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the embo journal 2010 29 184­195 www.embojournal.org 2010 european molecular biology organization all rights reserved 0261-4189/10 the embo journal social isolation stress induces atf-7 phosphorylation and impairs silencing of the 5-ht 5b receptor gene toshio maekawa1,6 seungjoon kim1,6,7 daisuke nakai1,2 chieko makino1,2,8 tsuyoshi takagi1 hiroo ogura3 kazuyuki yamada4 bruno chatton5 and shunsuke ishii1,2 1 laboratory of molecular genetics riken tsukuba institute tsukuba ibaraki japan 2university of tsukuba graduate school of comprehensive human sciences tsukuba ibaraki japan 3tsukuba research laboratories eisai co ltd ibaraki japan 4support unit for animal experiment research resources center brain science ´ institute bsi riken wako saitama japan and 5ecole superieure ´ de biotechnologie de strasbourg universite louis pasteur parc d innovation umr7100 cnrs-ulp strasbourg illkirch cedex france introduction atf-7 originally called atfa is structurally related to atf-2 hai et al 1989 maekawa et al 1989 gaire et al 1990 a member of the atf­creb family of transcription factors atf-2 atf-7 and cre-bpa nomura et al 1993 form a subfamily in the atf­creb family each of these three factors contains a transcription-activation domain consisting of a metal-finger structure and stress-activated protein kinase sapk phosphorylation sites and a b-zip type dna-binding domain various stresses including inflammatory cytokines activate sapks such as p38 and jnk davis 2000 which then phosphorylate atf-2 and activate its trans-activating capacity gupta et al 1995 livingstone et al 1997 van dam et al 1997 atf-7 is also phosphorylated by p38 but not by jnk de graeve et al 1999 atf-2 and atf-7 can form homodimers or heterodimers with jun and bind to camp response element cre 50 -tgacgtca-30 chatton et al 1994 atf-7 binds to mouse atfa-associated modulator mam which is a component of the eset complex de graeve et al 2000 wang et al 2003 as eset is a histone methyltransferase hmtase that converts lysine 9 of histone h3 h3-k9 from the dimethyl to the trimethyl form therefore atf-7 is thought to support gene silencing by inducing histone h3-k9 trimethylation two reports have suggested a role for atf-2 family transcription factors in epigenetic gene silencing the yeast homologue of atf-2 atf1 contributes to heterochromatin formation independently of the rnai machinery jia et al 2004 vertebrate atf-2 also interacts with the histone variant macroh2a which is enriched in the inactive x chromosome in female mammalian cells and functions to maintain gene silencing agelopoulos and thanos 2006 both atf-7 and atf-2 are ubiquitously expressed in various tissues including the brain takeda et al 1991 goetz et al 1996 atf-2 null mice die immediately after birth because of defects in respiration which appear to be caused by impaired proliferation of cytotrophoblasts in the placenta maekawa et al 1999 atf-2 heterozygotes are highly prone to mammary tumours in which the expression levels of maspin a tumour suppressor and gadd45a which is induced by hypoxic stress are decreased maekawa et al 2007 both these genes encode the regulators of apoptosis suggesting that defects in the apoptotic machinery are linked to the occurrence of mammary tumours in contrast the physiological role of atf-7 is unknown although the atf-2 and atf-7 double mutant exhibits embryonic lethality with abnormalities in the developing liver and heart breitwieser et al 2007 human neuropsychiatric disorders such as depression have multiple-risk factors including environmental and genetic factors a loss of social contact is one environmental factor that appears to be linked to both the onset and relapse 2010 european molecular biology organization many symptoms induced by isolation rearing of rodents may be relevant to neuropsychiatric disorders including depression however identities of transcription factors that regulate gene expression in response to chronic social isolation stress remain elusive the transcription factor atf-7 is structurally related to atf-2 which is activated by various stresses including inflammatory cytokines here we report that atf-7-deficient mice exhibit abnormal behaviours and increased 5-ht receptor 5b htr5b mrna levels in the dorsal raphe nuclei atf-7 silences the transcription of htr5b by directly binding to its 50 -regulatory region and mediates histone h3-k9 trimethylation via interaction with the eset histone methyltransferase isolation-reared wild-type wt mice exhibit abnormal behaviours that resemble those of atf-7-deficient mice upon social isolation stress atf-7 in the dorsal raphe nucleus is phosphorylated via p38 and is released from the htr5b promoter leading to the upregulation of htr5b thus atf-7 may have a critical role in gene expression induced by social isolation stress the embo journal 2010 29 184­195 doi:10.1038 emboj.2009.318 published online 5 november 2009 subject categories chromatin transcription neuroscience keywords 5-ht receptor atf-7 histone methylation social isolation stress corresponding authors t maekawa or s ishii laboratory of molecular genetics riken tsukuba institute 3-1-1 koyadai tsukuba ibaraki 3050074 japan tel þ 81 29 836 9031 fax þ 81 29 836 9030 e-mail maekawa@rtc.riken.jp or sishii@rtc.riken.jp 6 these authors contributed equally to this work 7 present address laboratory of veterinary reproduction kyungpook national university sankyuk-dong buk-gu daegu 702-701 south korea 8 present address division of molecular genetics department of physiology and cell biology kobe university graduate school of medicine kobe 650-0017 japan received 12 december 2008 accepted 8 october 2009 published online 5 november 2009 184 the embo journal vol 29 no 1 2010

