
 chronic stress is recognized to be a major risk factor for several psychiatric disorders , including anxiety and depression [ 13 ] . in the presence of an environmental stressor
, the body responds with major modifications of the normal homeostasis , including alterations of the hypothalamic - pituitary - adrenal ( hpa ) axis , the major neuroendocrine system activated by stressful experiences [ 4 , 5 ] .
activation of the hpa induced by stress culminates with the release of corticosterone ( cort ) from the adrenal glands .
these processes are finely controlled by neuronal activity of the hippocampus ( hpc ) and prefrontal cortex ( pfc ) , which exert a negative feedback on the hpa axis activation .
interestingly , it has been reported that both hyper- and hypoactivation of the hpa axis are associated with increased susceptibility to developing psychiatric disorders [ 6 , 7 ] . in order to better understand the etiology of depression and anxiety and to develop new treatments ,
several chronic stress methods , either physical or psychosocial , have been described to promote modifications in behavior , brain structure , and neuroendocrine system in rodents [ 812 ] . because psychiatric disorders in humans have been linked prevalently with social stress and/or reduced social interaction rather than physical stress [ 1316 ] , there is an increasing interest in developing chronic social stress paradigms for modeling psychiatric disorders in animal models .
different models of chronic social stress are able to induce various modifications in the behavior , brain function , and neuroendocrine system [ 1720 ] . while it has been extensively reported that social isolation ( si ) rearing induces long - lasting effects on behavior and brain structure in rodents , the behavioral consequences of si in adult animals , especially in mice , have been less investigated [ 2127 ] . moreover ,
chronic stress - induced behavioral modifications have been correlated with changes of neuroplasticity - related genes in different brain areas , including neurotrophic factors , metabotropic glutamate receptors , and immediate early genes [ 11 , 2831 ] . in particular ,
brain - derived neurotrophic factor ( bdnf ) , a brain plasticity marker , has been extensively investigated and associated with stress response , depression , and anxiety [ 3136 ] .
the bdnf gene is a complex gene , formed by at least eight 5 noncoding exons , each of which can be alternatively spliced to a common 3 protein - coding exon , to generate different transcripts [ 37 , 38 ] .
the complex structure of the bdnf gene allows the distinct bdnf splice variants to be differentially expressed in subcellular localizations , in specific brain regions and in response to distinct stimuli [ 35 , 36 , 3841 ] .
however , it is not known whether and how the bdnf splice variants are differentially regulated in the hpc and pfc of socially isolated mice . in this study , we assessed the effect of si stress on anxious- and depressive - like behaviors in adult male mice . moreover , we measured the plasma cort levels and the adrenal gland weight in socially isolated and grouped mice
. finally , we investigated whether the expression of total bdnf , bdnf splice variants , and other neuroplasticity - related genes ( mglur1 , mglur2 , mglur5 , c - fos , arc , and egr-1 ) were altered in the hpc and pfc of socially isolated compared to grouped adult male mice .
male c57bl/6j mice that are 8 weeks old were purchased from charles river ( calco , italy ) and allowed to acclimatize 1 week before being randomly divided in socially isolated ( si ) and group - housed ( gh ) groups .
mice were maintained in a standard 12  h light / dark cycle , temperature controlled room ( 21  1c ) , with access to food and water ad libitum .
all animal procedures were conducted according to current regulations for animal experimentation in italy and the european union and were approved by the italian ministry of health . to reduce the number of animals used , in agreement with the 3r guidelines
all experimental procedures involving animals were performed in accordance with the european community council directive 86/609/eec and were approved by italian legislation on animal experimentation ( decreto legislativo 116/1992 ) .
nine - week - old male mice were individually housed ( socially isolated : si ) or group - housed ( 4 mice per cage ; group - housed : gh ) in standard mouse cages .
mice were tested in the open field test ( oft ) on day 28 and in the tail suspension test ( tst ) on day 31 ( figure 1(a ) ) .
mice were sacrificed on day 33 and blood , adrenal glands , hpc , and pfc were harvested ( figure 1(a ) ) .
oft was conducted on day 28 as described in ieraci and herrera   with some modifications .
briefly , mice were placed in the center of a 40  40  cm square arena divided into central ( 20  cm  20  cm ) and peripheral areas , with 35  cm high walls , for 10  min .
the total distance traveled and the time spent in the central and peripheral areas were analyzed by a video - tracking system ( any - maze purchased by ugo basile , varese , italy ) .
briefly , mice were individually suspended by an adhesive tape , placed about 1  cm from the tip of the tail , on a horizontal bar 50  cm above from the bench top within a visually isolated area .
