The Effects of Nicotine on Cortical Excitability After Exercise

BRIEF REPORT The Effects of Nicotine on Cortical Excitability After Exercise A Double-Blind Randomized, Placebo-controlled, Crossover Study Thomas Zandonai, PhD,*† Fabio Pizzolato, PhD,‡ Enrico Tam, PhD,‡ Paolo Bruseghini, PhD,§ Cristiano Chiamulera, PharmD,|| and Paola Cesari, PhD‡ Abstract: Purpose: The use of smokeless tobacco/nicotine products is common Downloaded from http://journals.lww.com/psychopharmacology by BhDMf5ePHKbH4TTImqenVIu1/FN/J4kOClpFV9JzxSmVhCnePMnfqEls36wZcLTzKbyy+ViEFSo= on 10/13/2020 among athletes, but clear evidence for their positive or negative effect on sports performance is lacking. Nicotine is a psychoactive substance involved in numerous neuronal processes including cortical excitability. The aim of this study was to evaluate its effect on cortical excitability associated with aerobic exercise in nicotine-naive healthy volunteers. Methods: Ten nicotine-naive healthy volunteers were recruited for this double-blind, randomized, crossover study to compare the effect of snus (8 mg nicotine), an oral, smokeless tobacco product, to placebo on cortical excitability before and after aerobic exercise. Transcranial magnetic stimulation (TMS) was used to measure changes in corticomotor excitability (motor-evoked potentials, MEPs) and electromyography of leg muscles during maximal voluntary contractions (MVC) to assess changes in muscle contractions. Before and after aerobic exercise and with or without nicotine treatment, MEPs and MVCs were measured. Results: Analysis of TMS data showed lower motor cortex activation (lower MEP amplitude) after snus administration compared with placebo, whereas electromyography data showed no difference in muscle contraction between snus and placebo treatment. Conclusions: These findings suggest a general reduction in cortical excitability, without no relevant effect on physical performance. Key Words: nicotine, smokeless tobacco, cortical excitability, exercise, TMS (J Clin Psychopharmacol 2020;40: 495–498) orticospinal excitability is reduced during exercise.1 One of the best techniques to test the effects of exercise on corticospinal excitability is transcranial magnetic stimulation (TMS). This noninvasive technique stimulates the brain while recording by means of surface electromyography (EMG) the changes in the neural drive of the motor system during fatiguing exercise.2 The measurement units are motor-evoked potentials (MEPs) and MEP amplitude, which reflect the amount of motor system activation.2–5 A reduction in the amplitude of resting MEPs has been observed after muscle fatiguing due to voluntary contractions6,7 and exercise.8 To the best of our knowledge, few studies have investigated the effects of nicotine on cortical excitability and brain plasticity. In general, nicotine exerts different effects on cortical excitability C From the *Department of Pharmacology, Pediatrics and Organic Chemistry, Miguel Hernández University, Elche; †Mind, Brain and Behaviour Research Center (CIMCYC), Department of Experimental Psychology, Faculty of Psychology, University of Granada, Granada, Spain; ‡Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona; §Department of Molecular and Translational Medicine, University of Brescia, Brescia; and ||Neuropsychopharmacology Laboratory, Department of Diagnostic and Public Health, University of Verona, Verona, Italy. Received October 22, 2019; accepted after revision May 20, 2020. Reprints: Thomas Zandonai, PhD, Department of Pharmacology, Pediatrics and Organic Chemistry, Miguel Hernández University, Crta. Nacional, N-332. s/n, 03550 Sant Joan, Alicante, Spain (e‐mail: [email protected]). Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0271-0749 DOI: 10.1097/JCP.0000000000001246 between smokers and nonsmokers because of the smoking status and acute exposure to nicotine.9 Published findings are not consistent, however. One study showed enhanced motor cortex inhibition and reduced facilitation associated with the effects of chronic nicotine exposure on cortical excitability in smokers,10 whereas another reported that nicotine administration enhanced cortical facilitation.9 In studies involving nonsmokers, Grundey et al9 found that nicotine exposure enhanced cortical inhibition, and Swayne et al11 showed that nicotine increased and extended the facilitatory after-effects of intermittent theta burst stimulation in the motor cortex. Still other studies indicated a significant impact of nicotine on somatosensory change-related cortical responses and suggested that the effect of nicotine is common in the sensory system of nicotine-naive individuals.12 The effects of nicotine on exercise have attracted growing interest owing to its increasing use among athletes.13,14 Snus is a smokeless, oral tobacco product popular with athletes.14,15 Studies on its effects on exercise performance have produced mixed results for naive16 and snus users.17 The aim of the present study was to determine whether nicotine administration via a snus product reduces central excitability after exercise at 65% of maximal aerobic power (65% Wmax) until exhaustion. In addition, the effects of nicotine on peripheral muscle contraction capacity were measured by surface EMG on the vastus femoralis and tibialis anterior (TA) muscles. We hypothesized that nicotine intake might increase cortical inhibition in nonsnus users9 and that nicotine effects do not affect muscle contraction capacity during maximal strength exercise.9 MATERIALS AND METHODS Study Design In this double-blind, placebo-controlled study, we compared the effect of snus versus placebo on cortical excitability after exercise at a constant work rate of 65% Wmax until exhaustion. The local ethical committee of the Department of Neuroscience, Biomedicine and Movement, University of Verona (Italy) approved the study protocol. The study was conducted in accordance with the current Declaration of Helsinki guidelines, and all subjects gave their written, informed consent after being informed about the procedures and risks of the study. In the first session, a maximal incremental cycle ergometer exercise test was performed to estimate 65% of Wmax.16 In the second session, the subjects received transcranial magnetic stimulation (TMS) to measure changes in corticomotor excitability. They were asked to relax while TMS was applied over the scalp for 20 stimuli. They then performed six leg extensions at 100% of maximal voluntary contraction (MVC) while muscle activity was recorded by EMG. Finally, they performed exercise until exhaustion at their 65% of Wmax on a cycle ergometer.16 Before starting the exercise, they received an oral product with (snus) or without nicotine (placebo). The order of treatment (sessions on the second or third day) was generated by online software (random.org). At the end of the exercise, Journal of Clinical Psychopharmacology • Volume 40, Number 5, September/October 2020 www.psychopharmacology.com Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved. 495 Journal of Clinical Psychopharmacology • Volume 40, Number 5, September/October 2020 Zandonai et al the subjects repeated the TMS and MVC procedure. The third session was scheduled a minimum of 7 days after the second session to allow for washout and recovery. During the final session, the experimental procedure was the same as in the second session. Participants The study sample was 14 healthy male athlete nonsmokers and nonsnus users (mean age ± SD, 23.3 ± 5.1 years; height, 177.3 ± 5.5 cm; weight, 73.6 ± 6.5 kg; VO2max, 48.9 ± 8.1 mL/kg per min). Two dropped out because of injuries unrelated to the study and two others because of technical problems. The data of the remaining 10 subjects were entered in the final analysis. Five reported mild throat discomfort, moderate nausea, and dizziness associated with snus administration, whereas no adverse events were associated with the placebo. Measures Transcranial magnetic stimulation was delivered by a STM9000 magnetic stimulator (2 Tesla ATES-EB Neuro, Italy) using a figure-of-8 coil (diameter, 70 mm). Transcranial magnetic stimulation stimuli sequences were processed by Psychology Tools Eprime2 software (Pittsburgh, PA). Muscle activity was recorded and amplified on a Digitimer D360 8-channel and CED Power 1401 (Digitimer, Hertfordshire, England), bandpass-filtered (20 Hz–3 kHz), and sampled at 5 kHz. Electromyography was recorded with surface electrodes placed over the TA and the vastus lateralis (VL) muscles. The