BACKGROUND
differential formation of excitatory and inhibitory synapses is critical for the functional development of the central nervous system.
inhibitory synaptic networks mediated by γ-aminobutyric acid  transmission are formed in the embryonic stages and mature as a result of neural activity and experience during the postnatal period  <cit> .
a class of gabaergic interneurons is characterized by expression of calcium-binding protein, parvalbumin .
most pv-positive interneurons also express the  <dig> kda isoform of glutamate decarboxylase   <cit>  and have been suggested to be involved in various higher brain functions including emotion, anxiety, and learning and memory  <cit> .
the basolateral amygdala  plays a critical role in emotional responsiveness and anxiety  <cit>  and approximately 10%–15% bla neurons are gabaergic interneurons  <cit> .
of these, pv-positive neurons constitute approximately 50% of the interneuronal population and extensively colocalize with calbindin-d28k   <cit> .
these pv-positive interneurons are potent inhibitors of both the perisomatic and distal dendritic domains of principal glutamatergic neurons  <cit> .
consequently, excitability of the principal bla neurons is regulated by these gabaergic neurons  <cit> .
thus, pv-positive neurons are critical for the expression of anxiety-like behavior since activation of pv-positive neurons and pharmacological stimulation of the gabaergic neuronal system in bla can decrease emotional arousal and anxiety-like behavior  <cit> .
rearing in an enriched environment , i.e., housing conditions containing toys, tunnels, ladders, running wheel, and several animals living together, is generally believed to facilitate enhanced motor, sensory, and cognitive stimulation and also provide relatively increased social interaction than a standard environment .
extensive studies demonstrated significant effects of ee on brain plasticity and subsequent behavior in adulthood  <cit> .
in terms of behavioral aspects, rodents reared in ee exhibit improved recovery from dysfunctions following brain lesions  <cit> , enhanced learning and memory  <cit> , and altered emotional behavior, such as increased exploratory and decreased anxiety-like behavior  <cit> .
taken together, these studies suggest that rearing in ee may induce functional plasticity of the pv-positive neurons in bla.
however, no previous studies have investigated the effects of ee on pv-positive neurons in bla.
here, we investigated whether ee affected the number of pv-positive neurons in bla and anxiety-like behavior and analyzed the relationships between these changes.
methods
animals and housing conditions
fifteen male wistar rats  were used .
the rats were housed in se  or ee .
we have adopted the experimental design with the standard definition of ee, “a combination of complex inanimate and social stimulation” by van praag et al.
<cit> .
the ee was equipped with horizontal platforms and various toys such as a running wheel, tunnels, a climbing ladder, wooden blocks, a bridge, and a maze, as previously described  <cit> .
the spatial arrangement of the objects was changed and some of toys were replaced with new toys twice a week in the ee cage.
the rats were maintained under controlled temperature  and 12-h light/dark cycle , and food and water were available ad libitum.
all experiments and animal housing adhered to the guideline for care and use of laboratory animals of the institute of laboratory animal resources, national research council .
experimental procedures were approved by the ethical review board for animal experiments at the university of toyama .
open field test
five weeks after rearing in each condition, activity in an open field was evaluated.
the test was initiated by placing each subject in the center of an open field arena  of a circular open field .
the behavioral field was set on a black plastic sheet to obtain clear image contrast of the animals.
behavior was video recorded for  <dig> min using a digital video camera , and locomotive behavior was analyzed offline using a software program .
we analyzed % distance traveled in the center arena and the time spent in the center of the arena.
the % distance traveled in the center arena was defined as the following formula;  <dig> × /.
the total distance traveled was defined as the distance traveled in the whole open field.
beam walking test
two days after the open field test, beam walking ability  <cit>  was evaluated.
in brief, the rats were trained so as to walk on a wooden beam  elevated  <dig> mm above the floor three times.
after training, walking to traverse the beam  was video recorded  <dig> times  and walking time was calculated.
tissue preparation
principle staining procedures have been previously described in detail  <cit> .
in brief, the brains of the rats were obtained after perfusion with heparin saline solution under deep anesthesia with pentobarbital  and fixation with 4% paraformaldehyde in  <dig>  m phosphate buffer.
after fixation  and cryoprotection in 20% sucrose solution, coronal sections  were cut on a freezing microtome.
for stereological quantification,  <dig> sections each were processed for one staining each: one for nissl staining with cresyl violet, one for pv staining, one for calb, and two for other staining including double immunolabeling to identify the phenotype of pv-positive cells.
immunohistochemistry
for all staining, free-floating sections were rinsed  <dig> times in phosphate buffer saline  between each incubation step.
these sections were quenched for  <dig> min in 3% h2o2/20% methanol in pbs, and incubated in a blocking solution, 3% normal horse serum in pbs-t  for  <dig> min at room temperature.
for pv and calb immunohistochemistry, the sections were incubated overnight at 4°c with mouse anti-pv antibodies  or mouse anti-calb antibodies  in 1% blocking solution.
after rinsing, sections were incubated for  <dig> h at room temperature with biotinylated secondary antibodies  and then reacted with avidin biotin peroxidase complex .
the reaction was visualized with a detection solution .
cell counting and morphological analysis
for cell counting, the stereological methods  have been recommended  <cit> .
however, poor penetration of the calb immunostaining made it quite difficult to apply to the optical dissector method  <cit> .
therefore, we counted all somatic profiles contained in the optical sections 0– <dig> μm from the surface  <cit> .
images of the sections were obtained with a light microscope  equipped with a digital camera  or all-in-one fluorescence microscope system .
at the outset, we examined the penetration of the immunostaining for pv and calb in  <dig> μm thick sections.
stacks of serial optical sections  <dig> μm apart were obtained with all-in-one microscope system bz- <dig> under an objective lens .
we counted stained cells in the whole area of the basolateral complex of the amygdala in each section, including the lateral , basal , and accessory basal nuclei  of the amygdala.
