J Neurophysiol 111: 2656 –2664, 2014. First published March 26, 2014; doi:10.1152/jn.00778.2013.
Motor cortical disinhibition with baroreceptor unloading induced by orthostatic stress Vasiliy E. Buharin,1 Andrew J. Butler,1,2,3 and Minoru Shinohara1,3 1 School of Applied Physiology, Georgia Institute of Technology, Atlanta, Georgia; 2Department of Physical Therapy, Georgia State University, Atlanta, Georgia; and 3Rehabilitation R&D Center of Excellence, Atlanta Department of Veterans Affairs Medical Center, Decatur, Georgia
Submitted 30 October 2013; accepted in final form 21 March 2014
transcranial magnetic stimulation; motor cortex; autonomic nervous system
is a measure of the excitability of the excitatory and inhibitory interneurons within the motor cortex, which influence fine motor skill and neural plasticity. Interventions that can alter intracortical excitability are useful for neurophysiological research on motor control and for neurological treatment of individuals with movement disorders. Aside from being altered artificially by applications of exogenous neuromodulatory agents (e.g., for dopamine and norepinephrine; Ziemann 2003), intracortical excitability is physiologically modulated with hypoxia (Szubski et al. 2006), high altitude exposure (Miscio et al. 2009), and circadian rhythm (Lang et al. 2011). Since these physiological conditions commonly influence autonomic nervous activity, it is possible that intracortical excitability is modified with the neurophysiological processes involved in the control of autonomic nervous activity. In human studies, orthostatic stress by the lower body negative pressure (LBNP) procedure has been applied for enhancing sympathetic nerve activity through baroreceptor unloading, as a human model of acute central hypovolemia
INTRACORTICAL EXCITABILITY
Address for reprint requests and other correspondence: M. Shinohara, School of Applied Physiology, Georgia Inst. of Technology, 555 14th St. NW, Atlanta, GA 30332-0356 (e-mail: [email protected]). 2656
(Cooke et al. 2004; Davy et al. 1998). Our recent data show that corticospinal excitability for a resting muscle increases with LBNP (Buharin et al. 2013). However, the effects of orthostatic stress and subsequent baroreceptor unloading on intracortical excitatory and inhibitory pathways within the motor cortex in resting or contracting muscle are unknown. There is substantial indirect evidence that suggests that orthostatic stress and subsequent baroreceptor unloading may influence intracortical excitability because of potential increases in the function of neuromodulatory monoamines in the motor cortex (Clement et al. 1992; Devoto et al. 2005; Foote and Morrison 1987; Kaehler et al. 2000; Singewald and Philippu 1993) and the amount of somatosensory afferent input (Hjortskov et al. 2005; Kamibayashi et al. 2009). The purpose of this study was to examine the effects of orthostatic stress on intracortical excitatory and inhibitory pathways in the motor cortex for resting and contracting muscle. The potential intracortical pathways influenced by orthostatic stress may be inferred from previous findings demonstrating that increases in corticospinal excitability with orthostatic stress are observed only at higher intensities of transcranial magnetic stimulation (TMS) (Buharin et al. 2013). Since TMS of higher intensity recruits interneurons that generate later I waves (i.e., I-2 and I-3 waves) in the motor cortex (Di Lazzaro et al. 1998a), greater contribution of later I waves may lead to increased corticospinal excitability with orthostatic stress. Contribution of later I waves to corticospinal excitability can be increased by 1) increasing the activity of the intracortical excitatory glutamatergic pathway responsible for later I-wave generation or by 2) decreasing the activity of the intracortical inhibitory GABAAergic pathway that inhibits later I waves (Di Lazzaro et al. 1998b; Hanajima et al. 2002). In the present study, it was hypothesized that orthostatic stress for baroreceptor unloading would increase the activity of the intracortical excitatory glutamatergic pathway and/or decrease the activity of the intracortical inhibitory GABAAergic pathway in resting and contracting muscle. MATERIALS AND METHODS
Subjects. Sixteen healthy adults (19.8 ⫾ 1.5 yr of age; 6 women, 10 men) participated in the study on 2 days separated by ⬃4 wk. Women were tested during their follicular phase to avoid potential confounding effects of estrogen and progesterone (Minson et al. 2000). All subjects were right-handed, as confirmed with the Edinburgh handedness inventory (laterality quotient: 0.82 ⫾ 0.155) (Oldfield 1971). Volunteers reported no symptoms of chronically altered sympathetic nerve activity: no history of diabetes, cardiovascular problems, brain or nerve disorder, obesity, hypertension, or hypotension (Landsberg www.jn.org