measurements of the 6 trials performed during MVC and the 6 trials recorded at rest (REST) were averaged. The electrode positions were marked on the skin with an indelible marking pen to ensure the same location during data collection PRE and POSTexercise.2 The EMG measurements are termed TMS-EMG for cortical activity and MVC-EMG for muscle voluntary contraction. The tobacco product was a commercial Catch White Eucalyptus Portion Snus (Swedish Match) (1.0 g, nicotine 8 mg/portion) and the placebo was an Onico Peppermint (Swedish Match) (1.0 g). The boxes containing the products were coded and masked, so that neither the investigators nor the subjects were aware of the contents before starting exercise until completion of the analysis.16 Experimental Procedures PRE Exercise Phase Subjects were tested at the Laboratory Action and Perception, University of Verona, Italy. They were seated comfortably in front of a 21-in. LCD computer screen (resolution, 1680° 1050 pixels; viewing distance, 100 cm). The optimal scalp position was identified by means of a neuronavigator system (Softaxic, EMS, Bologna, Italy).18 Optimal scalp position was defined over the left primary motor cortex, and TMS was applied to elicit stable MEPs from TA and VL. The resting motor threshold (MT) was defined as the lowest stimulus intensity able to evoke 5 of 10 MEPs with an amplitude of at least 50 μV in the VL.19 Twenty transcranial stimuli (TMS-EMG), with at least 10 seconds pause between stimuli (Fig. 1A), were delivered to the relaxed muscles at an intensity of 1.2 MT. Peak-to-peak MEP amplitude was defined as the level of motor cortex excitability.5 The subjects were then asked to perform with their dominant lower limb six leg extensions at MVC-EMG. Electromyography was recorded over the TA and VL muscles. The interval between each MVC was 20 seconds. Visual feedback displayed on a computer screen cued the subjects when to execute the MVC (Contraction!) and when to relax (Relax!). The subjects then performed a 65% Wmax test after receiving either the snus or the placebo according to the randomization list. They kept the sachets in their mouth before starting the 65% Wmax test until the end of the exercise. During the test, they pedaled constantly until exhaustion, defined as the inability to maintain pedaling frequency (60 rpm) despite vigorous encouragement by the experimenters.16 POST Exercise Phase At the end of the 65Wmax test, the subjects returned to the Laboratory Action and Perception and repeated the same procedure as in the preexercise session (TMS-EMG and MVC-EMG). Subjects started the first MVC 35 ± 7 minutes after the end of the 65Wmax test. A flow diagram of the study protocol is shown in Figure 1. Data Analysis The measurements of brain activation were the peak-to-peak amplitudes of the MEPs induced by TMS on the TA and VL muscles and the EMG peaks recorded for the TA and VL muscleswere a measurement of muscle fatigue. The TMS-EMG and MVC-EMG data were normalized and scaled to a range of 0 and 1 because of elevated between-subject variability. This scaling allowed us to obtain a normal distribution of the data (checked with the Shapiro-Wilk test), while the homogeneity of variance was verified with Bartlett test. Values that exceeded 4 times the median were considered outliers and removed from the TMS-EMG data. FIGURE 1. Flow diagram of the study protocol. 496 www.psychopharmacology.com © 2020 Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved. Journal of Clinical Psychopharmacology • Volume 40, Number 5, September/October 2020 Within repeated-measures ANOVA was performed on the normalized data. The TMS-EMG data (primary motor cortex activity) were analyzed at 2 levels: treatment (snus/placebo)  2 levels muscles (VT and TA)  2 levels time (pre/post). The MVC-EMG data (maximun muscle contraction) were analyzed at 2 levels: treatment (snus/placebo)  2 levels muscles (VT and TA)  2 levels time (pre/post). Bonferroni multiple comparison was applied when required. Statistical significance was set at P less than 0.05. All data are reported as mean ± standard error of the mean (SE), and all analyses were performed using SPSS (version 20.0.0, SPSS-IBM, IBM Inc, Armonk, NY). The magnitude of the effect size followed Cohen d: small