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atf-7 and social isolation stress t maekawa et al of depression paykel et al 1980 long-term social isolation of rodents after weaning provides a model to study the behavioural consequences of loss of social interactions many of the symptoms induced by isolation rearing may be relevant to neuropsychiatric disorders rodgers and cole 1993 isolated animals are aggressive and exhibit anxietylike behaviours and increased locomotor activity rodgers and cole 1993 blanchard et al 2001 one of the typical abnormal behaviour in isolation-reared mice is a deficit in pre-pulse inhibition ppi of the acoustic startle response wilkinson et al 1994 in fact isolation-induced disruption of ppi has been used as a disease model in screening antipsychotic drugs in animal studies isolation stress changes the activity of brain neurotransmitters blanc et al 1980 blanchard et al 2001 in the case of acute stress several transcription factors including c-fos and corticosteroid receptors are activated and modulate multiple target genes kaufer et al 1998 however the transcription factors that are activated and the regulation of gene expression patterns in response to a chronic stress such as social isolation stress remain elusive in addition as the effect of social isolation stress on behaviour is long-lived this stress may cause epigenetic changes however the mechanism by which epigenetic change is caused by isolation stress remains unknown in this study we have demonstrated that atf-7-deficient atf-7À/À mice exhibit abnormal behaviours reminiscent of isolation-reared wild-type wt mice social isolation stress induced the phosphorylation of atf-7 and p38 in the dorsal raphe nuclei as well as a release of atf-7 from the promoter of the 5-ht receptor 5b htr5b gene leading to an impaired silencing of this gene results abnormal behaviours of atf-7À/À mice we generated atf-7À/À mice supplementary figure s1 and under pathogen-free conditions atf-7À/À mice appeared healthy until at least 12 months of age as atf-7 mrna is expressed at relatively high levels in parts of the brain goetz et al 1996 we examined various behaviours originally using wt and atf-7À/À littermate mice with a mixed cba 25 Â c57bl/6 75 genetic background and later using c57bl/6 congenic mice in the marble-burying test which is used to examine anxiety-related behaviours spooren et al 2000 atf-7À/À mice exhibited increased marble-burying behaviour compared with wt mice figure 1a and supplementary figure s2a in other tests of anxiety-related behaviours such as the amount of time spent in the centre of an open-field and the elevated plusmaze test spooren et al 2000 there was no significant difference between atf-7À/À and wt mice figure 1b and c and supplementary figure s2b atf-7À/À mice did exhibit a significant increase in the startle response to a pulse-alone stimulus figure 1d and supplementary figure s2c ppi in which the startle reflex response is attenuated by a pre-pulse is an important measure of sensorimotor gating geyer et al 1990 atf-7À/À mice displayed lower levels of ppi of the acoustic startle response figure 1e and supplementary figure s2d although the association between the startle response and ppi is not currently clear a negative correlation between the startle response and ppi in wt mice has been 2010 european molecular biology organization figure 1 abnormal behaviours in atf-7À/À mice wild-type wt þ þ and atf-7À/À c57bl/6 congenic mice were used for all assays data are mean±s.e.m a marble-burying test po0.01 n ¼ 10­12 for each group b center of the open-field test time spent in the center of the test apparatus is expressed as a percent of total time 10 min ns no significant difference n ¼ 13­16 for each group c elevated plus-maze test mice were observed in an elevated plus-maze for 5 min percentage of entries into open arms left and the time spent in open arms right are shown n ¼ 13­16 for each group d acoustic startle response amplitude of the startle response to a 120 db acoustic stimulus is shown n ¼ 13­16 for each group e pre-pulse inhibition of the acoustic startle response the response to a white noise stimulus of 120 db after a 20 ms pre-pulse warning stimulus 70 or 80 db is shown n ¼ 13­16 for each group reported egashira et al 2005 if these two phenomena are correlated in atf7À/À mice an increase in startle reactivity may lead to decreased ppi however we cannot exclude the possibility that atf-7 is independently involved in the modulation of startle response and its ppi the embo journal vol 29 no 1 2010 185

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atf-7 and social isolation stress t maekawa et al the atf-7À/À and wt mice responses were indistinguishable in other behavioural tests we examined spontaneous locomotor activity in a new environment by placing mice in an open-field chamber and monitoring their behaviour there was no significant difference in the locomotor activity of atf-7À/À and wt mice on the first and second day of the trials supplementary figure s3a and b we also examined motor coordination using a rotating rod treadmill overall the amount of time mice spent on the rotarod increased with training supplementary figure s3c the retention time of atf-7À/À mice on the rod was not significantly different from that of wt mice at 0 stationary 5 or 10 r.p.m in the footprint test there was no significant difference in the stride length and the step width between mutant and wt mice supplementary figure s3d in the forced swimming test there was also no difference between atf-7À/À and wt mice supplementary figure s4a we also examined spatial learning ability using the morris water maze task wt and atf-7À/À mice took similar lengths of time to reach the visual platform to escape from the water supplementary figure s4b thus indicating that atf-7À/À mice have normal vision motor function and escape behaviour in the water maze task mice were then trained in a hidden platform task in which mice search for a submerged platform to escape from the water atf-7À/À and wt mice took similar lengths of time over the 7 days of testing to locate the hidden platform supplementary figure s4c thus atf-7À/À and wt mice were able to learn the location of a hidden platform during the course of the trials we then carried out a probe test in which the platform is removed from the pool after completion of the hidden platform task and the trained mice are allowed to swim freely for 60 s the time spent in the target quadrant by atf-7À/À mice was similar to that of wt mice supplementary figure s4d these results indicate that a normal spatial learning ability is present in atf-7À/À mice the number of crossings of the hidden platform and the swimming distance of atf-7À/À