Neuronal adenosine A2A receptors signal ergogenic effects of caffeine

Caffeine is one of the most used ergogenic aid for physical exercise and sports. However, its mechanism of action is still controversial. The adenosinergic hypothesis is promising due to the pharmacology of caffeine, a nonselective antagonist of adenosine A1 and A2A receptors. We now investigated A2AR as a possible ergogenic mechanism through pharmacological and genetic inactivation. Forty-two adult females (20.0 ± 0.2 g) and 40 male mice (23.9 ± 0.4 g) from a global and forebrain A2AR knockout (KO) colony ran an incremental exercise test with indirect calorimetry (V̇O2 and RER). We administered caffeine (15 mg/kg, i.p., nonselective) and SCH 58261 (1 mg/kg, i.p., selective A2AR antagonist) 15 min before the open field and exercise tests. We also evaluated the estrous cycle and infrared temperature immediately at the end of the exercise test. Caffeine and SCH 58621 were psychostimulant. Moreover, Caffeine and SCH 58621 were ergogenic, that is, they increased V̇O2max, running power, and critical power, showing that A2AR antagonism is ergogenic. Furthermore, the ergogenic effects of caffeine were abrogated in global and forebrain A2AR KO mice, showing that the antagonism of A2AR in forebrain neurons is responsible for the ergogenic action of caffeine. Furthermore, caffeine modified the exercising metabolism in an A2AR-dependent manner, and A2AR was paramount for exercise thermoregulation.

Ergospirometry. Mice were accustomed to a single-lane treadmill (Panlab LE8710, Harvard apparatus) at speed 15 cm/s (10 min, slope 5°, 0.2 mA) with a 24 h interval between each habituation session (Fig. S1). The incremental running protocol started at 15 cm/s, with an increment of 5 cm/s every 2 min at 5° inclination 40 . The exercise lasted until running exhaustion, defined by the inability of the animal to leave the electrical grid for 5 s 40,44 .
We estimated the running and critical power output for a treadmill based on a standard conversion of the vertical work, body weight, and running speed 40,45,46 . Running power is the sum (Σ) of all stages of the exercise test, and critical power is the running work performed above VȮ 2 max.
Vaginal cytology. We evaluated the estrous cycle immediately after the exercise test, through 4-5 consecutive vaginal lavages (with 40-50 μL of distilled H 2 O) then mounted on gelatinized slides (76 × 26 mm) 47,48 . These procedures lasted no more than 3-5 min, and there were no significant time delays between behavioral experiments and fluid collection for vaginal cytology.
The vaginal smear was desiccated at room temperature and covered with 0.1% crystal violet for 1 min, then twice washed with 1 mL H 2 O and desiccated at room temperature 47,48 . The slides were mounted with Eukitt medium (Sigma-Aldrich) and evaluated under an optical microscope at 1x, 5x, and 20x (Zeiss Axio Imager 2). We evaluated three cell types for determining the estrous cycle: nucleated epithelial cells, cornified epithelial cells, and leukocytes. Cellular prevalence defined proestrus (nucleated), estrus (cornified), metestrus (all types in the same proportion), and diestrus (leukocytes) 47,48 . Thermal imaging. An infrared (IR) camera (FLiR C2, emissivity 0.95, FLiR Systems) placed overtop (25 cm height) of a plastic tube (25 cm diameter) was used to acquire a static dorsal thermal image 40,49,50 . IR images were taken immediately before and after exercise tests, namely at resting and recovery (Fig. 1H), respectively. IR images were analyzed with FLiR Tools software (Flir, Boston).
Statistics. Data  www.nature.com/scientificreports/ Statistical analyzes were performed according to an intention-to-treat principle using StatSoft, Inc. (2007). STATISTICA (data analysis software system), version 8.0. www.stats oft.com. ANOVA two-way was used to evaluate open field, VȮ 2 max, running power, and resting and recovery temperature, followed by Newman-Keuls post hoc test. The evolution of submaximal VȮ 2 , running power, respiratory exchange ratio (RER), and heating were evaluated by ANOVA for repeated measures followed by Bonferroni post hoc test. The differences were considered significant when P < 0.05.
