we have recently characterized a strain of glucocorticoid receptor ( gr ) heterozygous mice ( gr ) in which one gr allele has been ablated by a conventional knockout strategy , resulting in an approximately 50% reduction of gr protein levels in the brain . these animals represent a valuable model for an endophenotype of severe major depression characterized by a disinhibited hpa - system due to altered gr function . similar to these severely depressed patients , gr mice demonstrate a non - suppression of corticosterone plasma levels to dexamethasone and a pathological combined dex / crh test . furthermore , gr mice exhibit an altered neuroendocrine stress response with increased and prolonged corticosterone release after a stressor . behaviorally , gr mice show increased learned helplessness , a well - established analog of depression - like behavior in mice [ 4 , 20 ] . as a molecular correlate depressive phenotype we previously identified downregulation of bdnf at the protein level in hippocampus of gr mice . compromised adult neurogenesis has also been implicated in the pathophysiology of hippocampal aspects of mood disorders . importantly , the  neurotrophin hypothesis of depression fits well with current concepts emphasizing the negative impact of stress on hippocampal structure . stress robustly impairs progenitor proliferation and neogenesis of granule neurons in the adult hippocampal dentate gyrus [ 5 , 8 , 9 , 17 ] . similarly , impaired bdnf signaling has recently been shown to result in impaired neurogenesis and refractory responses to antidepressants . deletion of the bdnf receptor trkb in adult progenitors also alters newborn neuron integration into hippocampal circuits and increases anxiety - like behaviors . here , we investigated net hippocampal neurogenesis in gr mice either under control conditions or following repeated restraint stress . we speculated that neurogenesis may be compromised in this strain due to its depressive phenotype and concurrent bdnf downregulation . the generation of gr - heterozygous mice ( gr ) has been described in detail previously . briefly , the founders of gr mice were developed by using homologous recombination in embryonic stem cells as described earlier . gr mice were generated by crossing heterozygous c57bl/6n males ( backcrossed for > 10 generations ) with wild - type fvb / n females in order to obtain f1 hybrid mice with exactly the same background as previously used for behavioral and neurochemical characterization studies . male gr mice and wild - type littermate controls were 46  months old at the beginning of experiments . mice were housed individually in standard laboratory cages under a reversed 12  h:12  h light / dark cycle , with ad libitum access to food and water . all experiments were performed according to national and institutional guidelines and were approved by german animal welfare authorities . thymidine analog brdu ( 5-bromo-2-deoxyuridine ; sigma - aldrich , germany ) was administered intraperitoneally at a concentration of 50  mg / kg body weight once daily . restraint stress consisted of placing a mouse inside a plastic tube ( inner diameter : 26  mm ) for 1  h during the dark phase . the experiment was structured in a 2    2 design ( fig .  1 ) . briefly , all animals received daily brdu injections for a period of 7  days . additionally , two groups were subjected to 1  h of restraint stress after brdu injections . all experiments were performed according to the  principles of laboratory animal care ( nih publication no . 86 - 23 , revised 1985 ) and conformed to national and institutional guidelines.fig . n  =  910 animals per group and condition experimental paradigm : description of experimental procedures is given in n  =  910 animals per group and condition mice were deeply anesthetized with ketamine and transcardially perfused with 0.9% sodium chloride followed by 4% paraformaldehyde ( pfa ) in 0.1  m phosphate buffer . brains were dissected from the skulls , postfixed in 4% pfa at 4  c over night , and transferred into 30% sucrose for dehydration . brains were cut on a sliding microtome ( leica , bensheim , germany ) in the coronal plane in 40-m - thick sections and cryoprotected . sections were stained free floating with all antibodies diluted in tris - buffered saline containing 3% donkey serum and 0.1% triton x-100 . for brdu staining , dna was denatured in 2  n hcl for 30  min at 37  c . sections were then rinsed in 0.1  m borate buffer and washed in tris - buffered saline ( tbs ) . primary and secondary antibodies were diluted in tbs containing 0.1% triton x-100 and 3% donkey serum ( tbs+ ) . primary antibodies were applied