and wt mice was also similar during the probe trial supplementary figure s4e and f thus the performance of atf-7À/À mice in the hidden platform task was indistinguishable from wt mice upregulation of the htr5b gene in the dorsal raphe nucleus of atf-7 À/À mice atf-7 mrna was detected in the cortex the cerebellum the hippocampus and the brainstem including the medulla the pons and the midbrain of wt mice figure 2a but not of atf-7À/À mice supplementary figure s5 no obvious morphological abnormalities were found in these tissues in atf-7À/À mice data not shown western blotting indicated that atf-7 expression levels varied in these tissues figure 2b several studies have linked abnormal marbleburying behaviour to 5-ht function jenck et al 1998 further disruptions in ppi of the startle response are correlated not only with d2 dopamine and n-methyl-d-aspartate signalling systems but also with 5-ht geyer et al 2001 therefore we focused our attention on the dorsal raphe nuclei of the brainstem where much of the 5-ht in the brain is localized and relatively high levels of atf-7 are expressed to identify the atf-7 target genes in the brainstem that may have a role in the abnormal behaviour in atf-7À/À mice we performed a dna microarray analysis using rna from the 186 the embo journal vol 29 no 1 2010 brainstem of atf-7À/À and wt mice the results indicate that 25 genes were upregulated and 38 genes downregulated by more than two-fold by the loss of atf-7 of these atf-7 target genes the functions of 11 of the upregulated genes are known whereas the functions of only 7 of the downregulated genes have been reported among these genes only the htr5b and the ciliary neurotrophic factor receptor cntfr genes have been associated with neuronal function as the 5-ht system appeared to be associated with the abnormal behaviour of atf-7À/À mice as described above upregulation of the htr5b gene may be associated with the phenotype of atf-7À/À mice cntf is a cytokine that has neurotrophic and differentiating effects on cells in the central nervous system and the cntf­cntf receptor system affects motor neurons vergara and ramirez 2004 however there has been no report demonstrating a connection between the cntf system and anxiety-related behaviours therefore we focused our attention on the htr5b gene as htr5b is thought to act as an autoreceptor serrats et al 2004 its upregulation may lead to a decrease in the extracellular concentration of serotonin 5-ht there is abundant evidence for the role of decreased 5-ht in depression and anxiety disorders artigasa et al 1996 selective serotonin re-uptake inhibitors ssris which increase the extracellular concentration of 5-ht in the dorsal raphe nuclei are widely used as anti-depressant drugs htr5b mrna levels in the atf-7À/À brainstem were approximately 12-fold higher than in wt figure 2c in situ hybridization showed higher levels of htr5b mrna expression in the atf-7À/À dorsal raphe nuclei which also expressed a serotonin transporter mrna than in the wt figure 2d and supplementary figure s6 there appeared to be no obvious difference in the levels of htr5b mrna between wt and atf-7À/À mice in other regions including the hippocampus the habenular nucleus and the inferior olivary nucleus supplementary figure s7 among the many reported antagonists of 5-ht receptors methiothepin has a relatively high affinity for the 5-ht 5b receptor although it also binds to other 5ht receptors including the 5-ht 1a receptor boess and martin 1994 injection of methiothepin into atf-7À/À mice suppressed the increased marble-burying behaviour whereas saline-treated control atf-7À/À mice still exhibited increased marble-burying behaviour figure 2e furthermore methiothepin also alleviated the lower levels of ppi in atf-7À/À mice figure 2f these results suggest that increased expression of htr5b mrna in the atf-7À/À brainstem may at least partly contribute to their abnormal behaviour although loss of atf-7 could cause changes in the expression of other genes in various regions of the brain and also contribute to abnormal behaviours silencing of the htr5b gene by atf-7 via direct binding to its 5 0 -region analysis of the dna sequence in the 50 region of the mouse htr5b gene identified three cre-like sites at nucleotides À3374 À3340 and À2325 where þ 1 is the major transcriptional start site all of which have only a 1 or 2 bp difference from the consensus cre sequence figure 3a gel mobilityshift assays were carried out using nine dna probes which cover approximately 4.4 kbp of the 50 -region of htr5b and nuclear extracts from 293t cells that were transfected with a flag-tagged atf-7 expression plasmid or control 2010 european molecular biology organization

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atf-7 and social isolation stress t maekawa et al figure 2 increased levels of 5-ht receptor 5b htr5b mrna in the atf-7À/À brainstem a atf-7 mrna expression in various regions of the brain was examined by in situ hybridization with anti-sense and sense probes bar 100 mm b extracts 20 mg of protein from the indicated regions of wild-type wt or atf-7À/À brains were used for western blotting with anti-atf-7 the bands indicated by arrows are the atf-7 signals c real-time rt­pcr analysis of htr5b mrna levels using total rna from the brainstem n ¼ 4 po0.001 d htr5b mrna expression in the dorsal raphe nuclei was examined by in situ hybridization using probes for htr5b green and the serotonin transporter 5htt red cell nuclei were identified by dna staining using toto-3 blue the sections were examined by laser confocal microscopy and representative images are presented the panels at the right show the merged images bar 100 mm the white box indicates a subregion of each image that is presented at higher magnification below e f a 5-ht 5b receptor antagonist reduced the abnormal behaviour of atf-7À/À mice marble-burying behaviour e and pre-pulse inhibition ppi f of wt and atf-7À/À c57bl/6 congenic mice was examined after administration of either vehicle or methiothepin n ¼ 10­12 for each group in e and n ¼ 7 for each group in f po0.05 2010 european molecular biology organization the embo journal vol 29 no 1 2010 187

