Ethics

The study was conducted at the Turku PET Centre, University of Turku and Turku University Hospital (Turku, Finland) and followed the principles of the Declaration of Helsinki. The Ethics Committee of the Hospital District of South-West Finland approved the research protocol. The purpose and potential risks of the study were explained to the subjects each of whom gave written informed consent. This study was registered at clinicaltrials.gov under the number NCT02615756.

Subjects

In total 22 healthy men (age 26.1±4.9 years, range 21−36 years) with a variable exercise background participated in the study (Table 1). The inclusion criteria were male sex, age 18–65 years, and body mass index below 27 kg m−2. The exclusion criteria were a history of or current neurological or psychiatric disease, use of tobacco products or medication affecting the central nervous system, current or past excessive alcohol or substance abuse, any chronic illness resulting in disability in daily life, excessive or competitive athletics not consistent with common exercising habits, claustrophobia, and the presence of any ferromagnetic objects that would contraindicate magnetic resonance imaging. Laboratory tests, urinanalysis, and an ECG were obtained to assess health and the absence of psychoactive drugs.

Table 1 Study Participants (N=22) Full size table

Maximal Exercise Test

To determine the individual workload for the MICT exercise task, the subjects performed a maximal aerobic exercise test on a bicycle ergometer (Ergoline 800 s, VIASYS Healthcare, Germany) starting at 40 W and followed by 30 W increments every 2 min until volitional exhaustion. Ventilation and gas exchange were measured (Jaeger Oxycon Pro, VIASYS Healthcare, Germany) and reported as the mean value per minute. The highest 1-min value of oxygen consumption was expressed as the VO 2max . The maximal workload (Load max ) was calculated as the average workload during the last two minutes of the test and used as a measure of maximal performance. Metabolic thresholds were determined from the blood lactate concentration measured from capillary samples taken repeatedly during the test. The aerobic threshold corresponded the workload at which the blood lactate rose above the baseline level, and the anaerobic threshold the workload at which blood lactate began to accumulate rapidly.

Experimental Design

In total 10 participants underwent two consecutive PET studies—one at rest and another after completion of a MICT session. Twelve other participants underwent three PET studies—after MICT, after HIIT, and after rest (Supplementary Figure S1). A bolus injection of [11C]carfentanil was used. The participants fasted for 3 h before studies and were instructed to discontinue the use of caffeine containing products and not perform any physical exercise 24 h before the PET studies. For all participants, the order of the PET studies was randomized and all studies took part on separate days.

The MICT session consisted of 60 min of continuous aerobic cycling (Tunturi E85, Tunturi Fitness, Almere, The Netherlands) at workload in the middle between aerobic and anaerobic thresholds predetermined individually by the maximal exercise test (mean workload 164±42 W; 54±7% from Load max ; range 145–295 W; average heart rate during exercise 143±15 beats per min; 74±7% from maximal heart rate). All participants performed the MICT session successfully. Because of an unexpected delay with radiotracer supply, one of the participants had a slightly longer MICT session (77 min rather than the programmed 60 min).

The HIIT session consisted of 4 min of warm-up and five all-out cycling efforts (Monark Ergomedic 894E; Monark, Vansbro, Sweden) with 4 min of recovery, during which participants remained still or did unloaded cycling. Each sprint started with a few seconds of acceleration to maximal cadence, followed by a sudden increase of the load (7.5% of body weight) and maximal cycling for 30 s. Participants were familiarized with the HIIT protocol before HIIT session. Two participants could perform only three 30 s sprints: one was too exhausted and experienced knee pain, the other vomited after three bouts. Nevertheless, the data from these studies was included in the data analysis.

Heart rate was monitored during exercise (RS800CX; Polar Electro Ltd., Kempele, Finland). The blood lactate concentration was measured from venous samples before and within 1 min after completion of training (Lactate Pro; Arkray KDK, Kyoto, Japan). Music, television, or other technical devices were not available to the subjects during exercise. PET scanning began within 15–37 min after the completion of the exercise session, concomitantly with the administration of the radiotracer.

During the MICT and HIIT sessions the participants’ subjective degree of exertion was assessed with Borg’s Rating of Perceived Exertion (RPE) 6–20 scale and their subjective feelings of emotional valence (pleasant versus unpleasant) and arousal (calm versus excited) were assessed with the Self-Assessment Manikin (SAM) rating scale (Bradley and Lang, 1994) (Supplementary Figure S2). The RPE and SAM scales were administrated before, and at 15, 30, 45 and 60 min during MICT, and before HIIT and within 5 s after each 30 s sprint. Subjective feelings of pleasant versus unpleasant emotions were measured using the Positive and Negative Affect Schedule (PANAS) (Watson et al, 1988) and a visual analog scale (VAS; separate scales for tension, irritation, pain, exhaustion, satisfaction, motivation to exercise, euphoria, and energy) before and within 3 min after the end of each training session. After the HIIT session, three of the participants vomited and filled the questionnaires within 15 min after exercise completion. Euphoria and energy were added in the middle of the study, thus only 15 participants completed full VAS questionnaire.