the basolateral complex of the amygdala was divided into the  <dig> subareas; the lateral amygdala  and basolateral amygdala .
cell counting was performed for each animal in  <dig> anatomically matched-sections that referred to adjacent nissl staining sections  <cit> : rostrocaudal location at  anterior to posterior  − <dig> ,  ap − <dig> ,  ap − <dig> , and  − <dig>  mm from the bregma for pv-positive cell counting.
the sections adjacent to pv-staining were used for calb-positive cell counting.
pv and calb-positive cells were quantified separately in the se and ee rats and expressed as mean values ± sem  per section.
pv-immunoreactive cells were differentially evaluated according to diameters of positive cell bodies: the cells with large cell bodies more than  <dig> μm rectangular diameter  were defined as large-positive cells, and the cells with small cell bodies less than  <dig> μm rectangular diameter were defined as small-positive cells.
all differential counts were performed blind in randomized section.
morphological images were adjusted for brightness and contrast using adobe photoshop cs .
phenotype of pv-positive cells
for double-immunolabeling studies, the sections were incubated in the blocking solution, followed by overnight incubation of the sections for  <dig> days at 4°c with  <dig> primary antibodies simultaneously—rabbit anti-pv antibodies  and mouse anti-gad  <dig> antibodies .
the secondary antibodies were alexa fluor  <dig> goat anti-rabbit igg  and alexa fluor  <dig> goat anti-mouse igg .
the sections were mounted on mas-coated glass slides  in mounting medium with dapi .
immunofluorescent images were obtained using the confocal laser scanning microscope lsm  <dig>  and analyzed to determine whether pv-positive cells colocalize with gad- <dig> using software .
the sections for immunofluorescence examination were located at rostrocaudal levels from ap − <dig>  to ap − <dig>  mm from the bregma.
we confirmed that there was no non-specific labeling of cells when each primary antibody was omitted.
only double immunofluorescence-positive cells that also expressed dapi-positive fluorescence were identified in order to avoid counting of partial and insufficient somata.
statistical analysis
all values are expressed as mean values ± sem.
all statistical analyses were performed using the software package spss .
significant differences between the groups in behavioral and immunohistochemical analyses were evaluated using student’s t-test and two-way repeated measures anova.
the correlation between behavioral data and the number of small cells was analyzed using simple linear regression analysis.
differences were considered statistically significant at p <  <dig> .
RESULTS
number of pv and calb-positive cells in bla
to determine whether rearing in ee leads to an altered organization of inhibitory circuits in bla, we analyzed the number of cells expressing the calcium-binding proteins pv or calb.
immunohistochemical results for pv and calb in bla of the se rats were identical to those in previous studies  <cit> .
bla contained the highest density of pv-positive neurons , whereas only a few pv-positive neurons were observed in other areas of the amygdala .
the number of pv-positive neurons in bla was significantly higher in the rats reared in ee  than those reared in se   .
however, no significant difference was observed between the groups in the number of calb-positive neurons .
cellular characterization of the increased pv-positive cells
pv-positive cells consist of various sub-types with different somata sizes .
we separately counted the neurons according to cell sizes, large or small positive cells.
statistical comparisons indicated that there was no significant difference in the number of the large pv-positive cells  between the groups in la; there was no significant main effect of group  =  <dig> , p >  <dig> ) and no significant interaction between group and ap level  =  <dig> , p >  <dig> ) .
furthermore, there was no significant difference in the number of the large pv-positive cells between the groups in bla; there was no significant main effect of group  =  <dig> , p >  <dig> ) and no significant interaction between group and ap level  =  <dig> , p >  <dig> ) .
on the other hand, statistical analyses showed that ee significantly increased the number of the small pv-positive cells  in la; there was a significant main effect of group  =  <dig> , p <  <dig> ), but no significant interaction between group and ap level  =  <dig> , p >  <dig> ) .
furthermore, ee also significantly increased the number of the small pv-positive cells in bla; there were a significant main effect of group  =  <dig> , p <  <dig> ), and a significant interaction between group and ap level  =  <dig> , p <  <dig> ) .
post-hoc tests indicated that the number of the small pv-positive cells was significantly larger in ee than se in the all ap levels   <cit> .
according to our classification based on cell size, large positive cells exhibited  <dig> morphological features : one type was medium-to-large-sized multipolar neurons with a few longer, aspiny dendrites of varying thickness, and corresponded to type  <dig> cells; the other type had fusiform somata with elongated dendrites and corresponded to type  <dig> cells.
pv-positive small cells had small, roundish somata or multipolar stellate perikarya with a few shorter dendrites of approximately equal thickness, which corresponded to type  <dig> cells .
the morphological features of the pv-positive neurons according to our classification based on cell size were consistent with those according to the previously reported classification based on cell type   <cit> .
almost all pv-positive neurons in bla are gabaergic   <cit> .
however, in postmortem specimens of subjects with schizophrenia, only 55% pv mrna-positive neurons showed detectable levels of gad <dig> mrna in the prefrontal cortex  <cit> .
accordingly, ee and/or se may also affect co-expressing phenotype for gad <dig> in pv-positive cells.
therefore, to determine whether the increased population of pv-positive cells co-expressed gad <dig>   <dig> pv-positive cells of  <dig> se rats and  <dig> pv-positive cells of  <dig> ee rats were analyzed for colocalization with gad <dig> in the bla.
among the pv-positive cells ,  <dig>  ±  <dig> % in se rats and  <dig>  ±  <dig> % in ee rats, were immunoreactive for co-expression of gad <dig> .
no significant difference was observed in colocalization between the se and ee rats .
the pattern of colocalization did not differ in the counted areas  or in cell-type classification  .
thus, although the number of pv-positive small cells was increased in the ee rats, the results indicate that the morphological and immunohistochemical phenotypes of the pv-positive cells did not show significant difference between the se and ee rats.