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Buharin VE, Butler AJ, Shinohara M. Motor cortical disinhibition with baroreceptor unloading induced by orthostatic stress. J Neurophysiol 111: 2656 –2664, 2014. First published March 26, 2014; doi:10.1152/jn.00778.2013.—Unloading of the baroreceptors due to orthostatic stress increases corticospinal excitability. The purpose of this study was to examine the effects of baroreceptor unloading due to orthostatic stress on intracortical excitatory and inhibitory pathways in the motor cortex. With transcranial magnetic stimulation, measures of intracortical excitability for a hand muscle were tested on 2 days in healthy young adults. Lower body negative pressure (LBNP) of 40 mmHg was applied during one of the days and not during the Control day. During application of LBNP heart rate and the low-frequency component of heart rate variability increased, while mean arterial blood pressure was maintained. In the resting state, LBNP decreased short-interval intracortical inhibition (SICI) and had no effect on intracortical facilitation (ICF) or short-interval intracortical facilitation (SICF) compared with the Control day. During isometric contraction, no effects of LBNP were observed on tested measures of intracortical excitability including SICI, SICF, and cortical silent period. It was concluded that baroreceptor unloading due to orthostatic stress results in diminished intracortical inhibition, at least in the resting muscle.
INTRACORTICAL EXCITABILITY AND BAROREFLEX
Subjects did not have vision of their hands because of obstruction by the LBNP chamber. The experiment was conducted in an electrically shielded room. Surface electromyogram (EMG) was recorded with Ag-AgCl electrodes (E224A, IVM, Healdsburg, CA) placed on the skin overlying the right first dorsal interosseus in a belly-tendon montage (Buharin et al. 2013). One electrode was placed over the belly of the muscle, and the other was attached to the skin over the distal tendon after abrasion of the skin. A wet circumferential strap electrode (F-E10SG1, Grass Technologies, West Warwick, RI) was placed around the right wrist for a reference. EMG was differentially amplified 300 times and band-pass filtered between 15 and 2,000 Hz (Y03-000, MotionLabs). EMG data were sampled at 5,000 samples/s with an analog-to-digital converter (Power 1401, Cambridge Electronic Design). Contraction intensity was determined based on the EMG amplitude of the first dorsal interosseus during maximal voluntary isometric contraction. A rectified running average EMG with an averaging window of 0.175 s was used to provide visual feedback to subjects and to calculate the maximal EMG (EMGmax) of the muscle. With their right hand secured in the hand brace, subjects increased their EMG amplitude to maximum in a ramp fashion over 3 s and maintained it at maximum for 2 s before relaxing. Verbal instruction and encouragement were provided while the right hand of subjects was visually monitored. TMS intensity was adjusted based on MEP in the first dorsal interosseus. A figure-of-eight coil (Magstim second-generation double 70-mm remote coil, Magstim) was held over the left primary motor cortex at the optimum position (i.e., hot spot) for eliciting an MEP in the resting muscle of the right hand (Buharin et al. 2013). The coil was held with the handle pointing posteriorly at an angle of ⬃45° to the sagittal plane, yielding an E field perpendicular to the central sulcus (Brasil-Neto et al. 1992). A TMS coil navigation system (NDI TMS Manager, Northern Digital, Waterloo, ON, Canada) was used to maintain the coil position in three-dimensional space relative to the head. Continuous running visual feedback of the EMG was provided to the subject and experimenter to ensure relaxation or appropriate activation of the muscle when necessary. The resting motor threshold was determined as the smallest TMS intensity needed to elicit an MEP with peak-to-peak amplitude (PPamp) ⬎ 50 V in 5 of 10 consecutive stimulations in the relaxed muscle (Darling et al. 2006). Active motor threshold was assessed during the isometric contraction at 10% EMGmax (Ortu et al. 2008). Because of the difficulty in differentiating between MEP and background contraction EMG in single stimulation traces, the active motor threshold was defined as the largest TMS intensity that produced an EMG response ⬍50 V above background EMG activity in the triggered average of 10 consecutive rectified stimulation traces (Ortu et al. 2008). TMS paradigms were chosen to test whether orthostatic stress would increase the activity of the intracortical excitatory glutamatergic pathway and/or decrease the activity of the intracortical inhibitory GABAAergic pathway. During the Resting stage, activity of the intracortical later I-wave, GABAAergic, and NMDAergic pathways in the resting muscle were assessed with the paired-pulse TMS paradigms for short-interval intracortical facilitation (SICF) (Hanajima et al. 2002), short-interval intracortical inhibition (SICI) (Ziemann et al. 1996b), and intracortical facilitation (ICF) (Schwenkreis et al. 1999; Ziemann et al. 1998), respectively (Fig. 1). The test stimulus for the paired-pulse stimulation in the Resting stage was determined as the TMS intensity that produced an average MEP with PPamp of 1 mV in 10 consecutive stimulations in the relaxed muscle (Ortu et al. 2008). SICF was measured by delivering the TMS pulse at 0.9 ⫻ active motor threshold 1.5 ms after the suprathreshold test stimulus pulse (Ortu et al. 2008). For SICI and ICF, a subthreshold TMS pulse at 0.9 ⫻ active motor threshold was followed by the suprathreshold test stimulus pulse with an interval of 2 and 10 ms, respectively (Kujirai et al. 1993; Ortu et al. 2008). An interstimulus interval of 2 ms was