effect (0.1 to 0.3); intermediate effect (0.3–0.5); strong effect (≥0.5).20 RESULTS The MEP amplitude showed a downward trend after exercise (from 0.447 ± 0.041 mV to 0.346 ± 0.037 mV). The TMS-EMG showed a significant Snus-Placebo effect (F(1,9) = 12.991; P = 0.006; d = 2.403), indicating higher activation in the placebo than in the snus condition (0.425 ± 0.018 mV in the placebo and 0.368 ± 0.013 mV in the snus condition). None of the main factors and interactions were significant. Analysis of the MVC-EMG showed a significant main effect of muscles, indicating that the TA was more active than the VL (F(1,9) = 13.029; P = 0.006; d = 2.406). None of the other factors and interactions were significant. DISCUSSION This is the first study to investigate the effect of snus on cortical excitability. The main finding was that nicotine influences neuroplasticity in nicotine-naive individuals and increases cortical inhibition. Our findings are shared by Grundey et al9 who suggested that cortical inhibition could be mediated by nicotinic acetylcholine receptors (nAChR). Activation of nAChR induces inhibition or disinhibition of pyramidal cortical neurons,9 leading to neuronal inhibition and excitation depending on the cortical site, upregulation or downregulation of receptors, and mode of nicotine administration. In addition, other individual factors to be taken into account include tolerance to nicotine effects and/or changes in nAChR availability, which could influence the response to the nicotine absorbed through snus and induce cortical inhibition. The complexity of this process is reflected in the mixed results of previous studies on the effects of nicotine on cortical excitability in smokers versus nonsmokers.9–11,21 Studies involving nonsmokers suggested that the focusing effect of nicotine on plasticity might help to explain how it indirectly enhances cognitive performance.22 Furthermore, we found that snus intake might produce an early and transient cognitive improvement on decision-making cognitive tasks in abstinent snus users, presumably acting as a withdrawal relief.14 Despite the popular use of nicotine among athletes, its effect on exercise is not clear.16,17 Mundel et al23 tested leg extensor force and countermovement jump height after administration of 2 or 4 mg nicotine gum (vs placebo) in nonnicotine users. They found that leg extensor torque was improved with the low-dose nicotine compared with the placebo but that neither strength of the nicotine gum influenced countermovement jump height. We previously demonstrated that, despite the increase in cerebral and muscular tissue oxygenation, snus administration did not affect endurance exercise performance in healthy naive subjects.16 In the present study, we found no significant changes in muscle strength. This observation is shared by Morente-Sanchez et al,24 who measured handgrip strength and countermovement jump height 40 minutes after snus or placebo administration in amateur © 2020 Wolters Kluwer Health, Inc. All rights reserved. Nicotine Effects on Cortical Excitability footballers who were not habitual tobacco users; they reported that snus use did not affect either performance measure. Different results were found among snus users. Recently, we demonstrated that snus induces a significant increase (+13.1%) in time to exhaustion after 12-hour abstinence in nicotine-addicted athletes.17 We suggested that because muscle nAChRs are located in the neuromuscular junction of somatic muscle, nicotine can have a biphasic effect at the neuromuscular junction,25 where it exerts excitatory (acute after withdrawal) followed by inhibitory (chronic when tolerant) effects. The small sample size notwithstanding, the study provides additional data on the inhibitory effect of nicotine on cortical excitability in nonsmokers. The lack of improvement in MVC could be explained by a central fatigue component accumulated during aerobic exercise. Further studies on the effects of nicotine on cortical excitability are needed to clarify the effect, illustrate the underlying mechanisms involved, and determine whether sex-related effects exist. ACKNOWLEDGMENT The authors wish to thank Daniele Mori for EMG data analysis. 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