a 6  min test session was videotaped and an experimenter , blinded to the experimental groups , recorded the total immobility time ( defined as passive hanging without any movements except respiration ) . to assess whether chronic social isolation stress affects adrenal glands weight , si and gh mice
were sacrificed on day 33 and adrenal glands were removed and pruned from fat and weighed .
values are reported as the adrenal / total body weight ratio ( mg / g ) . for cort levels measure ,
trunk blood was collected on ice - cooled microcentrifuge tube containing edta 0.5  m ph 8.00 , and plasma was separated by centrifugation and stored at 80c .
cort levels were measured using the corticosterone elisa kit ( enzo life sciences , florence , italy ) according to the manufacturer 's instructions .
total rna from hpc and pfc was extracted using the direct - zol rna miniprep ( zymo research , purchased by euroclone , milan , italy ) according to manufacturer 's instructions and then quantified by absorption at a260  nm measured by uv spectrophotometry ( nanovue , ge healthcare europe gmbh , milan , italy ) .
cdna was synthesized from 1  g of dnase - treated total rna using the iscript kit ( biorad , milan , italy ) according to manufacturer 's instructions .
qpcr analysis of mrna expression levels was performed on a 7900ht fast pcr system ( applied biosystems , monza , italy ) and itaq universal sybr green supermix ( biorad ) , as previously described .
primers used are listed in table 1 and were either de novo designed or previously published elsewhere [ 35 , 44 ] .
pcr cycling conditions were 10  min at 95c , 40 cycles of 15  s at 95c , and 1  min at 60c .
relative expression of mrna for the target genes was performed by the comparative ct  ( ct ) method using -actin and gapdh as control reference genes .
western blot was performed as previously described ; briefly hpc and pfc were lysed in ice - cold ripa buffer ( 0.15  mm nacl , 0.05  mm tris_hcl , ph 7.2 , 1% triton x-100 , 1% sodium deoxycholate , and 0.1% sds ) with protease inhibitor cocktail ( sigma - aldrich , milan , italy ) .
protein concentration was determined by the quantum bicinchoninic protein assay ( euroclone ) and 15  g of proteins was separated on sds - page gels and transferred to a polyvinylidene difluoride membrane ( ge healthcare , amersham , milan , italy ) , followed by blocking in 5% milk dissolved in tris buffer saline - tween 20 ( tbst ) .
membranes were incubated with anti - bdnf ( 1  :  2,000 , rabbit polyclonal , alomone , jerusalem , israel ) or anti--actin ( 1  :  20,000 , mouse monoclonal , sigma ) antibodies . after washing with tbst
, filters were incubated with peroxidase - conjugated secondary anti - rabbit ( 1  :  3,000 , sigma - aldrich ) or with the fluorescent irdye secondary anti - mouse antibody ( li - cor , purchased from carlo erba reagents , milan , italy ) .
chemiluminescence and fluorescence membrane signals were scanned and quantified in an odyssey li - cor scanner ( carlo erba reagents ) .
statistical analyses were performed using graphpad prism 6 ( graphpad software , la jolla , ca ) .
statistical analyses were made using unpaired t - test or two - way analysis of variance ( anova ) , and lsd procedure was used for multiple comparison analysis .
pearson 's r correlations were performed to assess the correlation between cort levels , gene expression levels , and behavioral analyses ( tables 27 ) .
to assess whether social isolation induces modifications of emotional behavior in adult mice , 9-week - old male mice were randomly housed in group ( gh ) or socially isolated ( si ) for 4 weeks and then tested in the oft and in the tst to measure the anxious- and depressive - like behaviors , respectively ( figure 1(a ) ) .
si mice spent significantly less time in the center of the arena ( p = 0.029 ) ( figure 1(b ) ) and showed a tendency to a reduced distance travelled in the center ( p = 0.067 ) ( figure 1(c ) ) , indicating anxious - like behavior .
interestingly , si mice showed an increased level of total distance travelled in the oft as compared to the gh mice ( p = 0.007 ) suggesting a hyperactivity phenotype in si mice ( figure 1(d ) ) .
moreover , in the tst , the si exhibited a significantly higher period of immobility time compared to the gh mice ( p = 0.03 ) , indicating a depressive - like behavior in si mice ( figure 1(e ) ) . to study whether the behavioral changes induced by social isolation are associated with alterations of the hpa axis function , we measured the plasma cort levels and the adrenal glands weight , as an index of terminal phase of the hpa activation , in the si and gh mice .