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1986). On the testing days, heart rate and blood pressure in the resting state were confirmed to be normal. They did not perform extensive hand grip activity, exhibit skilled use of hands, report arthritis of the hands, or take any medication that may affect motor control or brain and nerve function. In addition, subjects were excluded if they had a family history of seizure or epilepsy, had skin allergies, were pregnant, were prone to severe headaches, or had metal in their head, excluding dental fillings (Rossi et al. 2011). To minimize the variability in the basal physiological level and responsiveness of sympathetic nerve activity across subjects, all experiments were conducted at 8 AM; participants abstained from food and drink, with the exception of water, for 10 h prior to the experiment (Berne et al. 1989). All subjects gave written informed consent. This study was approved by the Georgia Institute of Technology and Emory University Institutional Review Boards. Experimental approach. Intracortical excitability was assessed with TMS delivered over the motor representation area of the first dorsal interosseus muscle. Motor evoked potentials (MEPs) were recorded from the muscle in the right hand with orthostatic stress on the LBNP day and without orthostatic stress on the Control day. The experimental protocol of each subject was temporally comparable between the two days. On a given day, intracortical excitability was assessed at rest (Resting stage), followed by contraction of the first dorsal interosseus (Active stage) with a 10-min intermission (Intermission stage) in between. The LBNP vacuum was turned off during the Intermission stage. Effects of LBNP on intracortical excitability were interpreted from the differences of corresponding measures between the Control day and the LBNP day. The order of the two days was randomized across subjects. LBNP. The LBNP procedure in our previous study (Buharin et al. 2013) was employed to apply orthostatic stress and unload the baroreceptors. The participants lay supine with their lower body inside the LBNP chamber (1.2 m ⫻ 0.6 m ⫻ 0.5 m). Subjects wore a neoprene belt about their hips at the level of the iliac crest. An airtight flexible nylon cover was fit over the opening of the LBNP chamber to form a seal between the chamber and the belt. A commercial vacuum (model 3Z708B, Dayton Industrial, Dayton, OH) attached to the chamber was used to lower the pressure inside the chamber. This setup has been used repeatedly in cardiovascular studies by others (e.g., Davy et al. 1998) and in our previous study (Buharin et al. 2013). On the LBNP day, orthostatic stress was applied to unload aortic and carotid baroreceptors by gradually reducing the pressure in the chamber to ⫺40 mmHg relative to ambient pressure in 20 s. MEP testing commenced 30 s after application of LBNP. On the Control day, the vacuum remained on and the pressure was set to 0 mmHg during the recording of MEPs. LBNP of 40 mmHg is known to physiologically unload the baroreceptors, heighten sympathetic nerve activity, and increase heart rate and low-frequency content of heart rate variability (HRVLF), with little change in blood pressure (Buharin et al. 2013; Lee et al. 2004). Blood pressure at the brachial artery in the left arm and heart rate at the fingertip were monitored noninvasively (Cardiocap/5, GE Healthcare, Chalfont St. Giles, UK), and mean arterial blood pressure was recorded in each TMS block. Electrocardiogram (ECG; Cardiocap/5, GE Healthcare) was sampled at 10,000 samples/s with an analog-to-digital converter (Power 1401, Cambridge Electronic Design, Cambridge, UK) and data acquisition software (Spike2 v7.0, Cambridge Electronic Design). ECG was not obtained in one subject because of technical issues. The cardiovascular data were recorded during TMS as well as before delivery of TMS (Baseline stage) and during the Intermission stage. Intracortical excitability. Intracortical excitability was assessed with single- and paired-pulse TMS (Magstim 2002, Magstim, Whitland, UK) of the left primary motor cortex. The head was oriented in neutral position on a pillow. The arms of each subject lay at his/her sides with the right hand in pronation, in a wooden brace when producing isometric contractions or resting on the bed when not.