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atf-7 and social isolation stress t maekawa et al figure 3 binding of atf-7 to the 5-ht receptor 5b htr5b promoter region leads to silencing a presence of camp response element crelike sites in the 50 region of the mouse htr5b gene the cre-like sites in the 50 -region of mouse htr5b and nine dna probes used for gel mobility-shift assays are shown b c gel mobility-shift assays were performed using nuclear extracts prepared from 293t cells transfected with a flag-atf-7 expression vector or control empty vector the #2 and #4 dna probes were used as the probes in b in some lanes anti-flag or control igg was added in c oligonucleotides containing the two cre-like sites derived from probe #2 or the one cre-like site from probe #4 were used as probes in some lanes a 50-fold excess of competitor containing the same sequence as the probe wild-type wt or a mutated cre-like site was added free probe is indicated by a closed arrowhead whereas atf-7-bound dna is shown by an open arrowhead the atf-7-dna complex which was super-shifted by the anti-flag antibody is indicated by the arrow d atf-7 represses htr5b gene transcription rn46a cells were transfected with the indicated htr5b promoter-luciferase construct together with 1 þ 2 þ þ or 3 þ þ þ mg of the atf-7 expression plasmid or the control empty vector À and luciferase activity was measured values indicate mean±s.d n ¼ 3 po0.05 po0.01 empty vector when the #2 or #4 probes were used a retarded band was detected in extracts containing flag-atf-7 figure 3b these specific retarded bands were further shifted when an anti-flag antibody was added indicating that the bands contained flag-atf-7 in contrast no retarded bands were observed with the other probes supplementary figure s8 we then used 54 bp and 18 bp oligonucleotides containing the cre-like sites derived from the #2 and #4 probes respectively the retarded bands generated using either probe were competed out by excess amounts of unlabelled competitor oligonucleotide but not by competitors which contained mutated cre-like sites figure 3c these results indicate that atf-7 binds directly to the cre-like sites in the 50 -region of the mouse htr5b gene when a htr5b promoter-luciferase reporter containing the 4.4 kb 50 -region of the htr5b gene was cotransfected into rn46a cells which are derived from rat medullary raphe nucleus cells atf-7 inhibited luciferase expression by ap188 the embo journal vol 29 no 1 2010 proximately 70 figure 3d in contrast mutation of the three cre-like sites in this reporter relieved the atf-7dependent silencing these results suggest that atf-7 suppresses the transcription of htr5b through interaction with cre-like sites the results of chromatin immunoprecipitation chip assays using the brainstem chromatin and an anti-atf-7 antibody indicated that atf-7 bound to the region containing cre-like sites of the htr5b gene but not to the rna start site or the 2nd exon figure 4a further the binding of atf-7 to this region was not detected using the atf-7À/À brainstem chromatin binding of atf-2 to this region was also not detected figure 4b atf-7 mediates histone h3-k9 trimethylation of the htr5b promoter region by recruiting the eset hmtase the results of chip assays using the wt brainstem chromatin and anti-h3-k9m3 antibodies indicated that histone h3 in the 2010 european molecular biology organization

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atf-7 and social isolation stress t maekawa et al figure 4 binding of atf-7 to the 5-ht receptor 5b htr5b promoter is correlated with histone h3-k9 trimethylation a b chromatin immunoprecipitation chip assays were carried out using the brainstem of wild-type wt and atf-7À/À mice and anti-atf-7 a anti-atf-2 b or control igg a b extracted dna was amplified by real-time pcr using primers that cover the camp response element cre like sites the transcription start site or the 2nd exon of the htr5b gene the relative densities of bands are indicated and each bar represents the mean±s.d n ¼ 3 c generation of an rn46a cell line in which atf-7 levels are downregulated atf-7 kd-rn46a by expression of a small hairpin-type double-stranded rna nuclear extracts of the parental rn46a cells and the atf-7 kd-rn46a cells were used for western blotting to detect atf-7 d real-time rt­pcr analysis of htr5b mrna levels using rnas from the parental rn46a cells and atf-7 kd-rn46a cells values are mean±s.d n ¼ 3 e chip assays were carried out using anti-histone h3-k9m3 and parental rn46a cells or atf-7 kd-rn46a cells extracted dna was amplified by real-time pcr using primers that cover the cre-like sites the transcription start site or the 2nd exon of the htr5b gene the relative densities of the bands are indicated and each bar represents the mean±s.d n ¼ 3 po0.05 po0.01 po0.001 detected this result may indicate that htr5b gene transcription is repressed by histone methylation in most cells of the brainstem with the exception of the dorsal raphe nucleus in atf-7À/À mice to assess the role of atf-7 in histone h3-k9 methylation we generated an atf-7 knock-down kd rn46a cell line by expressing a small hairpin-type doublestrand rna the atf-7 level was approximately one-eighth that of the parental cell line figure 4c in kd-rn46a cells htr5b mrna levels increased approximately 2.3-fold compared with the parental cell line figure 4d in chip assays binding of atf-7 to the region containing the cre-like sites of the htr5b gene was detected in parental rn46a cells but not in the atf-7 kd-rn46a cells supplementary figure s9b binding of atf-2 to the same region was not detected supplementary figure s9c the degree of histone h3-k9 trimethylation in the region containing the cre-like sites or the 2nd exon of htr5b gene was lower in the kd-rn46a cells than in parental cells figure 4e thus atf-7 contributes to h3-k9 trimethylation at the htr5b gene promoter to investigate whether the eset hmtase is involved in the silencing of htr5b by atf-7 we examined atf-7­eset interactions by co-immunoprecipitation immunocomplexes prepared from rn46a cell lysates using anti-atf-7 contained eset whereas immunocomplexes prepared with control igg did not contain eset figure 5a the results of chip assays using the wt brainstem chromatin and anti-eset antibodies indicated that eset bound to the region