behavioral alteration between the rearing groups
if the increased pv-positive cells were integrated into the neuronal circuits of bla, some behavioral alterations would be induced because bla plays a critical role in the expression of anxiety-like behaviors under stressful situations  <cit> .
to validate this hypothesis, we compared behavioral phenotypes between the ee rats, which increased the number of pv-positive cells in bla, and se rats.
in the open field test, the total distance traveled was significantly shorter in the ee rats than the se rats .
in addition, separate analyses indicated that the distance traveled in the peripheral arena was also significantly shorter in the ee rats than the se rats , whereas the distance traveled in the center arena was significantly greater in the ee rats than the se rats .
although the ee rats exhibited decreased locomotion than the se rats, this trend was evident only in the peripheral arena.
thus, the % distance traveled in the center arena was significantly greater in the ee rats than the se rats .
furthermore, the time spent in the center arena significantly increased in the ee rats than the se rats .
in a previous report, ee rats showed a good performance in a beam walking task  <cit> .
in the present study, the ee rats also traversed the elevated narrow beam faster .
because the beam was narrow and elevated, the beam walking test was also anxiogenic.
therefore, performance in both anxiogenic tasks  should be correlated.
simple regression analysis revealed that walking time was significantly and negatively correlated with the % distance traveled in the center arena  =  <dig> , p <  <dig> , r =  <dig> ; figure 5a) and tended to be positively correlated with the total distance traveled  =  <dig> , p =  <dig> , r =  <dig> ).
relationships between behavioral alteration and increased number of pv-positive cells
the observed alterations of pv-positive cells in bla could affect behavior in the anxiogenic  tests.
figure 5b and c shows the relationships between performance in these behavioral tests and the number of pv-positive small cells in bla.
simple regression analyses revealed that the number of pv-positive small cells tended to be positively correlated with the % distance traveled in the center arena  =  <dig> , p =  <dig> , r =  <dig> ; figure 5b), and negatively correlated with the peripheral distance traveled  =  <dig> , p =  <dig> , r =  <dig> ).
furthermore, a simple regression analysis also revealed that the number of pv-positive small cells tended to be positively correlated with the time spent in the center arena  =  <dig> , p =  <dig> , r =  <dig> ).
on the other hand, the number of pv-positive small cells was not significantly correlated with the total distance traveled  =  <dig> , p >  <dig> , r =  <dig> ) nor with the center distance traveled  =  <dig> , p >  <dig> , r =  <dig> ).
notably, in the beam walking test, the number of pv-positive small cells was significantly and negatively correlated with walking time  =  <dig> , p <  <dig> , r =  <dig> ; figure 5c).
discussion
our results indicate that ee significantly affects anxiety-like behavior and pv-positive neurons in bla.
first, ee increased the number of pv-containing neuronal phenotype in bla, but not that of calb-containing neuronal phenotype.
this increase in the number of pv-positive neurons was evident only in the morphologically small neurons co-expressing gad <dig>  finally, the number of small pv-positive neurons in bla was negatively correlated with walking time in the beam walking test and tended to be positively correlated with activity in the center arena in the open field test.
effects of enriched environment on pv-positive neurons in bla
in the present study, the number of pv-positive neurons in bla was influenced by the environmental conditions during adolescence, while that of calb-positive neurons was unchanged.
a neuroanatomical study in bla reported that pv-immunoreactivity reached maturity  on postnatal day  <dig> at the same level of postnatal day  <dig>  <cit> .
in the present study, we divided the animals into  <dig> groups  from postnatal day  <dig>  and the animals were kept under same environment  for five weeks.
these findings suggest that the number of pv-positive cells was stable in se group, while pv-positive cells in ee increased during environmental intervention for five weeks.
furthermore, the postnatal maturation of pv-positive neurons was later than that of calb-positive neurons  <cit> , and some studies have reported partial phenotypic shift from pv-negative immunoreactive neurons to pv-positive neurons during postnatal development  <cit> .
these results further suggest that the increase in the number of pv-positive neurons as a result of ee might be partly attributable to the phenotypic shift into pv-positive expression.
since the number of calb-positive neurons was unchanged in the present study, two possibilities of the phenotypic shift could be raised; a shift from pv-negative and calb-negative neurons to pv-positive and calb-negative neurons or a shift from pv-negative and calb-positive neurons to pv-positive and calb-positive neurons.
exposure to ee and physical activity  induces hippocampal neural progenitor proliferation and neurogenesis  <cit> .
furthermore, ee has positive effects on neurogenesis in the subventricular zone in rodent damaged brains  <cit> .
these previous findings suggest that neurogenesis of pv-positive neurons might also contribute to pv-positive augmentation in the present study.
however, it is unlikely since bla is one of the non-neurogenic areas  <cit> .
neurogenesis throughout adulthood in the mammals has been clearly demonstrated at the two restricted brain areas, the subventricular zone of lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus.
although neural stem cells or progenitors can be isolated from many areas of adult nervous system, adult neurogenesis  has only been consistently found in these two areas by specific neurogenic niche, but not in bla  <cit> .
a previous study reported that ee did not increase the number of pv-positive neurons  <cit> .
the discrepancy may be partially attributable to the differences from our study: 1) the rearing conditions of the se rats in that study– <dig> rats, including both males and females were housed together  <cit> , while  <dig> male rats were housed in a single cage in the present study, 2) pv-positive neurons were counted in the hippocampus in that study  <cit> .
our findings of an increase in pv-positive neurons are consistent with the effects of physical exercise  on hippocampal pv-positive neurons during adolescence  <cit> .
enhanced motor and sensory experiences in the present study, in particular the use of a running wheel, may play a substantial role in modulation of pv-positive neurons in bla.
effects of enriched environment on anxiety-like behavior
in the present study, the % distance traveled in the center arena in the open field test was significantly greater in the ee rats .
however, the total distance traveled was decreased in the ee rats .