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SICF
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1 mV
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50 ms
used for SICI assessment to avoid contamination of SICI measurement by SICF peaks (Peurala et al. 2008). With this set of stimulation intensity and interval combinations, MEPs in response to test stimulation, SICF, SICI, and ICF were collected. TMS was delivered every 6 s. Seventeen MEPs were collected for each TMS paradigm in the Resting stage. Because of technical issues, SICF was not measured in one subject. During the Active stage, the primary variables of interest were activity of the intracortical GABAAergic and GABABergic pathways, assessed with cortical silent period (CSP) in the contracting muscle. CSP was measured during isometric contraction at 50% EMGmax to minimize variability of CSP due to the contraction strategy of subjects (Mathis et al. 1998) and to delineate potential effects of orthostatic stress on intracortical GABAAergic and GABABergic pathways (Kimiskidis et al. 2006). Subjects were instructed to ramp up to the 50% EMGmax contraction, to maintain the contraction through the ensuing stimulation, and to relax only when told to do so, at least 1 s after TMS (Mathis et al. 1998). Data from subjects who failed to follow this instruction were removed from analysis of 50% EMGmax data (2 subjects). CSP was measured in response to TMS at resting motor threshold (low-intensity TMS) and TMS at 2 ⫻ resting motor threshold (high-intensity TMS), separately, because the response to lowand high-intensity TMS is indicative of GABAAergic and GABABergic activity, respectively (Kimiskidis et al. 2006). For those subjects whose 2 ⫻ resting motor threshold was greater than the maximal stimulator output (n ⫽ 4), TMS at the maximal stimulator output was used to assess CSP with high-intensity TMS. TMS was delivered every 15 s. CSPs with low- and high-intensity TMS were collected in this order, and 12 responses were collected for each. Additionally, activity of the later I-wave and GABAAergic pathways of the active first dorsal interosseus were investigated with the paired-pulse protocols for SICF and SICI during isometric contraction at 10% EMGmax. While there was a concern that SICI may not be induced in contracting muscles (Ridding et al. 1995), SICI was
measured following the protocol of Ortu et al. (2008) that has observed SICI of 38% during isometric contraction with the first dorsal interosseus at 10% EMGmax. Note that activity of the intracortical NMDAergic pathway cannot be measured during muscle contraction because of the disappearance of ICF (Ortu et al. 2008). The test stimulus for paired-pulse stimulation in the Active stage was determined as the TMS intensity that produced a peak amplitude between 0.5 mV and 1 mV in the average of 10 consecutive rectified stimulation traces during 10% EMGmax contraction (Ortu et al. 2008). SICF was measured by delivering the TMS pulse at 0.9 ⫻ active motor threshold 1.5 ms after the suprathreshold test pulse (Ortu et al. 2008). For SICI, a subthreshold TMS pulse at 0.7 ⫻ active motor threshold was followed by a suprathreshold test stimulus pulse at 2 ms. The 2-ms interstimulus interval and conditioning stimulation were chosen to best analyze SICI without interference from SICF during voluntary contraction (Ortu et al. 2008; Peurala et al. 2008). Response to single-pulse TMS at 0.9 ⫻ active motor threshold was also measured to check whether the conditioning stimulations produced an MEP. MEPs in response to test stimulation were collected first, followed by SICF, SICI, and single-pulse TMS at 0.9 ⫻ active motor threshold, in random order, during 10% EMGmax contraction. TMS was delivered every 6 s, and subjects maintained the contraction level for the duration of each TMS paradigm block. Twelve MEP responses were collected for each TMS paradigm. Data reduction. The first two recordings in each TMS paradigm were discarded to control for possible startle responses. In the Resting stage, EMG recordings that showed prestimulus EMG activity above baseline 100 ms preceding TMS were discarded. The root mean square amplitude of the prestimulus EMG, the MEP PPamp, and the MEP area bounded by the MEP and the 0-mV axis (MEP area) were calculated for each response. The amplitude of the prestimulus EMG was calculated from data 100 ms preceding the application of TMS. The EMG in the period between 20 and 50 ms after application of TMS was used to measure MEP PPamp and MEP area. MEP PPamp
