Fatigue is a common sensation experienced during exercise and is currently understood to be a protective mechanism that prevents severe disruption of an organism's physical and physiological integrity (Ament and Verkerke, 2009; Noakes et al., 2004). Fatigue leads to either reduced power output or interruption of the exercise and can be modulated by, among other factors, exercise intensity/duration, nutritional status and physical training (Noakes et al., 2004; Blomstrand, 2006). The reduction in work capacity during prolonged physical exercise is considered to be a multi-factorial process (Noakes et al., 2004; Gandevia, 2001; Lambert et al., 2005) that includes a reduction of the central nervous system's ability to recruit skeletal muscles (Nybo and Nielsen, 2001).
Fatigue during prolonged exercise has been associated with the activity of the monoaminergic system in the brain (Coimbra et al., 2012; Meeusen and Roelands, 2010). An elevated ratio of the concentration of serotonin (5-hydroxytryptamine; 5-HT) to that of dopamine in the brain may cause fatigue either by increasing lethargy or by inducing a loss in central drive/motivation (Meeusen et al., 2006). In running rats, a central injection of tryptophan, which is the amino acid precursor for 5-HT synthesis, increases the heat storage rate and decreases mechanical efficiency, thereby accelerating fatigue during submaximal exercise (Cordeiro et al., 2014; Soares et al., 2003; 2004). The contribution of serotonergic function to fatigue mechanisms has also been investigated using pharmacological tools in human studies (Bridge et al., 2003; Marvin et al., 1997; Meeusen et al. 1997; 2001; Roelands et al., 2009). However, some of the tested drugs, such as buspirone, are non-selective agonists of 5-HT receptors; therefore, caution is required when interpreting the outcomes from these studies based on exclusive changes in the serotonergic system.
In contrast to buspirone, paroxetine is a potent and selective inhibitor of the 5-HT transporter (Nemeroff and Owens, 2003). To date, only three studies (Struder et al., 1998; Strachan et al., 2004; Wilson and Maughan, 1992) have used paroxetine to first increase central serotonergic activity and then investigate the association between brain 5-HT and fatigue, and their conclusions are contradictory. Wilson and Maughan (1992) and Struder et al. (1998) showed that 20 mg of paroxetine reduced the total exercise time (TET) by 19% and 17%, respectively. In contrast, Strachan et al. (2004), using the same dose, did not observe any difference in the TET. Therefore, the evidence generated from human studies regarding the role of brain 5-HT in the modulation of fatigue is still limited and inconclusive. One of the hypotheses that we assessed in the present study was that the dose used in the published studies (i.e., 20 mg of paroxetine) was not sufficient to consistently influence the serotonergic system and, consequently, physical performance. Therefore, we investigated the effects that were induced on physical performance by a higher dose of paroxetine (40 mg).
Furthermore the response of 5-HT receptors may be influenced by the aerobic capacity of an individual. Previous studies have suggested that 5-HT receptors are down-regulated in endurance-trained individuals relative to non-endurance-trained individuals (Broocks et al., 1999; Jakeman et al., 1994). In contrast, Dywer and Flynn (2002) did not observe alterations in the sensitivity of 5HT receptors in young men subjected to a short-term endurance training protocol. In addition to the contradictory findings, an important limitation of these studies (Broocks et al., 1999; Dywer and Flynn, 2002; Jakeman et al., 1994) is that the individuals were not exercising when they were challenged with the administration of 5-HT agonists. The sensitivity of 5-HT receptors during exercise was investigated in only one study (Dywer and Browning, 2000), which used experimental animals. Physical training decreased the ergolytic effect caused by the central administration of a 5-HT agonist, which suggests decreased sensitivity in trained animals. Because the evidence generated for a role of 5-HT in fatigue in rodents could not be consistently reproduced in human studies (Roelands et al., 2009), it is important to investigate whether the sensitivity of the 5-HT system is affected by aerobic capacity in exercising subjects.
Thus, in the present study, we investigated whether pre-existing differences in aerobic capacity modulate the response of the brain serotonergic system during cycling exercise. Our hypothesis was that individuals with higher aerobic capacities are less responsive to pharmacological activation of the serotonergic system and would, therefore, exhibit no changes on exercise performance induced by the ingestion of paroxetine. We also investigated whether the physical performance of young individuals subjected to moderate-intensity exercise is influenced by increasing doses of paroxetine.