this decrease in the total distance traveled was attributable to a decrease in the distance traveled only in the peripheral arena, whereas the distance traveled in the center arena was increased in the ee rats .
these results suggest that rearing in ee decreased the expression of anxiety-like behavior in the rats.
in the beam walking test, walking time was shorter in the ee rats .
in this test, the animals were tested under the anxiogenic environment, i.e., leaving them alone on the elevated narrow beam.
in humans, similar situations, such as unprotected, open, and elevated areas, can evoke panic and vestibular symptoms   <cit> .
these findings indicate the pathological link between anxiety and vestibular/balancing deficits  <cit> .
these deficits are conceptualized as stress-evoked sensorimotor disintegration  <cit> .
taken together, shorter walking time in the ee rats might be attributable to changes in emotional states.
in other words, decreased anxiety-like behavior in the ee rats ameliorated stress-evoked sensorimotor disintegration.
furthermore, walking time was significantly and negatively correlated with the % distance traveled in the center arena, thereby suggesting that anxiety-like behavior is negatively correlated with performance in the beam walking test .
these findings strongly suggest that shorter walking time in the ee rats was attributable to decreased anxiety-like behavior as a result of being reared in ee.
other previous data also supports this hypothesis.
first, the se rats could walk faster within a few days and achieved the same level as in the ee rats, suggesting that shorter walking time in the ee rats was not attributable solely to plastic changes in only the motor system.
second, in the training session, when the ee rats were placed on a beam just in front of a goal cage , they could return more quickly into the cage  <cit> , suggesting that the ee rats could recognize the novel situation and adapt more quickly than the se rats under the same anxiogenic conditions.
these findings suggest that the plastic changes occur primarily in the brain regions involved in the expression of anxiety.
functional roles of pv-neurons in anxiety-like behavior
the above behavioral changes were correlated with the number of pv-positive neurons in bla.
the number of pv-positive small cells in bla was negatively correlated with walking time in the beam walking test  and tended to be positively correlated with the % distance traveled in the center arena in the open field test .
because bla is one of the brain regions involved in the expression of anxiety, the data suggest that the increased number of pv-positive neurons decreased anxiety levels in the ee rats.
these results are consistent with those of a previous study  <cit>  in which one inbred rat line that exhibited lesser anxiety-like behavior in an open field and elevated plus-maze had more pv-positive neurons in bla than the other rat line that exhibited anxiety-like behavior.
on the other hand, mice lacking ß <dig> subunits of gaba-a receptors exhibited pronounced neurological and balance deficits as well as hyperemotionality  <cit> .
furthermore, antidepressants such as benzodiazepines ameliorate both anxiety and vestibular/balancing deficits  <cit> .
these results strongly support the hypothesis that the gabaergic subpopulation co-expressing pv in bla is specifically responsible, at least in part, for suppressing anxiety-like behavior in the open field and beam walking tests.
significance of rearing in enriched environment
our results indicate that the gabaergic system  in bla is augmented by ee.
deficits in pv-positive interneurons have been suggested to be associated not only with anxiety disorders  but also with other psychiatric disorders.
interneurons expressing pv exhibit the fast-spiking phenotype and have been suggested to be involved in gamma oscillations  <cit> .
disruption of gamma oscillation has been suggested to underlie psychiatric disorders such as schizophrenia  <cit>  and autism  <cit>  and may be attributable to deficits in gabaergic pv-positive interneurons  <cit> .
furthermore, adverse prenatal or early life events such as exposure to ethanol and maternal separation alter emotional responsiveness and the number of pv-positive interneurons in bla, medial septum, medial prefrontal cortex, and hippocampus  <cit> .
in addition, pv-positive interneurons in bla express excitatory 5-ht2a receptors  <cit> , and 5-ht inhibits glutamatergic projection neurons involved in anxiety behavior through this subtype of the interneurons  <cit> .
therefore, our results suggest that ee, even in adolescence, might be beneficial to disorders that are characterized by deficiencies in pv-positive neuronal networks.
CONCLUSIONS
the present results demonstrated that rearing in ee augmented a specific subpopulation of gabaergic neurons co-expressing pv in bla.
the ee rats showed lowered anxiety-like behavior in the open field test and quickly traversed the beam in the beam walking test.
significant correlation between increased numbers of pv-positive neurons in bla and changes in anxiety-like behavior suggest that plastic changes in pv-positive neurons induced alteration of these anxiety-like behaviors under anxiogenic situations.
furthermore, rearing conditions during adolescence critically affect the inhibitory neuronal networks of the pv-positive subpopulation in bla leading to behavioral plasticity in emotional responsiveness.
these results suggest that ee might be beneficial for certain psychiatric disorders in which gabaergic dysfunction is suspected.
abbreviations
gaba: γ-aminobutyric acid; pv: parvalbumin; calb: clbindin-d28k; bla: basolateral amygdala; la: lateral amygdala; gad67:  <dig> kda isoform of glutamate decarboxylase.
competing interests
there are no competing interests.
authors’ contributions
su and hn conceived and designed the experiments.
su, kt, and ns performed the experiments.
su and hn analyzed the data.
eh and ns contributed reagents/materials/analysis tools.
su, to, and hn wrote the paper.
all authors read and approved the final manuscript.
acknowledgments
this work was supported partly by jsps  asian core program, and the ministry of education, science, sports and culture, grant-in-aid for scientific research  .