containing cre-like sites of htr5b but not to the region containing the rna start site or the 2nd exon figure 5b further binding of eset to this region was not detected using the atf-7À/À brainstem chromatin similar results were also obtained in chip assays using the parental rn46a and atf-7 kd-rn46a cells figure 5c members of the atf-2 subfamily are activated in response to various stresses and tnf-a is one of the typical inflammatory cytokines which can activate atf-2 brinkman et al 1999 when rn46a cells were treated with tnf-a the level of htr5b mrna was gradually increased by approximately 2.5-fold at 8 h after tnf-a addition figure 5d phosphorylation of atf-7 at thr-53 and of atf-2 at thr-71 was also enhanced by tnf-a treatment figure 5e the gradual increase in atf-7 and atf-2 phosphorylation observed here is apparently different from the rapid and transient induction of atf-2 phosphorylation in response to osmotic stress and tgf-b treatment in non-neuronal cells in which atf-2 phosphorylation peaks at 30 min after treatment and then decreases sano et al 1999 tnf-a also rapidly induces phosphorylation of atf-2 within 1 h in non-neuronal cells brinkman et al 1999 thus the gradual response of the p38-atf-2/7 pathway to tnf-a may be characteristic of neurons furthermore the results of chip assays using tnf-a-treated rn46a cells indicate that atf-7 was released from the 50 -region of htr5b by tnf-a treatment figure 5f social isolation stress induces abnormal behaviours similar to those of atf-7À/À mice and htr5b expression defects in ppi attenuation of the acoustic startle response are induced by social isolation stress wilkinson et al 1994 although isolated animals exhibit a variety of phenotypes including aggressive behaviour blanchard et al 2001 this observation suggests that exposure of wt mice to social the embo journal vol 29 no 1 2010 189 region containing the cre-like sites the rna start site and the 2nd exon of htr5b is trimethylated at k9 supplementary figure s9a when the atf-7À/À brainstem chromatin was used similar levels of histone h3-k9 trimethylation were 2010 european molecular biology organization

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atf-7 and social isolation stress t maekawa et al figure 5 atf-7 recruits the eset hmtase to the 5-ht receptor 5b htr5b gene a co-immunoprecipitation of atf-7 and eset whole cell lysates of rn46a cells were immunoprecipitated with anti-atf-7 or control igg and the immunocomplexes were then analyzed by western blotting using anti-eset or anti-atf-7 antibodies b recruitment of eset to the htr5b gene by atf-7 chromatin immunoprecipitation chip assays were carried out using anti-eset and chromatin from wild-type wt or atf-7À/À brainstems extracted dna was amplified by real-time pcr using primers that cover the camp response element cre like sites the rna start site or the 2nd exon of the htr5b gene the relative densities of the bands are indicated and each bar represents the mean±s.d n ¼ 3 c chip assays were carried out using anti-eset and the parental rn46a or the atf-7 kd-rn46a cells extracted dna was amplified by real-time pcr using primers that cover the atf-7-binding sites of the htr5b gene n ¼ 3 d upregulation of htr5b mrna by tnf-a in rn46a cells real-time rt­pcr analysis of htr5b mrna was performed using rnas from rn46a cells treated with tnf-a for the indicated times n ¼ 3 e increase in phosphorylation of atf-7 in response to tnf-a nuclear extracts were prepared from rn46a cells treated with tnf-a for the indicated times and used for western blotting with antibodies that recognize the indicated proteins we have identified the atf-7 band by confirming that it was lost in the atf-7À/À whole brain nuclear extract supplementary figure s10 f release of atf-7 from the htr5b promoter by tnf-a treatment chip assays were carried out as described above using anti-atf-7 and rn46a cells treated with tnf-a for the indicated times n ¼ 3 po0.05 po0.01 isolation stress may cause abnormal behaviour and an increase in htr5b mrna levels in the dorsal raphe nuclei both of which were observed in atf-7À/À mice in fact wt mice exhibited increased marble-burying behaviour after 1 month of isolation rearing figure 6a furthermore after isolation rearing of wt mice for 1 month htr5b mrna levels in the brainstem increased approximately 12-fold figure 6b in situ hybridization indicated that the level of htr5b mrna in the dorsal raphe nuclei which also expressed serotonin transporter mrna was enhanced by isolation rearing figure 6c and supplementary figure s6c the result of chip assays using the brainstem from group or isolationreared mice indicated that the degree of histone h3-k9 trimethylation was not affected by isolation stress supplementary figure s11 this result may indicate that htr5b gene transcription is repressed by histone methylation 190 the embo journal vol 29 no 1 2010 in most brainstem cells with the exception of the dorsal raphe nucleus in isolation-reared mice when atf-7À/À mice were exposed to isolation stress enhancement of marble-burying behaviour was observed figure 6d however htr5b mrna levels in the atf-7À/À brainstem were not further increased by isolation stress figure 6e these results suggest that isolation stress induces abnormal marble-burying behaviour not only by induction of htr5b but also by changing the expression of other genes social isolation stress induces the phosphorylation of atf-7 and release of atf-7 from the htr5b gene we examined phosphorylated atf-7 p-atf-7 signals in the dorsal raphe nucleus of group and isolation-reared wt mice social isolation stress significantly increased the number of 2010 european molecular biology organization

