisphere .
these results suggest that the right cea is more strongly activated in response to the auditory 
 cs . in the present study ,
fear - conditioned subjects showed fear responses and an increment of fos expression in the amygdala simultaneously .
given that the amygdala 
 plays a critical role in fear responses [ 21 , 22 ] , we assume a causal relationship 
 between these 2 phenomena .
however , we can not deny an alternative possibility that the observed difference in fos expression was ascribed to the activation of the 
 neurons that are not related to fear responses , because we did not confirm the type of neurons that showed fos expression . to clarify this
however , the correlation was not significant possibly due to the small number of the subjects . therefore , further analyses 
 are crucial to clarify this point . based on the findings in the present and previous studies
, we hypothesize that the cea is the region of the amygdala that plays a lateralized role in auditory 
 fear conditioning . in the present study
, we found stronger activation in the right hemisphere in the cea , but not the bla .
in addition , in our previous study in 
 which we obtained results suggesting a lateralized role of the amygdala , the lesioned brain area included the cea . 

this hypothesis may also explain why previous studies have not reported a lateralized role of the amygdala . in rats with pre - training   or post - training [ 2 ,
20 ] electrical lesion of the unilateral 
 amygdala , differences in fear responses did not occur according to the lesioned side .
however , the lesions in these studies appear to have been placed mainly on 
 the bla , such that the damage to the cea was likely minimal . given that the cea mediates fear responses to the auditory cs independently from the bla , it is possible that the residual cea mediated fear responses , which prevented the observation of a lateralized role of the 
 amygdala in these studies . in contrast to auditory fear conditioning , contextual fear conditioning has been reported to be right amygdala dominant .
this may be attributable to the facts 
 that an intact basal amygdala is indispensable for contextual fear conditioning . in an above - mentioned study , freezing in response to a contextual cs
was reduced 
 when a post - training , but not pre - training , electrical lesion was placed mainly on the right as compared with the left bla .
specifically , rats received an electrical lesion of the entire 
 amygdala in one hemisphere and a small chemical lesion of each sub - nucleus of the amygdala in the contralateral hemisphere .
then , these rats underwent fear 
 conditioning and were re - exposed to the contextual cs .
the authors found that rats showed reduced freezing when the left amygdala and right bla were lesioned as 
 compared with when the right amygdala and left bla were lesioned .
given that the cea requires an intact bla , 
 specifically an intact basal amygdala , to mediate fear responses to a contextual cs , it is possible that lesioning the bla was sufficient to impair the function of the ipsilateral cea , which enabled us to observe the lateralized 
 role of amygdala in the present study .
consistent with this notion , fos expression in the basal amygdala and cea in response to the contextual cs was greater in 
 the right hemisphere than in the left hemisphere .
one possible explanation for the lateralized role of the amygdala is that the conditioning procedure induces more synaptic plasticity in the right cea than in 
 the left cea .
some of the neurons that induce fear responses receive both synapses 
 transmitting foot shock information and synapses transmitting the cs information .
the foot shock information and cs information strongly and weakly activate the 
 neurons , respectively .
when these activations occur simultaneously during fear conditioning , the synapses transmitting the cs information are strengthened , which 
 enable the cs alone to strongly activate the neuron . as a result
previous findings suggest that this synaptic plasticity can be more easily established in the right cea than in the left cea .
for example , neurons 
 in the right cea show greater activation to pain than those in the left cea .
in addition , pain activates the 
 extracellular signal - regulated kinases required for synaptic plasticity associated with auditory fear conditioning   in 
 the right , but not the left , cea .
therefore , during fear conditioning , it is possible that the pain caused by foot 
 shocks activates a greater number of neurons that induce fear responses in the right compared with the left cea , which increases the number of the neurons that 
 receive cs information simultaneously . as a result ,
the number of strengthened synapses in the right cea becomes higher than that in the left cea , leading to 
 right hemisphere dominance in fear responses to the cs . in summary
, we found that fos expression in the cea , but not the bla , was greater in the right hemisphere compared with the left hemisphere .
these results 
 suggest that the right cea shows a greater degree of activation in response to an auditory cs .
although lateralized brain function has been reported for both fear 
 conditioning and other phenomena , the biological significance of lateralization has yet to be clarified .
 pavlovian fear conditioning is an experimental procedure in which a conditioned stimulus ( cs ) acquires an ability to elicit fear responses . this type of 
 conditioning depends on the basolateral complex of the amygdala ( bla ) and/or central amygdala ( cea ) . 
 we previously found that rats showed reduced fear responses 
 to an auditory cs when they were subjected to a pre - training chemical lesion of the entire right amygdala as compared with the left amygdala . based on this 
 finding , we hypothesize that the bla and/or cea in the right hemisphere will be more strongly activated by an auditory cs than those in the left hemisphere . to 
 test this hypothesis , 
 we re - exposed fear - conditioned and non - conditioned rats to an auditory cs 1 day after fear conditioning . 
 we assessed fos expression in the 
 bla and cea in each hemisphere . 
 we found that fear - conditioned subjects showed fear responses , such as increased freezing and decreased walking , as well as 
 increased fos expression in the bla and cea . when we compared fos expression between hemispheres , fos expression in the cea , but not the bla , was greater in the 
 right hemisphere compared with the left hemisphere . 
 these results suggest that the right cea is more strongly activated by the auditory cs . 