Circulating levels of endocannabinoids respond acutely to voluntary exercise, are altered in mice selectively bred for high voluntary wheel running, and differ between the sexes

Abstract

The endocannabinoid system serves many physiological roles, including in the regulation of energy balance, food reward, and voluntary locomotion. Signaling at the cannabinoid type 1 receptor has been specifically implicated in motivation for rodent voluntary exercise on wheels. We studied four replicate lines of high runner (HR) mice that have been selectively bred for 81 generations based on average number of wheel revolutions on days five and six of a six-day period of wheel access. Four additional replicate lines are bred without regard to wheel running, and serve as controls (C) for random genetic effects that may cause divergence among lines. On average, mice from HR lines voluntarily run on wheels three times more than C mice on a daily basis. We tested the general hypothesis that circulating levels of endocannabinoids (i.e., 2-arachidonoylglycerol [2-AG] and anandamide [AEA]) differ between HR and C mice in a sex-specific manner. Fifty male and 50 female mice were allowed access to wheels for six days, while another 50 males and 50 females were kept without access to wheels (half HR, half C for all groups). Blood was collected by cardiac puncture during the time of peak running on the sixth night of wheel access or no wheel access, and later analyzed for 2-AG and AEA content by ultra-performance liquid chromatography coupled to tandem mass spectrometry. We observed a significant three-way interaction among sex, linetype, and wheel access for 2-AG concentrations, with females generally having lower levels than males and wheel access lowering 2-AG levels in some but not all subgroups. The number of wheel revolutions in the minutes or hours immediately prior to sampling did not quantitatively predict plasma 2-AG levels within groups. We also observed a trend for a linetype-by-wheel access interaction for AEA levels, with wheel access lowering plasma concentrations of AEA in HR mice, while raising them in C mice. In addition, females tended to have higher AEA concentrations than males. For mice housed with wheels, the amount of running during the 30min before sampling was a significant positive predictor of plasma AEA within groups, and HR mice had significantly lower levels of AEA than C mice. Our results suggest that voluntary exercise alters circulating levels of endocannabinoids, and further demonstrate that selective breeding for voluntary exercise is associated with evolutionary changes in the endocannabinoid system.

Keywords: Artificial selection; Locomotor activity; Reward; Sex differences; Training effect; Voluntary exercise.

Figure 1

Wheel running on day five of experiment. Values are LS means +/− standard error from SAS Proc Mixed. N = 90.

Figure 2

HCA on day 5 of experiment. See Table 1 for statistical results. Mice with wheel access always had reduced HCA (p = 0.0001), this reduction was greater in HR than in C mice (linetype by wheel access interaction, p = 0.0558), females always had higher HCA than males (p = 0.0047), and HR mice always had higher HCA than C mice (p = 0.007). Values are LS means (log₁₀ transformed) +/− standard error from SAS Proc Mixed. N = 190.

Figure 3

Levels of 2-AG in mouse plasma collected during peak activity on the 6^th night of wheel running. See Table 2 for statistical results. The three-way interaction among sex, linetype, and wheel access was statistically significant (p = 0.0408), with females also having lower levels than males (p = 0.0265). Values are LS means +/− standard error from SAS Proc Mixed. N = 189.

Figure 4

Levels of AEA in mouse plasma collected during peak activity on the 6^th night of wheel running. See Table 4 for statistical results, which indicated a linetype by wheel access interaction (p = 0.0628). Values are LS means +/− standard error from SAS Proc Mixed. N = 185.

Figure 5

Plasma AEA concentration from mice with wheel access as a function of the number of wheel revolutions in the 30 minutes prior to plasma sampling (N = 89). The number of wheel revolutions (transformed to the 0.4 power for statistical analyses [Table 5], but shown here as raw values) was a significant positive predictor of AEA values (p = 0.0043), home-cage was a negative predictor (p = 0.0383), and HR mice had lower levels than C mice after adjusting for these relationships (Table 5). The interactions between linetype and amount of wheel running or home-cage activity were not statistically significant (results not shown and these terms not included in final statistical model).

Figure 6

Levels of AEA in mouse plasma collected during peak activity on the 6^th night of wheel running, with amount of wheel running and home-cage activity used as covariates (see text), and including mice without wheels in the analysis by assigning them zero for wheel revolutions run. Values are LS means +/− standard error from SAS Proc Mixed, based on analyses presented in Table 6. N = 183.