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atf-7 and social isolation stress t maekawa et al of chip assays using the brainstem chromatin of group and isolation-reared wt mice and anti-atf-7 indicated that binding of atf-7 to the 50 -region of htr5b was lost by social isolation stress figure 7c these data suggest that social isolation stress induces a release of atf-7 from the htr5b promoter social isolation stress also significantly increased the number of neurons expressing phosphorylated active p38 in the dorsal raphe nuclei figure 7d discussion this study suggests that atf-7 may contribute to the formation of a heterochromatin-like structure in the htr5b promoter via histone h3-k9 trimethylation the effect of isolationrearing on behaviour is long-lived suggesting that epigenetic changes may have occurred in the absence of stress silencing of htr5b may be maintained via atf-7-mediated histone h3-k9 trimethylation phosphorylation of atf-7 may disrupt interactions with histone methyltransferase and enhance the association with co-activators containing histone acetyltransferase and/or histone demethylase leading to disruption of the heterochromatin-like structure the resulting transcriptionally active chromatin structure may be stable for a relatively long time mechanisms by which transcriptionally active memory can be modulated without affecting dna methylation remain elusive but a recent report showed that the epigenetic memory of an active state can be established by histone h3.3 deposition ng and gurdon 2008 although at present the mechanism by which isolation stress induces phosphorylation of atf-7 is unknown isolation stress increases the peripheral tissue levels of tnf-a wu et al 1999 which may move into the brain and be involved in the onset of depression connor and leonard 1998 both isolation stress and tnf-a increased atf-7 phosphorylation and htr5b mrna levels which are accompanied by a release of atf-7 from the htr5b promoter the mechanism by which the phosphorylation of atf-7 causes a release of atf-7 from target sites is unknown the phosphorylation-induced release of atf-7 from dna could be caused by changes in interactions between atf-7 and uncharacterized factors that enhance atf-7 affinity for dna atf-7 is highly homologous to atf-2 but there are differences between the two proteins in luciferase reporter assays atf-2 activated transcription from the cre-containing promoter but atf-7 did not even in the presence of active p38 this result suggests that atf-7 may have a role primarily in transcriptional repression as we have observed that atf-7 forms a heterodimer with atf-2 data not shown further study is required to compare the function of atf-7 and atf-2 homodimers and atf-7­atf-2 heterodimers our results suggest that upregulation of htr5b may partly contribute to the abnormal behaviour of atf-7À/À mice the rodent 5-ht5 receptor family consists of two receptors 5-ht 5a and 5b htr5a and htr5b which share 69 amino acid identity and have 23­34 homology with other 5-ht receptors plassat et al 1992 htr5a has been identified in mouse rat and human mouse and rat have a functional htr5b gene whereas the human coding sequence is interrupted by stop codons grailhe et al 2001 thus humans do not have a functional htr5b and therefore another subtype such as htr5a could be upregulated and has a role in response to social isolation stress in humans htr5a-deficient mice the embo journal vol 29 no 1 2010 191 figure 6 social isolation stress increases marble-burying behaviour and induces 5-ht receptor 5b htr5b mrna expression a marble-burying behaviour of group and isolation-reared wt mice was examined n ¼ 10­12 of c57bl/6 congenic mice for each group b real-time rt­pcr analysis of htr5b mrna was performed using rnas from the brainstem of group and isolationreared wt mice n ¼ 3 c htr5b mrna expression in the dorsal raphe nuclei of group and isolation-reared wt mice was examined by in situ hybridization as described in figure 2d d the groupreared wt and atf-7À/À mice and the isolation-reared atf-7À/À mice were used for marble-burying tests n ¼ 10­12 of c57bl/6 congenic mice for each group e real-time rt­pcr analysis of htr5b mrna was performed using rnas from the brainstems of the mice described in d n ¼ 3 po0.01 po0.001 neurons expressing p-atf-7 and p-atf-2 in the dorsal raphe nucleus figure 7a as the amino-acid sequence of the p38 phosphorylation site is highly conserved between atf-7 and atf-2 antibodies to p-atf-7 also recognize p-atf-2 however the results of western blotting using extracts from the brainstem indicated that the phosphorylation of atf-7 increased under social isolation stress conditions whereas the phosphorylation of atf-2 did not figure 7b the results 2010 european molecular biology organization

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atf-7 and social isolation stress t maekawa et al figure 7 social isolation stress induces atf-7 phosphorylation and release of atf-7 from the 5-ht receptor 5b htr5b promoter a brain sections containing dorsal raphe nuclei of group or isolation-reared wild-type wt mice were stained with antibodies which recognize p-atf-7 and p-atf-2 green or neun red a neuronal specific nuclear protein dna was stained with toto-3 blue the merged images are shown in the right panels the average number of neurons expressing p-atf-7 or p-atf-2 in three independent experiments is indicated by the bar graph±s.d b nuclear extracts were prepared from the brainstem of group or isolation-reared wt mice which were perfused with pfa and analyzed by sds­page after decrosslinking followed by western blotting with anti-p-atf-2/7 anti-atf-7 and anti-atf-2 the ratio of the density of each band in isolation-reared mice to that in group-reared mice is indicated in the bar graph c release of atf-7 from the htr5b promoter by isolation stress chromatin immunoprecipitation chip assays were carried out using chromatin from the brainstem of group or isolation-reared wt mice and anti-atf-7 extracted dna was amplified by real-time pcr using primers that cover the atf-7-binding sites of htr5b n ¼ 3 d brain sections containing dorsal raphe nuclei of group or isolation-reared wt mice were stained with antibodies that recognize p-p38 as described above the number of neurons expressing p-p38 is quantified at the right n ¼ 3 po0.05 po0.01 display increased exploratory activity when exposed to new environments suggesting that htr5a modulates the activity of neural circuits involved specifically in exploratory behaviour grailhe et al 1999 however there has been no report of a mouse knockout of htr5b isolation-rearing was recently reported to reduce 27.0­60.9 transcription of many postsynaptic 5-ht receptors in the prefrontal cortex and the 5-ht 1b 2a and 2c receptors in the hypothalamus and the midbrain whereas 