The increase in RER during the exercise test was lower in animals treated with caffeine (F 7,119 = 3.6, η 2 = 0.17, β = 0.97, P < 0.05, Fig. 3E), wild type, and fb-A 2A R KO. Resting and recovery core temperatures were similar in all groups (Figs. 3F and Fig. 3G). The exercise test did not change the core temperature (Fig. 3F'). However, exercise heated the mice's tail in a similar way between groups (F 1,22 = 102, η 2 = 0.69, β = 0.99, 95% CI 24.9-26.4, P < 0.05, Fig. 3G').  SCH 58261 and caffeine improved VȮ 2 max, running and critical power of wild type mice. These results are in line with the improved running time observed in caffeine-treated rats 16,26,53 and mice 19 . Further evidence for the ergogenic effect of caffeine is based on its ability to increase muscle power and endurance output in rodents [54][55][56][57][58] . For the first time, we demonstrated that the selective antagonism of A 2A R is ergogenic. Also, for the first time, we demonstrated that the genetic inactivation of A 2A R impaired the ergogenic effects of caffeine. Tissue-specific A 2A R KO selectively in forebrain neurons further allowed showing that these ergogenic effects of caffeine are due to the antagonism of A 2A R in forebrain neurons. Thus, we suggest that caffeine decreases central fatigue during exercise. Moreover, caffeine decreased RER in the submaximal stages of the exercise test, an effect also abrogated in A 2A R KO mice. However, exercise-induced core and tail hyperthermia were similar among animals treated with SCH 58261 or caffeine, except for A 2A R KO mice, suggesting possible A 1 R-A 2A R-mediated interactions 56,57 in the temperature control 51 .

Selective A 2A R antagonism is psychostimulant in males, not females.
We assessed the baseline motor behavior due to the motor nature of the running test, without any motor impairment found related to the different genotypes and treatments. Thus, the observed differences were not due to impaired animals' motor behavior. We also assessed the psychostimulant effects of caffeine and SCH 58261 34 . Notably, the effects of caffeine were abrogated in A 2A R KO mice, and SCH 58261 did not modify locomotion in female mice. These results corroborate the robust evidence showing the psychostimulant effects of caffeine in male rodents 58 . However, little is known about the role of sexual dimorphism in adenosine signaling [59][60][61][62][63] . The absence of a psychostimulating effect of SCH 58261 in females is on step ahead, in notable agreement with the reported ability of the anxiolytic effect of SCH 58261 in males [59][60][61] but not in females 60 . However, these differences did not disturb the ergogenic effects of SCH 58261 on females. Future studies will better understand sex differences in adenosine signaling, which was not the aim of this study.
The neuropharmacology of the ergogenic effects of SCH 58261 and caffeine. Adenosine is a potent purine that modulates CNS signaling and functions from its main A 1 R and A 2A R 21,23,29,62 . Here, caffeine (nonspecific A 1 R and A 2A R antagonist) and SCH 58261 (selective A 2A R antagonist) similarly increased the VȮ 2 max, running power, and critical power of exercising male and female mice. Most importantly, these ergogenic effects were abrogated by the selective deletion of A 2A R in forebrain neurons, which indicates the key role of CNS A 2A R as an ergogenic mechanism. The basal nuclei, namely the striatum, is the brain region with the highest density of A 2A R 34,35,37,63 , which prompts the hypothesis that the A 2A R antagonism in the basal ganglia might mediate the ergogenic effect of SCH 58261. In resting and running rodents, caffeine intake can result in a concentration of caffeine of 50 µM in the brain 19,64 . This concentration is close to the EC 50 of caffeine (40 µM) to antagonize A 1 R and A 2A R in the CNS 23 . Since caffeine was not ergogenic in fb-A 2A R KO mice, it is concluded that forebrain A 2A R signal the ergogenic effects of caffeine. This provides a direct demonstration of the involvement of neuronal A 2A R in the ergogenic effects of caffeine, as suggested by two previous reports showing that NECA prevented the ergogenic effects of caffeine in rats 16 and, conversely, that systemic caffeine reversed the poor running performance of NECA-treated rats 24 . Although nonselective, the pharmacological use of NECA demonstrated that adenosine receptors are crucial for the ergogenic effects of caffeine 16,26,53 . We now identified A 2A R, specifically located in forebrain neurons, as responsible for this ergogenicity of caffeine.