 differential formation of excitatory and inhibitory synapses is critical for the functional development of the central nervous system. inhibitory synaptic networks mediated by γ-aminobutyric acid transmission are formed in the embryonic stages and mature as a result of neural activity and experience during the postnatal period. a class of gabaergic interneurons is characterized by expression of calcium-binding protein, parvalbumin. most pv-positive interneurons also express the kda isoform of glutamate decarboxylase and have been suggested to be involved in various higher brain functions including emotion, anxiety, and learning and memory. the basolateral amygdala plays a critical role in emotional responsiveness and anxiety and approximately 10%–15% bla neurons are gabaergic interneurons. of these, pv-positive neurons constitute approximately 50% of the interneuronal population and extensively colocalize with calbindin-d28k. these pv-positive interneurons are potent inhibitors of both the perisomatic and distal dendritic domains of principal glutamatergic neurons. consequently, excitability of the principal bla neurons is regulated by these gabaergic neurons. thus, pv-positive neurons are critical for the expression of anxiety-like behavior since activation of pv-positive neurons and pharmacological stimulation of the gabaergic neuronal system in bla can decrease emotional arousal and anxiety-like behavior. rearing in an enriched environment, i.e., housing conditions containing toys, tunnels, ladders, running wheel, and several animals living together, is generally believed to facilitate enhanced motor, sensory, and cognitive stimulation and also provide relatively increased social interaction than a standard environment. extensive studies demonstrated significant effects of ee on brain plasticity and subsequent behavior in adulthood. in terms of behavioral aspects, rodents reared in ee exhibit improved recovery from dysfunctions following brain lesions, enhanced learning and memory, and altered emotional behavior, such as increased exploratory and decreased anxiety-like behavior. taken together, these studies suggest that rearing in ee may induce functional plasticity of the pv-positive neurons in bla. however, no previous studies have investigated the effects of ee on pv-positive neurons in bla. here, we investigated whether ee affected the number of pv-positive neurons in bla and anxiety-like behavior and analyzed the relationships between these changes. methods animals and housing conditions fifteen male wistar rats were used. the rats were housed in se or ee. we have adopted the experimental design with the standard definition of ee, “a combination of complex inanimate and social stimulation” by van praag et al.. the ee was equipped with horizontal platforms and various toys such as a running wheel, tunnels, a climbing ladder, wooden blocks, a bridge, and a maze, as previously described. the spatial arrangement of the objects was changed and some of toys were replaced with new toys twice a week in the ee cage. the rats were maintained under controlled temperature and 12-h light/dark cycle, and food and water were available ad libitum. all experiments and animal housing adhered to the guideline for care and use of laboratory animals of the institute of laboratory animal resources, national research council. experimental procedures were approved by the ethical review board for animal experiments at the university of toyama. open field test five weeks after rearing in each condition, activity in an open field was evaluated. the test was initiated by placing each subject in the center of an open field arena of a circular open field. the behavioral field was set on a black plastic sheet to obtain clear image contrast of the animals. behavior was video recorded for min using a digital video camera, and locomotive behavior was analyzed offline using a software program. we analyzed % distance traveled in the center arena and the time spent in the center of the arena. the % distance traveled in the center arena was defined as the following formula; × /. the total distance traveled was defined as the distance traveled in the whole open field. beam walking test two days after the open field test, beam walking ability was evaluated. in brief, the rats were trained so as to walk on a wooden beam elevated mm above the floor three times. after training, walking to traverse the beam was video recorded times and walking time was calculated. tissue preparation principle staining procedures have been previously described in detail. in brief, the brains of the rats were obtained after perfusion with heparin saline solution under deep anesthesia with pentobarbital and fixation with 4% paraformaldehyde in m phosphate buffer. after fixation and cryoprotection in 20% sucrose solution, coronal sections were cut on a freezing microtome. for stereological quantification, sections each were processed for one staining each: one for nissl staining with cresyl violet, one for pv staining, one for calb, and two for other staining including double immunolabeling to identify the phenotype of pv-positive cells. immunohistochemistry for all staining, free-floating sections were rinsed times in phosphate buffer saline between each incubation step. these sections were quenched for min in 3% h2o2/20% methanol in pbs, and incubated in a blocking solution, 3% normal horse serum in pbs-t for min at room temperature. for pv and calb immunohistochemistry, the sections were incubated overnight at 4°c with mouse anti-pv antibodies or mouse anti-calb antibodies in 1% blocking solution. after rinsing, sections were incubated for h at room temperature with biotinylated secondary antibodies and then reacted with avidin biotin peroxidase complex. the reaction was visualized with a detection solution. cell counting and morphological analysis for cell counting, the stereological methods have been recommended. however, poor penetration of the calb immunostaining made it quite difficult to apply to the optical dissector method. therefore, we counted all somatic profiles contained in the optical sections 0– μm from the surface. images of the sections were obtained with a light microscope equipped with a digital camera or all-in-one fluorescence microscope system. at the outset, we examined the penetration of the immunostaining for pv and calb in μm thick sections. stacks of serial optical sections μm apart were obtained with all-in-one microscope system bz- under an objective lens. we counted stained cells in the whole area of the basolateral complex of the amygdala in each section, including the lateral, basal, and accessory basal nuclei of the amygdala. the basolateral complex of the amygdala was divided into the subareas; the lateral amygdala and basolateral amygdala. cell counting was performed for each animal in anatomically matched-sections that referred to adjacent nissl staining sections: rostrocaudal location at anterior to posterior −, ap −, ap −, and − mm from the bregma for pv-positive cell counting. the sections adjacent to pv-staining were used for calb-positive cell counting. pv and calb-positive cells were quantified separately in the se and ee rats and expressed as mean values ± sem per section. pv-immunoreactive cells were differentially evaluated according to diameters of positive cell bodies: the cells with large cell bodies more than μm rectangular diameter were defined as large-positive cells, and the cells with small cell bodies less than μm rectangular diameter were defined as small-positive cells. all differential counts were performed blind in