On the day of the baseline study, the participants rested for 60 min before the studies without music, television, or mobile entertaining devices. PANAS and VAS were administrated before and after the scans.

Radiotracer Synthesis and PET Data Acquisition and Analysis

[11C]Carfentanil was synthesized by 11C-methylation of desmethyl carfentanil (sodium salt) with [11C]methyl triflate prepared from cyclotron-produced [11C]methane. The synthesis was performed according to the procedure previously published (Hirvonen et al, 2009) with minor modifications. An intravenous catheter was placed in the left arm of each study participant for blood sampling and radiotracer injection. The radioligand was administered at tracer doses with no expected pharmacological side effects. The specific radioactivity and the injected mass of the radioligand did not differ between the scans (Supplementary Table S1). After an intravenous injection of [11C]carfentanil (257±12 MBq, range 224–295 MBq, injected mass 0.26±0.17 μg, specific radioactivity 507±259 MBq/nmol), radioactivity in the brain was measured with a Philips Ingenuity PET/MR scanner for 51 min, using 13 frames, with in-plane resolution of 3.75 mm. A T1- weighted TR 25 ms, TE 4.6 ms, flip angle 30°, scan time 376 s MR images (1 mm3 voxel size) were acquired for anatomical reference. Radioactivity data acquisition was started concomitantly with the injection of the radiotracer.

To correct for head motion all the volumes of the dynamic PET scans were realigned to each other. A T1-weighted MR image was coregistered with the summed PET image and the occipital cortex was manually defined on the coregistered T1 image using the PMOD 3.3 software (PMOD Technologies, Zürich, Switzerland). Receptor availability was expressed in terms of BP ND , which is the ratio between specific and non-displaceable binding in the brain. A simplified reference tissue model (SRTM) was used to obtain non-displaceable binding potential estimates voxel-wise using occipital time activity curve as reference tissue input (Gunn et al, 1997). This outcome measure is not confounded by changes or differences in peripheral distribution. Specific binding of [11C]carfentanil is unaffected by changes in cerebral blood flow (Endres et al, 2003; Frost et al, 1989; Liberzon et al, 2002). The resulting parametric BP ND images were normalized to the Montreal Neurological Institute (MNI) space using deformation fields obtained by segmenting the T1-weighted images. Finally, normalized parametric images were smoothed with a Gaussian kernel of 7 mm full-width half-maximum. All preprocessing steps were carried out using the SPM8 software (www.fil.ion.ucl.ac.uk/spm/) run on Matlab R2012a (MathWorks), except for simplified reference tissue modeling, which was done using PMOD. Because of technical problems with the PET scanner, the PET-data following one MICT and one HIIT were subsequently found to be invalid and were excluded from the analysis.

Statistical Analyses

Within subject effects of MICT on MOR availability were tested with the paired t test for the whole group, since every subject completed rest and MICT scans (n=21, age 25.1 (8.6) years). The within subject effects of MICT and HIIT on MOR availability were tested with repeated measures one-way ANOVA for the subset of participants who completed all three studies (n=11, age 24.8 (9.1) years) (Supplementary Figure S1). The calculations were performed using the SPM8 software (www.fil.ion.ucl.ac.uk/spm/) running on Matlab R2012a (MathWorks). The threshold for statistically significant differences was set at p<0.05 for all assessments, false discovery rate corrected at cluster level. The Marsbar toolbox for SPM (http://marsbar.sourceforge.net/) was used to extract mean BP ND estimates from each activation cluster observed in the full-volume analysis.

The effects of physical exercise on RPE, valence and arousal measured during exercise, and positive and negative affect and VAS items measured before and after exercise, were analyzed using ANOVA for the participants who performed both exercise modes (MICT and HIIT, n=12) with IBM SPSS Statistics 21 for Mac OS X (IBM Corp., Chicago, IL, USA). To ensure that MICT-induced affective responses were similar between the group that performed only MICT (n=10) and the group that performed both MICT and HIIT (n=12), another set of general linear model analyses were performed using condition (pre/post) as a within-subject factor and group (only MICT; or both MICT and HIIT) as a between-subject factor. The associations of exercise-induced changes in mood responses and changes in MOR binding were assessed using an exploratory whole-brain analysis with statistical threshold set at p<0.05, false discovery rate corrected at cluster level.