Caffeine decreases RER in mice expressing peripheral A 2A R. Caffeine decreases the RER in submaximal exercise in humans 1,77,78 and rats 79 . For the first time, we provide evidence that this metabolic effect involves a modification of the A 2A R function. In the past, the inhibition of phosphodiesterase and increased intracellular calcium mobilization 2,7,8,80 were the proposed mechanisms. However, these proposals are inconsistent with pharmacological data: caffeine has an EC 50 for adenosine antagonism of 40 µM, 1,000 mM for phosphodiesterase inhibition, and 3,000 mM for Ca 2+ -triggered muscle contraction 23 . Higher caffeine concentrations cause toxicity (above 200 µM) and lethality (above 500 µM) 23 . Thus, biological effects for caffeine must be in the range below 100 µM. We have previously shown that caffeine reaches a plasma peak of 10 µM after caffeine intake (6 mg/kg) in running mice 19 . The metabolic effects of caffeine during exercise are currently associated with increased activity of the autonomic nervous system (ANS) 1,19,77,78 , including high blood adrenaline and lactate levels, tachycardia and increased blood pressure. However, we must recognize the limitations of lungbased RER measures and their effects on metabolism, due to the possible artifacts such as hyperventilation and disturbances in the acid-base balance. www.nature.com/scientificreports/ Adenosine receptors are crucial for exercise-induced hyperthermia. The temperature changes observed were dependent on sex and genotype. The exercise test improved VȮ 2 , an index of heat production 26, but the core temperature increased only in females. The tail temperature, an index of heat loss 26, increased in both sexes. These results are in line with previous results from our group 40 . Caffeine and SCH 58261 did not modify these thermal responses to the exercise test. In males, tail heating of fb-A 2A R KO mice was also similar to that of the wild type mice. However, the thermal response of the global A 2A R KO females was different, indicating a peripheral role of these receptors, known to regulate body temperature 51 . NECA (nonspecific A 1 R and A 2A R agonist) causes core hypothermia in rats and rabbits 26,76 , an effect inhibited by 8-phenyltheophylline (a potent and selective antagonist for A 1 R and A 2A R that crosses the blood-brain barrier, BBB) 76, but unaffected by 8-(p-sulfophenyl)theophylline (another potent adenosine receptor antagonist with little BBB penetration) 76, indicating a centrally-mediated effect. In the case of A 2A R, its role in regulating body temperature is controversial 81 . The selective A 2A R agonist 2-p-(2-carboxyethyl)-phenethylamino-5′-Nethylcarboxamidoadenosine-hydrochloride (CGS 21680) induces hyperthermia in rats 82 and mice 83 . We now show that SCH 58261 does not change resting and recovery temperature. This evidence suggests that the peripheral activation of A 2A R can induce hypothermia in rodents, but this mechanism does not seem to be physiologically engaged as gauged by the lack of effects of the pharmacological or genetic blockade of A 2A R. The previous data are from animals at rest-animals with CNS A 2A R deletion present normal hyperthermia response during exercise. However, global A 2A R KO displays a decreased response, even hypothermia, when treated with caffeine. Thus, A 2A R seems to undergo a gain of function in the periphery during exercise. This data reinforces the wellknown role of A 1 R in hypothermia 81 . Circulating adenosine levels increase during exercise 30,31 , and global A 2A R KO imbalance appears to increase the A 1 R role, signaling hypothermia even after exercise. These results are limited to the use of infrared temperature, as we have not measured rectal temperature due to interference (vaginal manipulation) in the evaluation of the estrous cycle of females. Also, we kept the same methodology in males.
conclusion. In summary, we have now demonstrated that A 2A R antagonism is a mechanism of action for ergogenicity, as SCH 58261 was ergogenic. Furthermore, we showed that the antagonism of forebrain A 2A R was the mechanism underlying the ergogenic effect of caffeine since caffeine was not ergogenic in fb-A 2A R KO. The use of selective A 2A R KO in forebrain neurons further reinforces the ergogenic role of caffeine in decreasing central fatigue, with possible involvement of decreased perceived exertion, pain, and mental fatigue in humans. Despite methodological limitations, our data further suggest that caffeine modified the exercising metabolism in an A 2A R-dependent manner and that A 2A R is essential for exercise thermoregulation.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.