randomized section. morphological images were adjusted for brightness and contrast using adobe photoshop cs. phenotype of pv-positive cells for double-immunolabeling studies, the sections were incubated in the blocking solution, followed by overnight incubation of the sections for days at 4°c with primary antibodies simultaneously—rabbit anti-pv antibodies and mouse anti-gad antibodies. the secondary antibodies were alexa fluor goat anti-rabbit igg and alexa fluor goat anti-mouse igg. the sections were mounted on mas-coated glass slides in mounting medium with dapi. immunofluorescent images were obtained using the confocal laser scanning microscope lsm and analyzed to determine whether pv-positive cells colocalize with gad- using software. the sections for immunofluorescence examination were located at rostrocaudal levels from ap − to ap − mm from the bregma. we confirmed that there was no non-specific labeling of cells when each primary antibody was omitted. only double immunofluorescence-positive cells that also expressed dapi-positive fluorescence were identified in order to avoid counting of partial and insufficient somata. statistical analysis all values are expressed as mean values ± sem. all statistical analyses were performed using the software package spss. significant differences between the groups in behavioral and immunohistochemical analyses were evaluated using student’s t-test and two-way repeated measures anova. the correlation between behavioral data and the number of small cells was analyzed using simple linear regression analysis. differences were considered statistically significant at p <.  number of pv and calb-positive cells in bla to determine whether rearing in ee leads to an altered organization of inhibitory circuits in bla, we analyzed the number of cells expressing the calcium-binding proteins pv or calb. immunohistochemical results for pv and calb in bla of the se rats were identical to those in previous studies. bla contained the highest density of pv-positive neurons, whereas only a few pv-positive neurons were observed in other areas of the amygdala. the number of pv-positive neurons in bla was significantly higher in the rats reared in ee than those reared in se. however, no significant difference was observed between the groups in the number of calb-positive neurons. cellular characterization of the increased pv-positive cells pv-positive cells consist of various sub-types with different somata sizes. we separately counted the neurons according to cell sizes, large or small positive cells. statistical comparisons indicated that there was no significant difference in the number of the large pv-positive cells between the groups in la; there was no significant main effect of group =, p > ) and no significant interaction between group and ap level =, p > ). furthermore, there was no significant difference in the number of the large pv-positive cells between the groups in bla; there was no significant main effect of group =, p > ) and no significant interaction between group and ap level =, p > ). on the other hand, statistical analyses showed that ee significantly increased the number of the small pv-positive cells in la; there was a significant main effect of group =, p < ), but no significant interaction between group and ap level =, p > ). furthermore, ee also significantly increased the number of the small pv-positive cells in bla; there were a significant main effect of group =, p < ), and a significant interaction between group and ap level =, p < ). post-hoc tests indicated that the number of the small pv-positive cells was significantly larger in ee than se in the all ap levels. according to our classification based on cell size, large positive cells exhibited morphological features: one type was medium-to-large-sized multipolar neurons with a few longer, aspiny dendrites of varying thickness, and corresponded to type cells; the other type had fusiform somata with elongated dendrites and corresponded to type cells. pv-positive small cells had small, roundish somata or multipolar stellate perikarya with a few shorter dendrites of approximately equal thickness, which corresponded to type cells. the morphological features of the pv-positive neurons according to our classification based on cell size were consistent with those according to the previously reported classification based on cell type. almost all pv-positive neurons in bla are gabaergic. however, in postmortem specimens of subjects with schizophrenia, only 55% pv mrna-positive neurons showed detectable levels of gad mrna in the prefrontal cortex. accordingly, ee and/or se may also affect co-expressing phenotype for gad in pv-positive cells. therefore, to determine whether the increased population of pv-positive cells co-expressed gad pv-positive cells of se rats and pv-positive cells of ee rats were analyzed for colocalization with gad in the bla. among the pv-positive cells, ± % in se rats and ± % in ee rats, were immunoreactive for co-expression of gad. no significant difference was observed in colocalization between the se and ee rats. the pattern of colocalization did not differ in the counted areas or in cell-type classification. thus, although the number of pv-positive small cells was increased in the ee rats, the results indicate that the morphological and immunohistochemical phenotypes of the pv-positive cells did not show significant difference between the se and ee rats. behavioral alteration between the rearing groups if the increased pv-positive cells were integrated into the neuronal circuits of bla, some behavioral alterations would be induced because bla plays a critical role in the expression of anxiety-like behaviors under stressful situations. to validate this hypothesis, we compared behavioral phenotypes between the ee rats, which increased the number of pv-positive cells in bla, and se rats. in the open field test, the total distance traveled was significantly shorter in the ee rats than the se rats. in addition, separate analyses indicated that the distance traveled in the peripheral arena was also significantly shorter in the ee rats than the se rats, whereas the distance traveled in the center arena was significantly greater in the ee rats than the se rats. although the ee rats exhibited decreased locomotion than the se rats, this trend was evident only in the peripheral arena. thus, the % distance traveled in the center arena was significantly greater in the ee rats than the se rats. furthermore, the time spent in the center arena significantly increased in the ee rats than the se rats. in a previous report, ee rats showed a good performance in a beam walking task. in the present study, the ee rats also traversed the elevated narrow beam faster. because the beam was narrow and elevated, the beam walking test was also anxiogenic. therefore, performance in both anxiogenic tasks should be correlated. simple regression analysis revealed that walking time was significantly and negatively correlated with the % distance traveled in the center arena =, p <, r =; figure 5a) and tended to be positively correlated with the total distance traveled =, p =, r = ). relationships between behavioral alteration and increased number of pv-positive cells the observed alterations of pv-positive cells in bla could affect behavior in the anxiogenic tests. figure 5b and c shows the relationships between performance in these behavioral tests and the number of pv-positive small cells in bla. simple regression analyses revealed that the number of pv-positive small cells tended to be positively correlated with the % distance traveled in