5-ht 6 receptor mrna levels increased 52.5 in the hippocampus bibancos et al 2007 htr5b expression was not examined the fold change in mrna level of these 5-ht receptors was much lower than the change in htr5b mrna described herein 12-fold we have also examined the expression level of 5-ht receptors other than 5b in atf-7À/À 192 the embo journal vol 29 no 1 2010 mice slight decreases in the 2a and 2c receptors in the hypothalamus and 1a and 1b receptors in the cortex were observed in atf-7À/À mice whereas the levels of 2a and 3a receptors in the cortex increased slightly supplementary figure s12 however the degree of these changes was much lower than the change in htr5b in the brainstem in response to social isolation rearing there was no difference in the htr5b mrna levels in the hypothalamus and in the cortex between wt and atf-7À/À mice thus the degree of change in the htr5b mrna levels in the brainstem in response to loss of atf-7 or social isolation stress is dramatic suggesting a unique role for htr5b however it is also likely that loss of atf-7 changes the expression of multiple genes in various regions of the brain that may also contribute to abnormal behaviours 2010 european molecular biology organization

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atf-7 and social isolation stress t maekawa et al materials and methods animals all mice used were 2­6-month-old males in the original behaviour tests immunohistochemistry rna analysis and chip assays the littermate mice which were generated by mating atf-7 heterozygotes with a mixed cba 25 Â c57bl/6 75 genetic background were used to confirm some behavioural abnormalities of atf-7À/À mice c57bl/6 congenic mice which were generated by backcrossing onto a c57bl/6 genetic background for 7 generations were used both wt and atf-7À/À mice were maintained in a temperature and humidity-controlled room with free access to food and water animals were maintained on a 12-h-light and 12-h-dark cycle lights on at 0800 h lights off at 2000 h for social isolation stress mice were group-housed until 1-month old and then housed individually for 1 month before the start of the experiments experiments were conducted in accordance with the guidelines of the animal care and use committee of the riken institute behavioural analysis marble-burying test the marble-burying behaviour tests were carried out using c57bl/6 congenic mice figures 1a and 2e or mice with a mixed cba 25 Â c57bl/6 75 genetic background supplementary figure s2a as described yamada et al 2002 the mice were placed individually in plastic cages 20 Â14 Â 22 cm3 for 30 min habituation trial and then returned to their home cages twelve clean coloured glass marbles 10 mm in diameter were evenly spaced 3­5 cm apart on 5 cm deep sawdust in the habituation cages mice were then re-introduced into these cages without food and water each test mouse was returned to the same cage in which they had been habituated the results of marble-burying behaviour were expressed as the number of marbles at least two-thirds buried within 30 min acoustic startle response the acoustic startle response was measured using specific startle chambers o hara co in figure 1d and e or sr-lab san diego instruments in supplementary figure s2c and d the startle chamber consisted of a plexiglas cylinder 3.5 or 3.8 cm in diameter resting on a sensor block or on a plexiglas frame in a sound-attenuated ventilated enclosure acoustic bursts were presented through a loudspeaker mounted 25 or 29 cm above the cylinder a piezoelectric transducer mounted below the sensor-block/frame detected motion of the animal in the cylinder stabilimeter readings were rectified and recorded by a microcomputer and interface ensemble o hara co or san diego instruments one mouse was placed in each chamber and allowed to acclimate for 10 min and then the experimental session was started background noise was set at 65 db white noise throughout both the acclimation period and the session in a session 10 trials for three types of stimuli each total of 30 trials were given in pseudo-random order after three initial startle-stimuli 20-ms burst of 120 db white noise which were given to avoid the effect of high responses to initial stimulations in the experiments one type of trial was a pulse-alone p alone trial which involved a 20-ms burst of 120 db white noise and the other two types of trials were pre-pulse and pulse pp70 p and pp80 p trials in which a 20-ms burst of 70 or 80 db white noise respectively was followed by a 20-ms burst of 120 db white noise 100 ms later the inter-trial intervals averaged 40 s 20­60 s and were pseudo-randomized the startle response was measured for 300 ms figure 1d and e or 100 ms supplementary figure s2c and d from the beginning of pulse presentation and the largest value was defined as the startle amplitude the startle amplitudes of the animal in response to repetitions of each trial type were averaged across the session the experimental schedule was controlled by a microcomputer methiothepin treatment methiothepin mesylate salt sigmaaldrich 0.1 mg/kg weight and saline otsuka were administered by intra-peritoneal injection tests were conducted 1 h after drug administration histological analysis and immunohistochemistry tissues were fixed by perfusion with 4 pfa dehydrated and embedded in paraffin sections 5 mm were stained with hematoxylin and eosin according to standard procedures frozen sections 2010 european molecular biology organization 10 mm were used for immunohistochemistry for indirect immunofluorescent staining anti-p71-atf-2 922 cell signaling anti-p180/p182-p38 mapk 4631 cell signaling anti-neun mab377 chemicon and toto-3 invitrogen were used the frozen sections were washed twice with tris-buffered saline 144 mm nacl 10 mm tris­hcl ph 7.6 and incubated overnight at 41c with primary antibody biotin-conjugated anti-rabbit igg antibody served as the secondary antibody and was incubated at room temperature for 2 h and further incubated with streptavidin alexa fluor 488 molecular probes and alexa fluor 546 anti-mouse igg at room temperature for 2 h luciferase reporter assay the htr5b promoter-luciferase reporter in which a 4.4 kb mouse htr5b promoter dna fragment from À4182 to þ 243 was linked to