in the following concentrations : anti - brdu ( rat , 1:500 ; harlan seralab , indianapolis , in ) , anti - neun ( mouse , 1:100 ; chemicon , temecula , ca ) , anti - s100 ( rabbit , 1:2500 ; swant , bellinzona , switzerland ) . immunohistochemistry followed the peroxidase method with biotinylated secondary antibody ( jackson immunoresearch laboratories , west grove , pa ) , abc elite reagent ( vector laboratories , burlingame , ca ) and diaminobenzidine ( dab ; sigma ) as chromogen . for immunofluorescence fitc- , rhodredx- or fluorescent sections were coverslipped in polyvinyl alcohol with diazabicyclooctane ( dabco ) as an anti - fading agent . cell counts were determined in one - in - six series of sections covering the entire hippocampus in its rostrocaudal extension as described previously [ 13 , 14 ] . briefly , cells located in the granule cell layer and adjacent subgranular zone , defined as a two - cell body - wide zone of the hilus along the base of the granule cell layer were counted . phenotypic analysis of brdu - labeled cells was performed using a spectral confocal microscope ( tcs sp2 ; leica , nussloch , germany ) . appropriate gain and black level settings were determined on control slices stained with secondary antibodies alone . two - way anova was followed by fisher s post hoc test , where appropriate . all values are given as mean    standard error of the mean ( sem ) . the generation of gr - heterozygous mice ( gr ) has been described in detail previously . briefly , the founders of gr mice were developed by using homologous recombination in embryonic stem cells as described earlier . gr mice were generated by crossing heterozygous c57bl/6n males ( backcrossed for > 10 generations ) with wild - type fvb / n females in order to obtain f1 hybrid mice with exactly the same background as previously used for behavioral and neurochemical characterization studies . male gr mice and wild - type littermate controls were 46  months old at the beginning of experiments . mice were housed individually in standard laboratory cages under a reversed 12  h:12  h light / dark cycle , with ad libitum access to food and water . all experiments were performed according to national and institutional guidelines and were approved by german animal welfare authorities . thymidine analog brdu ( 5-bromo-2-deoxyuridine ; sigma - aldrich , germany ) was administered intraperitoneally at a concentration of 50  mg / kg body weight once daily . restraint stress consisted of placing a mouse inside a plastic tube ( inner diameter : 26  mm ) for 1  h during the dark phase . the experiment was structured in a 2    2 design ( fig .  1 ) . briefly , all animals received daily brdu injections for a period of 7  days . additionally , two groups were subjected to 1  h of restraint stress after brdu injections . all experiments were performed according to the  principles of laboratory animal care ( nih publication no . 86 - 23 , revised 1985 ) and conformed to national and institutional guidelines.fig . n  =  910 animals per group and condition experimental paradigm : description of experimental procedures is given in mice were deeply anesthetized with ketamine and transcardially perfused with 0.9% sodium chloride followed by 4% paraformaldehyde ( pfa ) in 0.1  m phosphate buffer . brains were dissected from the skulls , postfixed in 4% pfa at 4  c over night , and transferred into 30% sucrose for dehydration . brains were cut on a sliding microtome ( leica , bensheim , germany ) in the coronal plane in 40-m - thick sections and cryoprotected . sections were stained free floating with all antibodies diluted in tris - buffered saline containing 3% donkey serum and 0.1% triton x-100 . for brdu staining , dna was denatured in 2  n hcl for 30  min at 37  c . sections were then rinsed in 0.1  m borate buffer and washed in tris - buffered saline ( tbs ) . primary and secondary antibodies were diluted in tbs containing 0.1% triton x-100 and 3% donkey serum ( tbs+ ) . primary antibodies were applied in the following concentrations : anti - brdu ( rat , 1:500 ; harlan seralab , indianapolis , in ) , anti - neun ( mouse , 1:100 ; chemicon , temecula , ca ) , anti - s100 ( rabbit , 1:2500 ; swant , bellinzona , switzerland ) . immunohistochemistry followed the peroxidase method with biotinylated secondary antibody ( jackson immunoresearch laboratories , west grove , pa ) , abc elite reagent ( vector laboratories , burlingame , ca ) and diaminobenzidine ( dab ; sigma ) as chromogen . for immunofluorescence fitc- , rhodredx- or cy5-conjugated secondary antibodies were all used at a concentration of 1:250 . fluorescent sections were