the center arena =, p =, r =; figure 5b), and negatively correlated with the peripheral distance traveled =, p =, r = ). furthermore, a simple regression analysis also revealed that the number of pv-positive small cells tended to be positively correlated with the time spent in the center arena =, p =, r = ). on the other hand, the number of pv-positive small cells was not significantly correlated with the total distance traveled =, p >, r = ) nor with the center distance traveled =, p >, r = ). notably, in the beam walking test, the number of pv-positive small cells was significantly and negatively correlated with walking time =, p <, r =; figure 5c). discussion our results indicate that ee significantly affects anxiety-like behavior and pv-positive neurons in bla. first, ee increased the number of pv-containing neuronal phenotype in bla, but not that of calb-containing neuronal phenotype. this increase in the number of pv-positive neurons was evident only in the morphologically small neurons co-expressing gad finally, the number of small pv-positive neurons in bla was negatively correlated with walking time in the beam walking test and tended to be positively correlated with activity in the center arena in the open field test. effects of enriched environment on pv-positive neurons in bla in the present study, the number of pv-positive neurons in bla was influenced by the environmental conditions during adolescence, while that of calb-positive neurons was unchanged. a neuroanatomical study in bla reported that pv-immunoreactivity reached maturity on postnatal day at the same level of postnatal day. in the present study, we divided the animals into groups from postnatal day and the animals were kept under same environment for five weeks. these findings suggest that the number of pv-positive cells was stable in se group, while pv-positive cells in ee increased during environmental intervention for five weeks. furthermore, the postnatal maturation of pv-positive neurons was later than that of calb-positive neurons, and some studies have reported partial phenotypic shift from pv-negative immunoreactive neurons to pv-positive neurons during postnatal development. these results further suggest that the increase in the number of pv-positive neurons as a result of ee might be partly attributable to the phenotypic shift into pv-positive expression. since the number of calb-positive neurons was unchanged in the present study, two possibilities of the phenotypic shift could be raised; a shift from pv-negative and calb-negative neurons to pv-positive and calb-negative neurons or a shift from pv-negative and calb-positive neurons to pv-positive and calb-positive neurons. exposure to ee and physical activity induces hippocampal neural progenitor proliferation and neurogenesis. furthermore, ee has positive effects on neurogenesis in the subventricular zone in rodent damaged brains. these previous findings suggest that neurogenesis of pv-positive neurons might also contribute to pv-positive augmentation in the present study. however, it is unlikely since bla is one of the non-neurogenic areas. neurogenesis throughout adulthood in the mammals has been clearly demonstrated at the two restricted brain areas, the subventricular zone of lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus. although neural stem cells or progenitors can be isolated from many areas of adult nervous system, adult neurogenesis has only been consistently found in these two areas by specific neurogenic niche, but not in bla. a previous study reported that ee did not increase the number of pv-positive neurons. the discrepancy may be partially attributable to the differences from our study: 1) the rearing conditions of the se rats in that study– rats, including both males and females were housed together, while male rats were housed in a single cage in the present study, 2) pv-positive neurons were counted in the hippocampus in that study. our findings of an increase in pv-positive neurons are consistent with the effects of physical exercise on hippocampal pv-positive neurons during adolescence. enhanced motor and sensory experiences in the present study, in particular the use of a running wheel, may play a substantial role in modulation of pv-positive neurons in bla. effects of enriched environment on anxiety-like behavior in the present study, the % distance traveled in the center arena in the open field test was significantly greater in the ee rats. however, the total distance traveled was decreased in the ee rats. this decrease in the total distance traveled was attributable to a decrease in the distance traveled only in the peripheral arena, whereas the distance traveled in the center arena was increased in the ee rats. these results suggest that rearing in ee decreased the expression of anxiety-like behavior in the rats. in the beam walking test, walking time was shorter in the ee rats. in this test, the animals were tested under the anxiogenic environment, i.e., leaving them alone on the elevated narrow beam. in humans, similar situations, such as unprotected, open, and elevated areas, can evoke panic and vestibular symptoms. these findings indicate the pathological link between anxiety and vestibular/balancing deficits. these deficits are conceptualized as stress-evoked sensorimotor disintegration. taken together, shorter walking time in the ee rats might be attributable to changes in emotional states. in other words, decreased anxiety-like behavior in the ee rats ameliorated stress-evoked sensorimotor disintegration. furthermore, walking time was significantly and negatively correlated with the % distance traveled in the center arena, thereby suggesting that anxiety-like behavior is negatively correlated with performance in the beam walking test. these findings strongly suggest that shorter walking time in the ee rats was attributable to decreased anxiety-like behavior as a result of being reared in ee. other previous data also supports this hypothesis. first, the se rats could walk faster within a few days and achieved the same level as in the ee rats, suggesting that shorter walking time in the ee rats was not attributable solely to plastic changes in only the motor system. second, in the training session, when the ee rats were placed on a beam just in front of a goal cage, they could return more quickly into the cage, suggesting that the ee rats could recognize the novel situation and adapt more quickly than the se rats under the same anxiogenic conditions. these findings suggest that the plastic changes occur primarily in the brain regions involved in the expression of anxiety. functional roles of pv-neurons in anxiety-like behavior the above behavioral changes were correlated with the number of pv-positive neurons in bla. the number of pv-positive small cells in bla was negatively correlated with walking time in the beam walking test and tended to be positively correlated with the % distance traveled in the center arena in the open field test. because bla is one of the brain regions involved in the expression of anxiety, the data suggest that the increased number of pv-positive neurons decreased anxiety levels in the ee rats. these results are consistent with those of a previous study in which one inbred rat line that exhibited lesser anxiety-like behavior in an open field and elevated plus-maze had more pv-positive neurons in bla than the other rat line that exhibited anxiety-like behavior. on the other hand, mice lacking ß subunits of gaba-a receptors exhibited pronounced neurological and balance deficits as well as hyperemotionality. furthermore, antidepressants