The endocannabinoid, 2-AG, and its congener, 2-OG, were quantified in lipid extracts of the prefrontal cortex, hippocampus and amygdala (). The effects of FST exposure on each lipid were analyzed using two-way ANOVA with relationship to the presence or absence of a swim session (i.e. with or without; “exposure”) and the day of the swim protocol (i.e. one or two; “day”) as the two main factors. There were no significant differences in 2-AG content between any of the groups in the three brain regions analyzed according to two-way ANOVA () [prefrontal cortex (day F1,21 = 3.1, p>0.05, exposure F1,21 = 0.2, p>0.05 and interaction F1,21 = 0.003, p>0.05), hippocampus (day F1,20 = 2.2, p>0.05, exposure F1,20 = 0.01, p>0.05 and interaction F1,20 = 0.2, p>0.05) and amygdala (day F1,20 = 2.3, p>0.05, exposure F1,20 = 1.5, p>0.05 and interaction F1,20 = 1.4, p>0.05)].
2-AG (A) and 2-OG (B) concentrations were determined in the brain regions indicated using lipid extraction followed by isotope dilution, LC/MS. Bars are the mean of 5–7 mice, vertical lines represent SEM. *p<0.05 two-way ANOVA.
2-OG content was determined in the same lipid extracts (). Interestingly, the content of 2-OG showed a distinct heterogeneity among the brain regions examined. One way ANOVA of the data from the control mice indicates a significant difference among the groups (F2,14 = 5.4, p<0.05) and Tukey's Multiple Comparison test indicates that the concentration of 2-OG in the prefrontal cortex is significantly greater than in the amygdala (q=4.6, p<0.05). Two-way ANOVA of the data indicates a significant effect of day on 2-OG content in the prefrontal cortex (day F1,21 = 7.8, p<0.05). There was neither a significant effect of swim exposure (exposure F1,21 = 0.006, p>0.05) nor an interaction between day and swim exposure (F1,21 = 0.006, p>0.05) on the 2-OG measurements. A similar trend for 2-OG to decrease 24 hours after the initial swim exposure was observed in the amygdala (day F1,20 = 3.8, p=0.07, exposure F1,20 = 1.0, p>0.05 and interaction F1,20 = 0.13, p>0.05) and hippocampus (day F1,20 = 2.4, p=0.14, exposure F1,20 = 0.02, p>0.05 and interaction F1,20 = 0.24, p>0.05).
We examined the correlations between 2-AG and 2-OG concentrations in each brain region and each treatment group (). 2-AG and 2-OG concentrations were not correlated in any brain region examined in control mice; there was a significant correlation in the prefrontal cortex from animals killed immediately after swim one, that correlation is dependent upon one animal that had very high amounts of both lipids (). Interestingly, a significant, positive relationship between 2-OG and 2-AG concentrations is present in the amygdala of mice killed 24 hours after the first swim exposure ().
Correlations between 2-AG and 2-OG contents in each treatment group and brain region.
Figure 3 2-AG and 2-OG data from individual mice determined in the prefrontal cortex immediately after exposure to a single swim (A) and determined in amygdala harvested from mice 24 hours after exposure to a single swim. Correlation coefficients are shown in (more ...)
Serum corticosterone concentrations were significantly higher after the swim exposure than before and this change was not affected by the number of swims according to two-way ANOVA (; day F1,21 = 0.04, p>0.05, exposure F1,21 = 112.1, p< 0.0001 and interaction F1,21 = 0.01, p>0.05).
Figure 4 (A) Serum corticosterone from all mice that received swims; ***p<0.0001, significant main effect between before and after swim by two way ANOVA. (B) Correlation between serum corticosterone and prefrontal cortical 2-AG in mice killed immediately (more ...)
After the second swim exposure, there was a significant, positive correlation between serum corticosterone concentrations and 2-AG concentrations in the prefrontal cortex (; p<0.05, Pearson r = 0.79), hippocampus (; p<0.05, Pearson r = 0.76) and amygdala (; p<0.05, Pearson r = 0.87). After the first swim exposure there was no correlation between serum corticosterone and 2-AG content in any brain region examined (). There was no correlation between serum corticosterone obtained before the first or second FST exposure and 2-AG content in any brain region examined. Serum corticosterone does not correlate with 2-OG concentrations in any of these three brain regions under any of the four experimental conditions ().
Correlations between circulating corticosterone and 2-AG or 2-OG concentrations in each treatment group and brain region.
The mean times immobile during the first and second FST were compared using an unpaired t test. As expected, mice exhibited significantly more immobility during swim two than swim one when all the mice were compared (; t25 = 2.7, p<0.05). In the mice that received both swim exposures (i.e. group 4), paired t test also confirmed significantly more immobility during swim two than swim one (; t6 = 4.2, p<0.01). There was no correlation between immobility during swim one or two and serum corticosterone concentrations measured immediately after swim exposure (). The change in immobility was calculated in the mice that were exposed to both swim sessions (group 4). There was no correlation between the change in immobility and serum corticosterone concentrations measured immediately after the second swim exposure. There were no significant correlations between immobility during swim one, two or the change in immobility and monoacylglycerol content in any brain region examined ().
Figure 5 Swim stress immobility in C57Bl/6N male mice exposed to one swim or two swims separated by 24 hours. (A) Immobility in all mice that received swims; *p<0.05, statistically different by unpaired t test. (B) Immobility in mice that received both (more ...)
Correlations among immobility during swim one, swim two or the change in immobility and circulating corticosterone or monoacylglycerol concentrations