4. Results
4.3 Impact of enrichment on “normal” anxiety-related behavior animals
4.2.3 Meta-analysis of coping style
Enrichment does not improve depression-like behavior or alter coping style, respectively.
To assess the overall effect of EE on coping style, we averaged the z-scores from TST and FST to obtain a “depression score” (fig. 35) comprising nine parameters in total: latency to first immobile episode, number of immobile episodes and percent time spent immobile for both, TST and FST, as well as number of struggling episodes, latency to first struggling and percent time struggling for FST. There was a significant effect of housing on depression score (F(1,71) = 6.697; p≤0.05), without an effect of sex (F(1,71) = 1.23; p=0.269) or an interaction effect of housing x sex (F(1,71) = 1.23; p=0.269). Fig. 34. shows that the significant effect of housing did not survive Tukey’s post-hoc test for males (p=0.840) and females (p=0.145), respectively.
factors p-value for main effect:
housing ≤0.05
sex 0.146
housing x sex 0.146
Males showed a reduction of 0.24 and females of 0.05 SDs below the mean of controls indicating a small effect size.
4.3 Impact of enrichment on “normal” anxiety-related behavior animals
p≤0.001), latency to enter inner zone (F(1, 35) = 7.387; p≤0.01), percent time spent in inner zone (F(1, 35) = 6.097; p≤0.05), percent distance travelled in inner zone (F(1, 35) = 6.333;
p≤0.05) and total entries inner zone (F(1, 35) = 11.584; p≤0.01) without an effect of sex or an interaction of housing x sex.
parameter measured p-value for main effect of:
housing sex housing x sex
total distance travelled ≤0.001 0.592 0.833
latency to enter inner zone 0.010 0.723 0.904
percent time spent in inner zone 0.019 0.271 0.623
percent distance travelled in inner zone 0.017 0.240 0.484
total entries inner zone 0.002 0.481 0.696
Fisher post-hoc tests reveal for males a significant increase in percent distance travelled and percent time spent in inner zone as well as shorter latency to enter (p≤0.05 for all) and
“more entries” (p≤0.01) into it (fig. 36). EE Females merely show a trend (p=0.10) to enter the inner zone with a shorter latency in relation to SE controls. These parameters indicate a significant anxiolytic effect for males but not females.
To assess the overall anxiolytic effect of EE, we calculated z-open field exactly as described before for HAB mice. 2-way ANOVA shows a significant effect of housing (F(1, 35) = 4.484; p≤0.05) without effects of sex or an interaction of both.
Fig. 36: OF points towards an anxiolytic effect of EE for males but not females with significant increases in percent distance travelled (A) and percent time spent inner zone (B), total entries into (C) and a reduced “latency to enter inner zone” (D). N (males) = 12 SE and 11 EE; N (females) = 8 SE and EE
Factors P-value for main effect:
housing 0.041
sex 0.427
housing x sex 0.427
Fisher post-hoc test confirmed a significant anxiolytic effect for males (p≤0.05) but not females (p=0.398) with 0.53 and 0.24 SDs below SE housed controls (fig. 37).
Importantly, we still observe an anxiolytic effect though z-open field corrects for existing differences in total distance travelled, thereby excluding confounding effects of locomotion on anxiety-related behavior.
For EPM, 2-way ANOVA detected a trend of housing (p=0.076) with EE animals entering percent open arms more often compared to controls. This trend did not survive Fisher post-hoc test for males (p=0.258) and females (p=0.161). There is neither an effect of housing, sex or interaction of both for percent time spent on open arms, nor total distance travelled.
Parameter measured P-value for main effect of:
Housing Sex Housing x sex
total distance travelled 0.811 0.402 0.741
percent entries open arms 0.076 0.871 0.712
percent time spent on open arms 0.115 0.957 0.533
As expected, there is no overall effect of housing (F(1, 35) = 0.01; p=0.115), sex (F(1, 35) = 0.444; p=0.511) or housing x sex (F(1, 35) = 0.444; p=0.511) on z-EPM indicating no anxiolytic effect of enrichment captured by parameters of this behavioral test (fig. 38). EE males are 0.1 SDs above, whereas females are 0.03 SDs below their respective SE controls.
Fig. 37: Z-open field corroborates the anxiolytic effect of enrichment for males without decreasing anxiety-related behavior for females. N (males) = 12 SE and 11 EE; N (females) = 8 SE and EE
LD corroborates the anxiolytic effect of OF by an effect of housing on percent time spent in light compartment (F(1, 35) = 34.75; p≤0.001) and entries into light compartment (F(1, 35) = 7.842; p≤0.01) as well as a trend for percent distance travelled in light compartment (F(1, 35)
= 3.722; p=0.062) and total distance travelled (F(1, 35) = 3.726; p=0.062).
Parameter measured P-value for main effect of:
Housing Sex Housing x sex
total distance travelled 0.062 0.084 0.356
latency to enter light compartment 0.841 ≤0.001 ≤0.001
entries into light compartment 0.008 0.304 0.279
percent distance travelled in light compartment 0.062 0.122 0.059 percent time spent in light compartment ≤0.001 0.356 0.020 Fisher post-hoc tests reveal a significant increase in percent time spent in light compartment for both, EE males (p≤0.01) and females (p≤0.001) as well as decreased latency to enter the light compartment for both sexes (p≤0.01 and p≤0.05 for males and females, respectively). Enriched compared to standard housed females performed significantly more entries into the light compartment and travelled more percent distance in the light compartment (p≤0.05 for both), whereas males do not exhibit a difference for both (p=0.193 and p=0.988, respectively) (fig. 39).