such as benzodiazepines ameliorate both anxiety and vestibular/balancing deficits. these results strongly support the hypothesis that the gabaergic subpopulation co-expressing pv in bla is specifically responsible, at least in part, for suppressing anxiety-like behavior in the open field and beam walking tests. significance of rearing in enriched environment our results indicate that the gabaergic system in bla is augmented by ee. deficits in pv-positive interneurons have been suggested to be associated not only with anxiety disorders but also with other psychiatric disorders. interneurons expressing pv exhibit the fast-spiking phenotype and have been suggested to be involved in gamma oscillations. disruption of gamma oscillation has been suggested to underlie psychiatric disorders such as schizophrenia and autism and may be attributable to deficits in gabaergic pv-positive interneurons. furthermore, adverse prenatal or early life events such as exposure to ethanol and maternal separation alter emotional responsiveness and the number of pv-positive interneurons in bla, medial septum, medial prefrontal cortex, and hippocampus. in addition, pv-positive interneurons in bla express excitatory 5-ht2a receptors, and 5-ht inhibits glutamatergic projection neurons involved in anxiety behavior through this subtype of the interneurons. therefore, our results suggest that ee, even in adolescence, might be beneficial to disorders that are characterized by deficiencies in pv-positive neuronal networks.  the present results demonstrated that rearing in ee augmented a specific subpopulation of gabaergic neurons co-expressing pv in bla. the ee rats showed lowered anxiety-like behavior in the open field test and quickly traversed the beam in the beam walking test. significant correlation between increased numbers of pv-positive neurons in bla and changes in anxiety-like behavior suggest that plastic changes in pv-positive neurons induced alteration of these anxiety-like behaviors under anxiogenic situations. furthermore, rearing conditions during adolescence critically affect the inhibitory neuronal networks of the pv-positive subpopulation in bla leading to behavioral plasticity in emotional responsiveness. these results suggest that ee might be beneficial for certain psychiatric disorders in which gabaergic dysfunction is suspected. abbreviations gaba: γ-aminobutyric acid; pv: parvalbumin; calb: clbindin-d28k; bla: basolateral amygdala; la: lateral amygdala; gad67: kda isoform of glutamate decarboxylase. competing interests there are no competing interests. authors’ contributions su and hn conceived and designed the experiments. su, kt, and ns performed the experiments. su and hn analyzed the data. eh and ns contributed reagents/materials/analysis tools. su, to, and hn wrote the paper. all authors read and approved the final manuscript. acknowledgments this work was supported partly by jsps asian core program, and the ministry of education, science, sports and culture, grant-in-aid for scientific research.
these parvalbumin-positive neurons had small, rounded soma and co-expressed the glutamate decarboxylase. the results suggest that rearing in the enriched environment augmented parvalbumin-positive specific neurons in the basolateral amygdala, which induced behavioral plasticity that was reflected by a decrease in anxiety-like behavior in anxiogenic situations. after weeks of rearing, the ee rats showed decreased anxiety-like behavior in an open field than the se rats. immunohistochemical tests revealed that the number of parvalbumin-positive neurons significantly increased in the basolateral amygdala of the ee rats than that of the se rats, while the number of calbindin-d28k-positive neurons did not change. anxiety-like behavior in the open field was significantly and negatively correlated with walking time in the beam-walking test. furthermore, the number of parvalbumin-positive small cells in the basolateral amygdala tended to positively correlate with emergence in the center arena of the open field and negatively correlated with walking time in the beam walking test. furthermore, the number of parvalbumin-positive small neurons in the basolateral amygdala was negatively correlated with walking time in the beam walking test and tended to be positively correlated with activity in the center arena in the open field test. in particular, alteration of parvalbumin-expressing neurons, a gamma-aminobutyric acid ergic neuronal subpopulation, has been suggested to be associated with psychiatric disorders. under another anxiogenic situation, in a beam walking test, the ee rats more quickly traversed an elevated narrow beam. here we investigated whether rearing in enriched environment could modify the expression of parvalbumin-positive neurons in the basolateral amygdala and anxiety-like behavior. rearing in the enriched environment augmented the number of parvalbumin-containing specific inhibitory neuron in the basolateral amygdala, but not that of calbindin-containing neuronal phenotype. early life experiences including physical exercise, sensory stimulation, and social interaction can modulate development of the inhibitory neuronal network and modify various behaviors.
 early life experiences including physical exercise, sensory stimulation, and social interaction can modulate development of the inhibitory neuronal network and modify various behaviors. in particular, alteration of parvalbumin-expressing neurons, a gamma-aminobutyric acid ergic neuronal subpopulation, has been suggested to be associated with psychiatric disorders. here we investigated whether rearing in enriched environment could modify the expression of parvalbumin-positive neurons in the basolateral amygdala and anxiety-like behavior.  three-week-old male rats were divided into two groups: those reared in an enriched environment and those reared in standard cages. after weeks of rearing, the ee rats showed decreased anxiety-like behavior in an open field than the se rats. under another anxiogenic situation, in a beam walking test, the ee rats more quickly traversed an elevated narrow beam. anxiety-like behavior in the open field was significantly and negatively correlated with walking time in the beam-walking test. immunohistochemical tests revealed that the number of parvalbumin-positive neurons significantly increased in the basolateral amygdala of the ee rats than that of the se rats, while the number of calbindin-d28k-positive neurons did not change. these parvalbumin-positive neurons had small, rounded soma and co-expressed the glutamate decarboxylase. furthermore, the number of parvalbumin-positive small cells in the basolateral amygdala tended to positively correlate with emergence in the center arena of the open field and negatively correlated with walking time in the beam walking test.  rearing in the enriched environment augmented the number of parvalbumin-containing specific inhibitory neuron in the basolateral amygdala, but not that of calbindin-containing neuronal phenotype. furthermore, the number of parvalbumin-positive small neurons in the basolateral amygdala was negatively correlated with walking time in the beam walking test and tended to be positively correlated with activity in the center arena in the open field test. the results suggest that rearing in the enriched environment augmented parvalbumin-positive specific neurons in the basolateral amygdala, which induced behavioral plasticity that was reflected by a decrease in anxiety-like behavior in anxiogenic situations. environmental enrichmentcalbindincalcium binding proteinsgabaanxietyamygdala 