Stress effects on anxiety-related behavior
In the light/dark exploration test, there was a significant effect of strain (F6,102=31.73, p<.01), but no main effect of stress or a strain x stress interaction, for time out of the shelter. Despite the absence of an interaction effect, our a priori hypothesis that strains would differ in their response to stress led us to perform post hoc comparisons between stressed and non-stressed groups. DBA/2J spent significantly less time out of the shelter than non-stressed controls (). Conversely, stressed C57BL/6J spent significantly more time out of the shelter than non-stressed C57BL/6J (). Under non-stressed conditions, 129S1, A/J, BALB/cByJ, and BALB/cJ spent significantly less time, and FVB/NJ spent more time, out of the shelter than C57BL/6J.
Basal and post-stress anxiety-like behavior in 7 strains
There was also a significant strain x restraint stress interaction (F6,102=3.21, p<.01) for shelter exits during the first 5 min. Post hoc analysis showed that stressed DBA/2J and FVB/NJ made significantly fewer exits than non-stressed DBA/2J and FVB/NJ controls, while stressed C57BL/6J made significantly more exits than C57BL/6J controls (). 129S1, A/J, BALB/cByJ, and BALB/cJ showed no change in shelter exits after stress - likely due to low shelter exits under non-stressed conditions; where all strains except FVB/NJ made significantly fewer exits than C57BL/6J.
There was a significant effect of strain (F6,102=19.96, p<.01) but not stress, and no strain x stress interaction for latency to first exit the shelter. Planned post hoc comparisons showed that C57BL/6J had a significantly shorter latency to exit than non-stressed counterparts, except FVB/NJ and DBA/2J (). Non-stressed 129S1, A/J, BALB/cByJ, and BALB/cJ were significantly slower to exit the shelter than non-stressed C57BL/6J.
Stress did not affect any measure during the last 5 min of the test. There were significant strain effects for shelter exits (F6,102=35.02, p<.01) and time out of the shelter (F6,102=52.01, p<.01). On both measures, non-stressed 129S1, A/J, BALB/cByJ, and BALB/cJ had lower scores than non-stressed C57BL/6J (data not shown).
In the elevated plus-maze, stressed C57BL/6J mice spent significantly more time in the open arms (non-stressed C57BL/6J=6.9 ±1.4 mean ±SEM, stressed C57BL/6J=14.2 ±3.0; non-stressed DBA/2J=3.3 ±1.1; stressed DBA/2J=4.3 ±1.1; t=2.33, df=18, p<.05, n=8–11/strain/stress) and made significantly more open arm entries (non-stressed C57BL/6J=3.3 ±0.6, stressed C57BL/6J=5.2 ±0.7; non-stressed DBA/2J=1.8 ±0.5; stressed DBA/2J=2.4 ±0.4; t=2.11, df=18, p<.05) and head-dips (non-stressed C57BL/6J=17.6 ±1.9, stressed C57BL/6J=25.1 ±2.4; non-stressed DBA/2J=5.8 ±1.0; stressed DBA/2J=6.1 ±1.1; t=2.46, df=18, p<.05) than non-stressed C57BL/6J, while stress did not affect any of these behaviors in DBA/2J. Open arm time (t=3.15, df=35, p<.01), open arm entries (t=3.41, df=35, p<.01) and head-dips (t=7.66, df=35, p<.01) were all significantly lower in DBA/2J than C57BL/6J, irrespective of stress. There was a non-significant trend for more closed arm entries in stressed relative to non-stressed mice of both strains (non-stressed C57BL/6J=16.4 ±0.9, stressed C57BL/6J=19.6 ±1.4; non-stressed DBA/2J=13.0 ±0.8; stressed DBA/2J=15.4 ±1.2). Finally, there was a significant effect of stress (F1,32=40.81, p<.01), but not genotype and no interaction, for body weight in this cohort. Stress significantly reduced body weight regardless of strain (non-stressed C57BL/6J=0.9 ±0.2 g mean ±SEM, stressed C57BL/6J=−0.6 ±0.1; non-stressed DBA/2J=0.6 ±0.3; stressed DBA/2J=−1.1 ±0.3).
Stress effects on body weight and depression-related behavior
There was a significant strain x stress interaction for change in body weight over the 10 day restraint period (F6,123=3.87, p<.01). Post hoc tests showed that, with the exception of 129S1, all strains had a significant reduction in body weight after restraint, as compared to the weight gain over the same period in non-stressed mice ().
Basal and stress-induced depression-related phenotypes in 7 strains
There was a significant effect of strain (F6,98=58.24, p<.01) and stress (F1,98=5.61, p<.05) but no strain x stress interaction for percent immobility in the FST. Planned post hoc analysis found that stressed BALB/cByJ showed significantly less immobility than non-stress BALB/cByJ counterparts, but no other strain showed a change in behavior after stress (). Under baseline conditions, DBA/2J and FVB/NJ showed significantly less immobility than C57BL/6J ().
There was a significant stress x strain interaction (F6,152=4.82, p<.01) for corticosterone levels. Post hoc analysis showed that, regardless of whether mice had been repeatedly restrained, swim stress significantly increased corticosterone in all strains as compared to a non-swim/non-restrained baseline group (). Moreover, swim stress produced significantly higher corticosterone levels in restrained C57BL/6J and DBA/2J than in non-restrained counterparts. A maximal (‘ceiling’) response under non-restrained conditions may have prevented detection of similar increases in restrained A/J, BALB/cJ and BALB/cByJ. However, contrasting with all other strains, restraint actually produced a blunted corticosterone response to swim stress in 129S1.