interestingly , the levels of plasma cort were markedly reduced in the si mice compared to gh mice ( p = 0.002 ) ( figure 2(a ) ) . moreover
, a negative pearson 's correlation was specifically detected between the immobility time measured in the tst and the plasma cort levels ( r = 0.6367 ; p = 0.0107 ; table 2 ) .
a two - way anova showed a significant overall effect for the size of the adrenal glands ( f(1,26 ) = 5.704 ; p = 0.0245 ) , with the left adrenal gland heavier than the right one ( figure 2(b ) ) , consistent with the data reported in literature [ 46 , 47 ] .
a further fisher 's lsd post hoc analysis revealed a significant difference between the right and left adrenal gland for the gh mice ( p = 0.03 ) , but not for the si mice ( p = 0.19 ) ( figure 2(b ) ) .
these differences were not due to changes in body weight ( p = 0.45 ) ( figure 2(c ) ) . to investigate the possible molecular mechanisms underlying the social isolation stress - induced behavioral changes , we evaluated the mrna levels of different genes involved in neuronal plasticity in the hpc and pfc of si and gh mice .
firstly , we measured the mrna levels of total bdnf , which is known to modulate stress - induced behavioral changes , and bdnf splice variants 1 , 2 , 3 , 4 , 6 , 7 , and 8 .
we found that bdnf-7 transcript was significantly and selectively downregulated in both the hpc and the pfc of si mice , compared to gh mice ( hpc : p = 0.028 ; pfc : p = 0.01 ) ( figure 3 ) , while no significant differences were revealed for total bdnf and all the other bdnf transcripts analyzed ( figure 3 ) . protein levels analysis by western blot revealed only a trend for reduction of pro - bdnf in the hpc ( p = 0.082 ; figure 3(c ) ) and mature bdnf in the pfc ( p = 0.085 ; figure 3(d ) ) .
interestingly , we have observed a significant positive correlation between the plasma cort levels and the bdnf-7 mrna levels measured in the hpc , with only a trend in the pfc ( hpc : r = 0.5552 ; p = 0.0317 ; pfc : r = 0.4933 ; p = 0.0522 ; table 3 ) .
total bdnf mrna levels in the hpc and bdnf-7 mrna levels in the pfc positively correlate with the percentage of time spent in the of center ( total bdnf : r = 0.5459 ; p = 0.0353 ; bdnf-7 : r = 0.5886 ; p = 0.021 ; table 4 ) .
moreover , we found a trend for correlation between the cortical bdnf-7 levels and the total distance travelled in the of ( r = 0.5107 ; p = 0.0517 ; table 6 ) .
several lines of evidence suggest that metabotropic glutamate receptors are involved in stress - related disorders and in the mechanisms of action of antidepressants [ 4850 ] .
thus , we sought to investigate whether behavioral modifications induced by si stress correlated with changes in the expression of mglur1 , mglur2 , and mglur5 in the hpc and pfc .
interestingly , we found that mglur1 and mglur2 mrna levels were significantly reduced in the pfc of si mice compared to gh mice ( mglur1 : p = 0.012 ; mglur2 : p = 0.038 ; figure 4(b ) ) , while no differences were revealed for mglur5 mrna levels in the pfc and for all three mrna levels in the hpc between si and gh mice ( figures 4(a)-4(b ) ) .
however , only mglur1 in the pfc showed a positive correlation with the percentage of time spent in the of center ( r = 0.5886 ; p = 0.021 ; table 4 ) and the distance travelled in the of center ( r = 0.5886 ; p = 0.021 ; table 5 ) .
immediate early genes ( iegs ) are genes rapidly expressed upon different stimuli and play important roles in synaptic plasticity .
however , constitutive changes in iegs expression have been reported also after chronic stress and antidepressant treatments [ 5254 ] . therefore , to investigate whether si induced alterations in the iegs expression , we evaluated the mrna levels of c - fos , arc , and egr-1 in the hpc and pfc of si and gh mice .
we found that mrna levels of c - fos , arc , and egr-1 were decreased in both hpc and pfc of si mice compared to gh mice ( hpc : c - fos : p = 0.0092 ; arc : p = 0.016 ; egr-1 : p = 0.027 ; pfc : c - fos : p = 0.0099 ; arc : p = 0.029 ; egr-1 : p = 0.69 ) ( figures 4(c)-4(d ) ) .