coverslipped in polyvinyl alcohol with diazabicyclooctane ( dabco ) as an anti - fading agent . cell counts were determined in one - in - six series of sections covering the entire hippocampus in its rostrocaudal extension as described previously [ 13 , 14 ] . briefly , cells located in the granule cell layer and adjacent subgranular zone , defined as a two - cell body - wide zone of the hilus along the base of the granule cell layer were counted . phenotypic analysis of brdu - labeled cells was performed using a spectral confocal microscope ( tcs sp2 ; leica , nussloch , germany ) . appropriate gain and black level settings were determined on control slices stained with secondary antibodies alone . two - way anova was followed by fisher s post hoc test , where appropriate . all values are given as mean    standard error of the mean ( sem ) . net neurogenesis in the hippocampus was analyzed 4  weeks after a 7-day course of single daily intraperitoneal injections of brdu . in this particular injection paradigm , brdu counts reflect a combination of initial proliferation and subsequent survival of newly generated cells . briefly , gr mice and gr littermate controls were subjected either to the control condition ( undisturbed in home cage ) or to the stress condition ( 1  h of restraint stress directly after intraperitoneal injection of brdu ) . two - way anova revealed that brdu cell counts were significantly reduced as an effect of stress and as an effect of gr genotype ( detailed results of the anova statistics are presented in figs . 2a and 3 ; analysis of 37 animals in total , n  =  910 animals / group ) . the majority of cells showed co - labeling with mature neuronal marker neun or astrocytic marker s100 ( figs . 2b and 4 ) with no further significant effect of either experimental condition or of genotype.fig .  2quantitative assessment of net neurogenesis . a the number of brdu+ cells was analyzed 4  weeks after a 7-day series of once - daily brdu injections . treatment condition ( control versus stress condition : f1,33  =  10.2 , p  =  0.003 ) and a significant effect of factor genotype ( f1,33  =  4.5 , p  =  0.04 ) on brdu cell counts with no significant interaction between factors ( genotype    treatment interaction : f1,33  =  0.8 , p  =  0.39 ) . * p  <  0.05 for the effect of stress within gr genotype , p  <  0.05 for the effect of genotype within the  stress groups . b phenotypic analysis of brdu+ cells did not reveal differences in neuronal ( neun ) or astrocytic ( s100 ) marker expression among experimental groupsfig . 3brdu+ cells in the dentate gyrus are reduced as an effect of gr genotype and as an effect of stress . the four images show brdu - stained cells ( dab ) in representative hippocampal sections of animals from the respective experimental groups . a gr mice in the control condition . b gr mice in the stress condition . c gr mice in the control condition . d gr mice in the stress conditionfig . 4phenotypic analysis a confocal image of a newly generated astrocyte in the granule cell layer expressing mature astrocytic marker s100 ( blue ) and brdu ( red ) . emission intensities measured along the lines drawn in the larger image ( a ) are shown in the bottom and right panels . furthermore , insets in a show the cell investigated in single channels with separate wavelengths . b newly generated neuron labeled with brdu ( red ) and expressing neun - immunoreactivity ( green ) . c brdu - positive cell ( red ) expressing neither neuronal marker neun ( green ) nor astrocytic marker s100 ( blue ) . scale bars in a , b , and c 25  m quantitative assessment of net neurogenesis . a the number of brdu+ cells was analyzed 4  weeks after a 7-day series of once - daily brdu injections . treatment condition ( control versus stress condition : f1,33  =  10.2 , p  =  0.003 ) and a significant effect of factor genotype ( f1,33  =  4.5 , p  =  0.04 ) on brdu cell counts with no significant interaction between factors ( genotype    treatment interaction : f1,33  =  0.8 , p  =  0.39 ) . * p  <  0.05 for the effect of stress within gr genotype , p  <  0.05 for the effect of genotype within the  stress groups . b phenotypic analysis of brdu+ cells did not reveal differences in neuronal ( neun ) or astrocytic ( s100 ) marker expression among experimental groups brdu+ cells in the dentate gyrus are reduced as an effect of gr genotype and as an effect of stress . the four images show brdu - stained cells ( dab ) in representative hippocampal sections of animals from the respective experimental groups . a gr mice in the control condition . b gr mice in the stress condition . c gr mice in the control condition . d gr