Genome-wide corticolimbic gene expression
Non-stressed DBA/2J and C57BL/6J differentially expressed 1786 probes (1524 known genes) in amygdala, 2540 probes (2154 known genes) in hippocampus, and 1712 probes (1491 known genes) in the vmPFC. A large proportion of the same genes was differentially expressed between strains in either 2 or all 3 regions (). For full list see Supplemental Spreadsheet 1
Basal and post-stress corticolimbic gene expression in C57BL/6J and DBA/2J
Analyzing the effect of stress as a function of strain, stress produced a similar number of upregulations and downregulations in the amygdala of DBA/2J and C57BL/6J (). Stress caused twice as many changes in the C57BL/6J than DBA/2J hippocampus (mainly upregulations). C57BL/6J showed more downregulations than upregulations in vmPFC after stress, while DBA/2J showed the opposite pattern. For all 3 regions, very few (~2%) of the stress-induced expression changes in C57BL/6J and DBA/2J involved the same genes.
Functional classification of stress-sensitive genes revealed enrichment in synaptic plasticity (e.g., glutamate receptors) and ion transport genes in C57BL/6J, and enrichment in genes related to nervous system development, programmed cell death, and myelination in DBA/2J (Supplemental Spreadsheet
RT-PCR confirmed significantly lower expression of hippocampal Gria1 and vmPFC Chrna4 and Rgs2, and higher expression of hippocampal Bdnf and prefrontal Comt, in non-stressed C57BL/6J as compared to non-stressed DBA/2J (all p<.05 by t-test) (). In C57BL/6J, stress significantly reduced amygdala expression of Grik1 and Gal, and hippocampal Nr4a2, and increased expression of amygdala Grin1 and vmPFC Chrna4, Drd1a and Per2. In DBA/2J, stress significantly increased amygdala Per1, hippocampal Dbp and prefrontal Per2, and decreased hippocampal Homer1. Array showed higher basal expression and stress-induced upregulation of Atp1a2 in C57BL/6J, while RT-PCR showed the reverse effect, likely due to differentially spliced variants.
Amygdala NMDAR neuronal signaling and metaplasticity
NMDAR-mediated eEPSC decay time (as measured by weighted tau) was significantly affected by strain (F1,30=23.67, p<.05) but not stress, reflecting significantly longer decay time in DBA/2J than C57BL/6J, irrespective of stress (non-stressed C57BL/6J=97.1 ±5.7, stressed C57BL/6J=104.7 ±12.5; non-stressed DBA/2J=181.6 ±24.3; stressed DBA/2J=172.2 ±17.2; n=6–12/strain/stress).
There was a significant pulse x strain x stress interaction for normalized eEPSC amplitude at all 3 stimulation frequencies: 10 Hz (F8,240=2.32, p<.05), 20 Hz (F8,240=3.61, p<.01) and 40 Hz (F8,240=2.31, p<.05). Post hoc analysis showed that eEPSC amplitude at the lower (10 Hz) stimulation frequency was unaltered by stress in C57BL/6J, but was significantly increased by stress in DBA/2J at later pulses (i.e., 6, 8–10) (). After 20 Hz stimulation, stress significantly increased amplitudes (pulses 7–10) in DBA/2J, but not C57BL/6J (). Amplitudes were not significantly altered by stress after 40 Hz stimulation, probably due to saturation (). Under non-stressed conditions, amplitude was significantly lower in DBA/2J than C57BL/6J after 10 Hz (pulses 5–10) and 20 Hz (pulses 8–10) but not 40 Hz.
Amygdala NMDAR neuronal signaling and metaplasticity in C57BL/6J and DBA/2J
Stress in NR2A and GluR1 null mutants
In NR2A null mutants, there were significant genotype x stress interactions for shelter exits (F1,37=6.08, p
<.05) and time out of the shelter (F1,37=14.12, p
<.01) during the first 5 min. Post hoc
comparisons showed that non-stressed −/− exhibited a trend for more time out of the shelter than +/+, consistent with data in other assays (Boyce-Rustay and Holmes, 2006
). Stress produced a significant, C57BL/6J-like, increase in shelter exits and time out of the shelter in +/+, but had no effects on exits and significantly decreased time out of the shelter in −/−, as compared to non-stressed −/− (). There was a trend for shorter latency to exit after stress, but no main effects or interaction (). There was a significant effect of stress (F1,29=64.83, p
<.01), but not genotype and no interaction, for body weight. Stress significantly reduced body weight regardless of genotype ().
Stress-induced anxiety-like behavior in NR2A null mutants
In GluR1 null mutants, there was a significant effect of stress but no stress x genotype interaction for time out of the shelter (F1,37=9.13, p<.01) and shelter exits (F1,37=9.77, p<.01). Stress significantly increased both measures irrespective of genotype (). −/− made significantly more shelter exits than +/+ (main genotype effect: F1,37=8.04, p<.01). Neither stress nor genotype affected latency to exit the shelter. There was a significant effect of stress (F1,37=18.12, p<.01), but not genotype and no interaction, for body weight. Stress significantly reduced body weight regardless of genotype ().
Stress-induced anxiety-related behavior in GluR1 null mutants
NR2A null mutant amygdala neuronal dendritic morphology and spine density
There was a significant effect of genotype (F1,12=4.90, p<.05) and branch order (F3,12=94.10, p<.05), but no interaction, for spine density. −/− had a significantly (~30%) lower spine density than +/+ regardless of branch order (). Overall amount of dendritic material, dendritic branch number and length, either in all branches or terminal branches only, was unaffected by genotype ().
Amygdala dendritic morphology and neuronal spine density in NR2A null mutants