a significant positive correlation was found for c - fos and arc mrna levels in the hpc and pfc and the percentage of time spent in the of center ( hpc : c - fos : r = 0.6627 ; p = 0.0071 ; arc : r = 0.536 ; p = 0.0395 ; pfc : c - fos : r = 0.5797 ; p = 0.035 ; arc : r = 0.5826 ; p = 0.0227 ; table 4 ) .
in the present study , we have found that si adult male mice spend less time in the center and are hyperactive in the oft . additionally , si mice showed higher immobility time in the tail suspension test compared to gh mice .
although several studies have reported that si rearing induces hyperactivity and anxious- and depressive - like behavior in rodents , only a few studies have addressed the consequences of si in adult rodents , especially in mice [ 21 , 23 , 5559 ] .
our findings suggest that social deprivation may be deleterious not only during childhood and adolescent period but also during adulthood in male mice
. moreover , our results are consistent with previous data showing that stress induces hyperactivity in rodents in response to exposure to novel environments , such as an open field [ 52 , 6063 ] .
the reduced plasma cort levels measured in si adult mice are paralleled by the decreased difference in weight between the left and right adrenal glands , suggesting that this hypofunction may be due to morphological changes in the hpa axis .
although stress is usually correlated with hpa hyperactivity , our results are in line with previous reports showing reduced levels of cort in si adult male rat and female mice [ 55 , 56 ] . moreover ,
lower adrenal activity in response to chronic stress was also reported in social defeat animal models .
interestingly , reduced basal levels of cort have been reported in patients with posttraumatic stress disorder ( ptsd ) , even decades after the traumatic events [ 6567 ] .
clinical studies have also reported lower cort levels in some patients at risk for ptsd and in healthy subjects living under constantly stressful environments [ 68 , 69 ] .
intriguingly , it has been reported that rats with reduced basal levels of cort display increased anxious - like behavior and low extinctions rates of conditioned fear , behavioral disturbances that model some aspect of ptsd . delayed and incomplete contextual fear extinction has been also described in si adult mice .
altogether these lines of evidence suggest that social isolation stress in adult mice could be a suitable model to study the behavioral and molecular alterations related to ptsd . however , further studies will be necessary to fully characterize this model . the results showing that si stress in adult male mice downregulated the expression of iegs are consistent with previous reports showing that social isolation rearing reduced iegs in rats [ 52 , 71 , 72 ] .
iegs are defined by their capability to be quickly transcribed without new protein synthesis , activated by transcription factors that are regulated by phosphorylation .
we measured the levels of gene expression 2 days after the tst to limit the possibility that behavioral tests affected the gene expression .
therefore , the downregulation of iegs observed here likely reflects an adaptation to the chronic si stress used , rather than the behavioral manipulation effects .
however , we can not exclude that the downregulation observed is due to the sum of social isolation and behavioral test manipulations .
we found that si stress decreased arc expression in both of the hpc and pfc .
it has been previously reported that arc mrna is delivered to dendrites , where it is specifically accumulated at recently activated synapses and locally translated into protein .
our finding that reduced arc expression correlated with increased anxiety- and depression - like phenotypes in si adult mice is consistent with previous data showing that arc expression is higher in the low - anxious sprague - dawley rats compared to the highly anxious hooded pvg strain and that chronic antidepressant treatment increased arc expression [ 54 , 75 ] .
altogether , these data suggest that arc may have a direct role in the synaptic plasticity responses after activation and in behavioral processes , coupling neuronal activity with structural remodeling and functional changes [ 76 , 77 ] .
interestingly , egr-1 is a transcriptional factor directly controlling the expression of arc that has also been associated with some form of synaptic plasticity and may be required for the development of late long - term potentiation and behavioral responses [ 7880 ] .
the present results , showing that both egr-1 and arc are decreased in hpc and pfc , may suggest that arc is downregulated as a consequence of stress - induced reduction of egr-1 expression .
remarkably , we found that bdnf-7 transcript was selectively reduced in both hpc and pfc of si mice , while the levels of total bdnf and all other exons were not changed . as a rule , bdnf-7 mrna levels
are lower compared to the most abundant bdnf transcripts in the brain ( 1 , 2 , 4 , and 6 ) ; therefore the reduction of bdnf-7 levels observed in si mice is probably not enough to significantly reduce the total expression of bdnf ( which includes all transcripts ) .
however , the bdnf-7 transcript is more efficiently translated to protein , compared to more highly expressed bdnf transcripts 1 , 4 , and 6   and is one of the few bdnf transcripts to be present in the dendrites in basal conditions .
it has been reported that synaptic plasticity controls local dendritic bdnf translation and that alterations in this pathway may compromise the long - lasting spine plasticity and lead to behavioral dysfunctions .