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Fig. 1. Representative recordings of motor evoked potentials (MEPs) in response to transcranial magnetic stimulation (TMS) recorded from the first dorsal interosseus muscle in 1 subject on the Control day. Traces represent different TMS protocols (top), a single MEP in the Resting stage (middle), and an average of 10 MEPs in the Active stage (bottom) without lower body negative pressure (LBNP). Test, single-pulse test stimulation; SICF, short-interval intracortical facilitation; SICI, short-interval intracortical inhibition; ICF, intracortical facilitation. Vertical scale is for middle and bottom rows.
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INTRACORTICAL EXCITABILITY AND BAROREFLEX
Unless otherwise stated, data are presented as means ⫾ SD in the text and tables and as means ⫾ SE in the figures. RESULTS
Subject characteristics. Basic subject characteristics, including EMGmax, resting motor threshold, active motor threshold, and test stimulation MEP and intensity for Resting and Active stages were not different between the LBNP and Control days (Table 1). No effects of day order were observed for these variables. Cardiovascular measurements. No effects of day order were observed for heart rate, HRVLF, or mean arterial blood pressure. With the application of 40 mmHg LBNP on the LBNP day, both heart rate and HRVLF were increased while mean arterial blood pressure was not significantly different compared with the Control day (Fig. 2). There were main effects of Day (P ⬍ 0.01), Stage (P ⬍ 0.01), and their interaction (P ⬍ 0.01) on heart rate. Heart rate was greater on the LBNP day when 40 mmHg LBNP was applied in Resting [by 11 beats per min (bpm), P ⬍ 0.01] and Active (by 12 bpm, P ⬍ 0.01) stages, respectively, compared with the Baseline and Intermission stages of the same day. Main effects of Day (P ⬍ 0.05), Stage (P ⬍ 0.05), and their interaction (P ⬍ 0.01) were detected for HRVLF. HRVLF was greater with the application of LBNP on the LBNP day by 48% in the Resting stage (P ⬍ 0.01) and by 40% in the Active stage (P ⬍ 0.01) compared with the Baseline and Intermission stages of the same day. There was no significant difference in heart rate or HRVLF between days during the Baseline or Intermission stage. There was only a main effect of Stage (P ⬍ 0.05) on mean arterial blood pressure. Mean arterial blood pressure was slightly greater at the end of data collection (Active stage, 88.6 ⫾ 7.3 mmHg when collapsed across days) compared with the beginning of data collection (Baseline stage, 86.1 ⫾ 6.8 mmHg, P ⬍ 0.05). Intracortical excitability in the Resting stage. No significant effects of day order were observed for any of the intracortical excitability measures in the Resting stage. Root mean square amplitude of the prestimulus EMG was not significantly difTable 1. Neuromuscular characteristics in first dorsal interosseus muscle used for TMS protocol on LBNP and Control days LBNP EMGmax, mV 0.95 ⫾ 0.32 RMT, %* 44.3 ⫾ 7.61 AMT, %* 32.7 ⫾ 8.09 Test stimulation during Resting stage TMS intensity, %* 55.6 ⫾ 12.52 TMS intensity, % RMT 122.5 ⫾ 11.23 MEP PPamp, mV 1.0 ⫾ 0.36 MEP area, mV·ms 3.4 ⫾ 1.31 Test stimulation during Active stage TMS intensity, %* 39.8 ⫾ 8.64 MEP peak, mV 0.9 ⫾ 0.26 MEP area, mV·ms 7.2 ⫾ 2.30
Control 0.95 ⫾ 0.29 44.7 ⫾ 7.15 32.4 ⫾ 5.75 53.9 ⫾ 10.04 118.7 ⫾ 9.44 1.2 ⫾ 0.37 4.7 ⫾ 2.00 38.5 ⫾ 7.93 0.8 ⫾ 0.31 7.2 ⫾ 2.69
Values are means ⫾ SD. TMS, transcranial magnetic stimulation; LBNP, lower body negative pressure; EMGmax, electromyogram amplitude during maximal voluntary contraction; RMT, resting motor threshold; AMT, active motor threshold; MEP, motor evoked potential; PPamp, peak-to-peak amplitude. *Expressed relative to maximal stimulator output. No significant difference observed between LBNP and Control days.