the luciferase gene was constructed the mutant construct containing mutated cre-like sites was constructed by replacing the 42-bp region containing the two cre sites upstream À3374bÀ3333 with the 6-bp sequence gagctc of a saci linker whereas the 8-bp sequence of the third cre site downstream À2325bÀ2318 was replaced by the 8-bp sequence aacgcgtt of a mlui linker to generate the atf-7 expression vector pact-atf-7 the human atf-7 cdna was inserted into the chicken cytoplasmic b-actin promotercontaining vector rn46a cells were cultured in dulbecco s modified eagle s medium dmem and f-12 ham d8062 sigma-aldrich 1:1 mixture supplemented with 10 fbs at 331c in 5 co2 rn46a cells were transfected using lipofectamine plus invitrogen with 0.1 mg of the htr5b promoter-luciferase reporter various amounts of the atf-7 expression plasmid or the control empty vector 0­3 mg and 1 mg of the internal control pras-b-gal in which the b-galactosidase gene was linked to the human c-ha-ras promoter at 48 h post-transfection luciferase activity was measured and normalized for transfection efficiency by b-galactosidase activity chip assays the brain tissues were prepared essentially as described by tsankova et al 2004 the brainstem was removed by gross dissection minced into b0.3 mm pieces and immediately crosslinked in 1.5 formaldehyde for 15 min at room temperature after addition of glycine to a final concentration of 0.125 m to quench the crosslinking reaction the chromatin was solubilized and extracted with lysis buffer and sheared to 400­600 bp fragments by sonication chip assays were carried out essentially as described by jin et al 2006 immunoprecipitation was carried out for 4­10 h at 41c with anti-atf-7 2f10 or 1a7 anti-histone h3 k9-m3 ab8898 abcam anti-eset upstate #07-378 or normal mouse or rabbit igg as negative controls the immunocomplexes were washed and incubated at 651c in 100 ml of ip elution buffer 1 sds 0.1 m nahco3 250 mm nacl 200 mg/ml proteinase k 10 mm dtt to release proteins the free precipitated dna was further purified using a qiaquick pcr purification kit qiagen and eluted in 30 ml sterile water eluted dna samples were used for real-time pcr 7500 real time pcr system applied biosystems chip assays using rn46a cells were carried out essentially as described by jin et al 2006 the primers and taqman probes qiagen used for amplification are described in supplementary table s1 co-immunoprecipitation assay for co-immunoprecipitation assays of endogenous atf-7 and eset rn46a cells were lysed by mild sonication in netn buffer 20 mm tris­hcl ph 8.0 1 mm edta 0.5 np40 400 mm nacl lysates were immunoprecipitated using anti-atf-7 1a7 or control igg immunocomplexes were resolved on 10 sds polyacrylamide gels and analyzed by western blotting with anti-eset upstate #07-378 supplementary data supplementary data are available at the embo journal online http www.embojournal.org acknowledgements we thank n saito for the rn46a cell line the staff of the research resources center of the riken brain science institute for dna array analysis and members of the experimental animal division of the the embo journal vol 29 no 1 2010 193

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atf-7 and social isolation stress t maekawa et al riken tsukuba institute for maintaining the mice this work was supported in part by grants-in-aid for scientific research from the ministry of education culture sports science and technology of japan conflict of interest the authors declare that they have no conflict of interest references agelopoulos m thanos d 2006 epigenetic determination of a cell-specific gene expression program by atf-2 and the histone variant macroh2a embo j 25 4843­4853 artigasa f romeroa l de montignyb c blierb p 1996 acceleration of the effect of selected antidepressant drugs in major depression by 5-ht1a antagonists trends neurosci 19 378­383 bibancos t jardim dl aneas i chiavegatto s 2007 social isolation and expression of serotonergic neurotransmissionrelated genes in several brain areas of male mice genes brain behav 6 529­539 ´ blanc g herve d simon h lisoprawski a glowinski j tassin jp 1980 response to stress of mesocortico-frontal dopaminergic neurones 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maekawa t sudo t seino y imura h saito n tanaka c ishii s 1991 expression of the cre-bp1 transcriptional regulator binding to the cyclic amp response element in central nervous system regenerating liver and human tumors oncogene 6 1009­1014 2010 european molecular biology organization 194 the embo journal vol 29 no 1 2010

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atf-7 and social isolation stress t maekawa et al tsankova nm kumar a nestler ej 2004 histone modifications at gene promoter regions in rat hippocampus after acute and chronic electroconvulsive seizures j neurosci 24 5603­5610 van dam h wilhelm d herr i steffen a herrlich p angel p 1997 atf-2 is preferentially activated by stress-activated protein kinases to mediate c-jun induction in response to genotoxic agents embo j 14 31798­31811 vergara c ramirez b 2004 cntf a pleiotropic cytokine emphasis on its myotrophic role brain res brain res rev 47 161­173 wang h an w cao r xia l erdjument-bromage h chatton b tempst p roeder rg zhang y 2003 mam facilitates conversion by eset of dimethyl to trimethyl lysine 9 of histone h3 to cause transcriptional repression mol cell 12 475­487 wilkinson ls killcross ss humby t hall fs geyer ma robbins tw 1994 social isolation in the rat produces developmentally specific deficits in prepulse inhibition of the acoustic startle response without disrupting latent inhibition neuropsychopharmacology 10 61­72 wu w yamaura t murakami k ogasawara m hayashi k murata j saiki i 1999 involvement of tnf-a in enhancement of invasion and metastasis of colon 26-l5 carcinoma cells in mice by social isolation stress oncol res 11 461­469 yamada k wada e yamano m sun yj ohara-imaizumi m nagamatsu s wada k 2002 decreased marble burying behavior in female mice lacking neuromedin-b receptor nmr-r implies the involment of nmb/nmb-r in 5-ht neuron function brain res 942 71­78 2010 european molecular biology organization the embo journal vol 29 no 1 2010 195

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