taken together , these lines of evidence strongly suggest that bdnf-7 may be rapidly translated in the dendritic compartment upon synaptic activation and that reduction of bdnf-7 levels induced by si stress may contribute to the behavioral impairments observed in si adult mice . our results showing that si stress decreased the levels of mglur1 and mglur2 in the pfc are in line with previous data reporting a significant attenuation in mglur function in the pfc of si reared rats .
remarkably , it has been also reported that chronic unpredictable stress ( cus ) reduces mglur2 expression and that upregulation of mglur2 reverses the anxious- and depressive - like behaviors caused by cus [ 48 , 50 ] .
furthermore , a possible role of mglur2 in controlling hyperactivity has been described as well .
moreover , injecting a mglur2/3 agonist locally into the pfc blocks the amphetamine - induced hyperactivity .
altogether , these data indicate that downregulation of mglur2 levels in the pfc of si mice may contribute to the behavioral impairments observed .
although previous studies have reported behavioral and synaptic plasticity deficits in the mglur1 null mice [ 87 , 88 ] and mglur1/5 agonist reduced the anxiety - like behaviors of dba/2 mice , the possible role of mglur1 in the pfc has not been specifically investigated yet .
further studies will be necessary to assess whether the expression of mglur1 in the pfc contributes to the behavioral changes induced by si .
we specifically found a reduction of mglur1 and mglur2 in the pfc but not in the hpc .
however , since we have analyzed gene expression in the entire hpc , without subdivision of the different regions ( dorsal and ventral hippocampus , dentate gyrus , ca1 , and ca3 ) , it is possible that variations in specific regions may be attenuated from the expression in the other regions and therefore not detected by our analyses .
further studies will be necessary to uncover whether changes of the mglur1 and mglur2 expression in specific regions of the hpc are actually induced by social isolation .
nevertheless , differential modulation of gene expression in different brain areas is quite common and it has been described for several genes and environmental manipulations .
although we have found significant correlations between behavioral impairments , cort plasma levels , and gene expression levels in the hpc and pfc , these findings are not sufficient to clearly establish a direct causal effect between these molecular changes and the behavioral alterations .
further studies using both molecular and pharmacological approaches , to specifically modulate the expression of these genes in hpc and pfc , will be necessary .
another limitation of the study is the lack on of time - dependent effects of si .
it would be interesting in future studies to investigate the shortest period of si that is able to induce behavioral and molecular changes in adult mice and whether a longer period of isolation is able to worsen the phenotype that we have reported here .
moreover , while we can not rule out that the molecular changes observed in si mice are a consequence of the combination of the social isolation paradigm and the behavioral tests , our results show that neuroplasticity - related genes are differentially modulated in gh and si mice .
overall , in the present study we showed that si stress in the adult male mice resulted in anxiety- and depression - like phenotypes .
additionally , we showed that behavioral modifications correlated with decrease of plasma cort levels and downregulation of neuroplasticity - related genes in hpc and pfc .
in particular , the selective reduction of the bdnf-7 transcript in both brain areas of si mice for the first time links this dendrite - resident bdnf isoform with synaptic plasticity and the behavioral consequences of si . further work to explore the role of this bdnf transcript is warranted .
 stress is a major risk factor in the onset of several neuropsychiatric disorders including anxiety and depression . 
 although several studies have shown that social isolation stress during postweaning period induces behavioral and brain molecular changes , the effects of social isolation on behavior during adulthood have been less characterized . 
 aim of this work was to investigate the relationship between the behavioral alterations and brain molecular changes induced by chronic social isolation stress in adult male mice . 
 plasma corticosterone levels and adrenal glands weight were also analyzed . 
 socially isolated ( si ) mice showed higher locomotor activity , spent less time in the open field center , and displayed higher immobility time in the tail suspension test compared to group - housed ( gh ) mice . 
 si mice exhibited reduced plasma corticosterone levels and reduced difference between right and left adrenal glands . 
 si showed lower mrna levels of the bdnf-7 splice variant , c - fos , arc , and egr-1 in both hippocampus and prefrontal cortex compared to gh mice . finally , si mice exhibited selectively reduced mglur1 and mglur2 levels in the prefrontal cortex . altogether 
 , these results suggest that anxious- and depressive - like behavior induced by social isolation stress correlates with reduction of several neuroplasticity - related genes in the hippocampus and prefrontal cortex of adult male mice . 
1. Introduction
2. Materials and Methods
3. Results
4.