mice in the stress condition phenotypic analysis a confocal image of a newly generated astrocyte in the granule cell layer expressing mature astrocytic marker s100 ( blue ) and brdu ( red ) . emission intensities measured along the lines drawn in the larger image ( a ) are shown in the bottom and right panels . furthermore , insets in a show the cell investigated in single channels with separate wavelengths . b newly generated neuron labeled with brdu ( red ) and expressing neun - immunoreactivity ( green ) . c brdu - positive cell ( red ) expressing neither neuronal marker neun ( green ) nor astrocytic marker s100 ( blue ) . here we show that depression - prone gr mice exhibit a significant net reduction of adult hippocampal neurogenesis . gr mice represent a particularly valuable animal model of depression because they reflect hpa system dysregulation which is a hallmark of a subgroup of depressive disorders in humans . our observation is in line with the concept that neurogenesis has a role in the etiopathogenesis of mood disorders and contributes to the therapeutic actions of antidepressants [ 6 , 16 , 22 ] ; but see also [ 10 , 19 , 24 ] . a number of current studies have provided novel insights into the link between depression - related behaviors and altered neurogenesis . in particular , recent work has shown that  learned safety , as opposed to learned helplessness is associated with increased survival of new neurons and increased bdnf levels in hippocampus and that conversely , ablation of neurogenesis retards safety learning . several other recent studies also demonstrate a link between bdnf signaling , neurogenesis , and behavior [ 1 , 15 , 21 ] . in particular , ablation of bdnf receptor trkb in neural precursor cells results in impaired neurogenesis . when exposed to chronic antidepressant treatment or wheel - running , furthermore , ablation of trkb also renders the animals behaviorally insensitive to antidepressive treatment in depression- and anxiety - like paradigms . lack of trkb in adult progenitors also results in disturbed organization of basic synaptic connections of newly generated neurons and impaired neurogenesis - dependent long - term potentiation , accompanied by compromised survival of newly generated cells and increased anxiety - like behaviors . reduced net hippocampal neurogenesis in gr mice as reported here fits well with these reports . importantly , we have previously demonstrated a significant reduction of bdnf protein concentrations in hippocampus of gr mice . however , it should be noted that the reduction in neurogenesis in gr mice which emerged in this comparatively large experiment was only moderate . this may support the notion that in addition to reduced neurogenesis , other mechanisms may also contribute prominently to the depression - related behavioral phenotype of gr mice . in summary , we here demonstrate reduced net adult hippocampal neurogenesis in gr mice , a novel genetic mouse model of affective disorders . our study provides further correlative evidence for a link between hippocampal neurogenesis , glucocorticoids , bdnf , and depression .
glucocorticoid receptor ( gr ) heterozygous mice ( gr+/ ) represent a valuable animal model for major depression . 
 gr+/ mice show a depression - related phenotype characterized by increased learned helplessness on the behavioral level and neuroendocrine alterations with hypothalamo - pituitary - adrenal ( hpa ) axis overdrive characteristic of depression . 
 hippocampal brain - derived neurotrophic factor ( bdnf ) levels have also been shown to be reduced in gr+/ animals . 
 because adult hippocampal neurogenesis has been implicated in the pathophysiology of affective disorders , we studied here the effects of the gr+/ genotype on neurogenesis in vivo . in a 2    2 design , gr+/ mice and gr+/+ littermate controls 
 were either subjected to 1  h of restraint stress or left undisturbed in their home cages after intraperitoneal injection of brdu . 
 stress exposure and brdu injections were performed once daily for 7  days and neurogenesis analyzed 4  weeks later . 
 brdu cell counts were significantly reduced as an effect of gr+/ genotype and as an effect of stress . 
 majority of the brdu+ cells showed co - labeling with mature neuronal marker neun or astrocytic marker s100 with no further significant effect of either experimental condition or of genotype . in sum , this results in reduced neurogenesis in gr+/ mice which is further repressed by restraint stress . 
 our results , thus , reinforce the link between reduced neurogenesis , stress , neurotrophins , and behavioral symptoms of and susceptibility to depression .