Sixteen healthy male young volunteers participated in this study. Although all the subjects were physically active, none of them had previous cycling experience or engaged in any training protocol for one year prior to the experiments. The volunteers were divided into two groups according their aerobic capacity: a group with eight volunteers who had the lowest aerobic capacity (LAC; 38-46 ml-kg-1 *min-1 range) and another group with eight volunteers who had the highest aerobic capacity (HAC; 54-62 ml.kg-1.min-1 range). According to the Guidelines proposed by De Pauw et al. (2013), we could classify the LAC volunteers as Performance Level 1 subjects and the HAC volunteers as Performance Level 3 subjects. The age, anthropometric characteristics, maximal power output (MPO) and maximal oxygen uptake (VO2max) of the subjects are presented in Table 1.
This study was approved by the Ethics Committee of the Universidade Federal de Minas Gerais and conducted according to the standards described by the Brazilian National Health Council (Resolution 196/96) and by the Declaration of Helsinki (2008). All subjects provided informed written consent to participate in the experiments.
Initial testing occurred one week before the first experimental trial and included body composition assessment and V[O.sub.2max] testing. The V[O.sub.2max] was measured during a protocol with continuous gas-exchange measurements (Cosmed K4[b.sup.2], Rome, Italy) on an incremental cycle ergometer (Monark, model 824-E, Varberg, Sweden). Maximal heart rate ([HR.sub.max]) and MPO were also recorded. [HR.sub.max] was considered to be the HR at the voluntary termination of exercise. MPO was calculated using the equation proposed by Kuipers et al. (1985).
The percent body fat was calculated from estimations of the corporal density (Heyward and Stolarczyk, 1996), which, in turn, was estimated from three measurements of skin-fold thickness (Jackson and Pollock, 1978). Before participating in the study, each volunteer received recommendations to do the following: 1) avoid taking any medication throughout the period of the experiments; 2) abstain from alcohol, caffeine or heavy physical exercise, particularly with the inferior limbs, for 24 h before any experimental trial; and 3) record the dietary intake on the day before the first trial and replicate this intake on the day prior to the subsequent experimental trials.
During the experiments, the ambient temperature and relative humidity were controlled at 21.40 [+ or -] 0.03[degrees]C and 64.9 [+ or -] 0.4%, respectively. The order of the experimental trials was randomized and counterbalanced, and the trials were separated by at least 1 week. All experiments were conducted at the same time of day in a double-blind manner. Each volunteer was subjected to four exercise trials with the following drug conditions: placebo (PLA) and various doses of paroxetine (10, 20 and 40 mg). The placebo consisted of 20 mg of cellulose.
On the day of the experiments, the volunteers were instructed to consume a standardized breakfast (~ 620 kcal) at 8:00 a.m. at home. Between breakfast and arrival at the laboratory, the volunteers were asked to not eat. They arrived at the lab at 11:00 a.m., and their left forearm vein was catheterized. The volunteers' left hand and forearm were immersed in water at 42-44[degrees]C for 8 min to allow for arterialization of the venous blood (Hadjicharalambous et al., 2008). Following this procedure, a 21 G cannula was introduced into a superficial vein on the dorsal surface of the heated hand. The indwelling catheter was kept patent by flushing it with a small volume of heparinized saline after sampling. Then, the volunteers were allowed to rest in a supine position for 30 min, and the first blood sample was collected (8 ml) as follows: 4 ml in a tube containing NaF/[K.sub.3]-EDTA and 4 ml in a tube containing a coagulation activator. After this procedure, the indwelling catheter was withdrawn, and the volunteers ingested a capsule containing placebo/paroxetine at 12:00 p.m. and were taken to a cafeteria where they had lunch ([approximately equal to] 850 kcal). Placebo and paroxetine were prepared in gelatin capsules that had similar appearance and stored in generic packs identified with a code. All the capsules were previously prepared by a pharmacist from another lab to ensure the double blind fashion of the present study.
After lunch, at 2:30 p.m., the volunteers returned to the laboratory and drank 500 ml of water to ensure that they were well hydrated before commencing exercise. At 3:00 p.m., the volunteers ate a standardized snack ([approximately equal to] 490 kcal) and were subjected to the same previously described procedure for the insertion of the venous catheter. All of the meals were prescribed by a nutritionist. The volunteers then put on the experimental clothes (shorts, socks and tennis...