Fig. 38: Z-elevated plus maze depicts no difference between enriched and standard housed mice. N (males) = 12 SE and 11 EE; N (females) = 8 SE and EE
To evaluate the overall anxiolytic effect of enrichment, we calculated the “emotionality score”, as described earlier, by averaging z-scores of OF, EPM and LD. 2-way ANOVA revealed a trend of housing (F(1, 36) = 3.726; p≤0.05), indeed confirming an anxiolytic effect of enrichment for “normal” anxiety-related mice, too.
Factors P-value for main effect:
housing 0.062
sex 0.084
housing x sex 0.356
Tukey post-hoc revealed a significant anxiolytic effect of enrichment by 0.38 SDs below the mean of SE males (p≤0.05). Females do not differ significantly, but EE mice are 0.16 SDs below the mean of their controls (fig. 40).
Fig. 39: EE reduces anxiety-related behavior significantly for males with females exhibiting an ever stronger anxiolytic effect compared to males. Enrichment reduces latency to enter light compartment (A) and increases entries (B), percent time (C) and percent distance travelled (D) into light compartment. N (males) = 12 SE and 11 EE; N (females) = 8 SE and EE
To analyze whether the distribution is shifted towards a medium or strong effect size, we performed a χ²-test with a 2x3 factorial design as described before. Indeed, males show a shift of effect size, i.e. an anxiolytic effect, from a small to a medium effect size (χ² = 19.17; p≤0.001). Thus, effect size of enriched males is shifted by 25% from small to medium.
Comparison χ² Degree of freedom P-value Cramer’s V
male SE vs. EE 19.17 2 p≤0.001 0.3096
female SE vs. EE 0 2 p=1.000 0
male vs. female EE 12.65 2 p=0.002 0.2515
male vs. female. SE 0.89 2 p=0.641 0.0667
Cramer’s V indicates a medium effect size for EE in relation to SE males. Standard housed groups do not differ significantly, whereas EE males in contrast to females show an anxiolytic effect (fig. 41).
Fig. 40: An anxiolytic effect for males, not females seems to be elicited by EE. N (males) = 12 SE and 11 EE; N (females) = 8 SE and EE
Fig. 41: Male NABs show an anxiolytic effect after EE depicted as a shift of effect size from small to medium. N (males) = 12 SE and 11 EE; N (females) = 8 SE and EE
Similar to HABs, the overall anxiolytic effect of NABs seems to be more pronounced for enriched-housed males compared to females. This entails that EE in relation to SE females do not differ significantly anymore and the shift of effect size observed for male EE NABs is medium in contrast to a high shift seen merely for male EE HABs.
4.4.2. Impact of EE on exploratory behavior
Enrichment increases exploratory behavior as indicated by a significantly increased number of rearings.
Differences in exploration might entail altered locomotion, which in turn is a confounding factor of anxiety. Thus, we counted rearings performed during OF and LD to assess the impact of exploration on locomotion and thus anxiety-related behavior. We do not observe a significant effect of housing (F(1, 35) = 2.679; p=0.111), sex (F(1, 35) = 1.176; p=0.286) or an interaction of housing x sex (F(1, 35) = 0.184; p=0.670) on rearings performed during OF.
In contrast, 2-way ANOVA reveals a significant effect of housing (F(1, 35) = 16.137;
p≤0.001) for LD, with EE mice performing significantly more rearings.
Factors P-value for main effect:
housing ≤0.001
sex 0.127
housing x sex 0.867
Fisher post-hoc test confirmed an increased number of rearings for males and females (fig.
42; p≤0.01 for both). To finally assess the effect of EE on exploration, we calculated
“exploratory score” as described before for HABs by averaging z-scores of rearings for OF and LD. 2-way ANOVA revealed a significant effect of housing (F(1, 36) = 13.675; p≤0.001) without effects of sex or an interaction of both factors.
Factors P-value for main effect:
housing ≤0.001
sex 0.648
housing x sex 0.766
Fig. 42: Exploratory behavior is significantly increased for both sexes after environmental enrichment. N (males) = 12 SE and 11 EE; N (females) = 8 SE and EE
Tukey post-hoc tests corroborated a significant increase in rearings for enriched-housed males (p≤0.05) and a trend for females (p=0.065) compared to controls. Males and females were 0.82 and 1.01 SDs above the mean of their respective standard-housed counterparts (fig. 43), therewith pointing toward an increased exploratory behavior after EE.
4.3.3 Effect of environmental enrichment on coping style and anhedonia EE does not alter coping style in NABs.
We performed TST to evaluate the impact of EE on coping style or depression-like behavior, respectively. We observe a trend of housing (F(1, 34) = 4.089; p=0.051), but no significant effect of sex (F(1, 34) = 1.718; p=0.199) or housing x sex (F(1, 34) = 0.377;
p=0.544) on latency to first immobile episode. This trend does not survive Fisher post-hoc test for both sexes (p=0.251 and p=0.108 for males and females, respectively). Thus, EE does not ameliorate depression-like behavior or alter coping style, respectively (fig. 44).
Fig. 43: Exploratory score confirms an increased exploration for both sexes, with males performing 0.82 and females 1.01 SDs more rearings related to respective controls. N (males) = 12 SE and 11 EE; N (females) = 8 SE and EE
Fig. 44: Coping style is not significantly altered when NABs were housed in an EE (B).
N (males) = 12 SE and EE; N (females) = 6 SE and 8 EE
4.4 Impact of environmental enrichment on anxiety-related behavior of outbred CD1