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and area of each response were averaged together within each paradigm block and expressed relative to the corresponding values for test stimulation. Effects of orthostatic stress on intracortical excitability were judged from differences in MEP ratios that were calculated from the measurements made on the same date. During the Active stage, collected recordings were inspected for comparable muscle activation. At each contraction level, those recordings whose amplitude of the prestimulus EMG fell 2 standard deviations outside of the mean were discarded. The recordings collected during subthreshold TMS at 0.9 ⫻ active motor threshold were averaged together. To observe whether subthreshold TMS produced an MEP during 10% EMGmax contraction, the PPamp of the prestimulus EMG and the PPamp between 20 and 50 ms after TMS were calculated. PPamp was chosen 1) to take into account possible fluctuations in the prestimulus EMG and 2) to not underestimate the MEP response. All other recordings were rectified and averaged together within each paradigm block. The automated cumulative sum method was used to measure the CSP objectively (King et al. 2006). The start and end of the CSP were defined as the times, after the MEP, when the average rectified EMG fell below and increased back up to the prestimulus EMG level, respectively. Each CSP was expressed in milliseconds and, in addition, normalized to the peak amplitude and area of the accompanying MEP (Orth and Rothwell 2004). The normalized value for CSP with high-intensity TMS was not available in one subject because of a technical issue in the EMG gain for measuring MEP size. The MEP peak amplitude and MEP area of paired-pulse stimulations were calculated and expressed relative to the corresponding values for test stimulation. As measures of sympathetic nerve activity, heart rate and heart rate variability were assessed from the ECG recordings taken during Baseline, Resting, Intermission, and Active stages. All ECG recordings used in calculation of heart rate variability were ⬎2 min in duration. From the ECG recording, all R-wave peaks were identified, marked, and visually inspected to rule out artifacts. Then the power spectrum of the R-to-R interval was calculated. HRVLF was defined as low-frequency (0.05– 0.15 Hz) power expressed relative to the power in total frequencies (0.05– 0.50 Hz) and was used as a measure of sympathetic nerve activity (Task Force of European Society of Cardiology and North American Society of Pacing and Electrophysiology 1996). Statistical analysis. In this within-subject study design, the main independent variable was Day (Control vs. LBNP). The main dependent variables for intracortical excitability were the appropriate ratios of MEP PPamp, MEP area, and MEP peak amplitude of SICI, ICF, and SICF to the corresponding values for test stimulation and the absolute and normalized durations of CSP. EMGmax, resting motor threshold, active motor threshold, test stimulation intensity for Resting and Active stages, prestimulus EMG from the Resting stage, and the main dependent variables were individually tested for difference between the two days with a two-tailed paired-samples t-test. To test whether the subthreshold stimulation produced motor activity, PPamp of the unrectified prestimulus EMG was tested against the PPamp within the MEP period of the unrectified response to the subthreshold TMS at 0.9 ⫻ active motor threshold during 10% EMGmax contraction, with a two-tailed paired-samples t-test. Effects of LBNP on heart rate, mean arterial blood pressure, and HRVLF were each tested with a two-factor (day, stage) analysis of variance (ANOVA) with repeated measures, where factor stage had four levels: Baseline, Resting, Intermission, and Active. Significant main effects and interaction of all ANOVAs were further tested with the Bonferroni post hoc test. To test the effects of experiment day order, the data were reorganized such that the independent variable Day reflected either day 1 or day 2 of the experiment, and the same statistical analyses were repeated. An ␣ level of 0.05 was used for all significance testing. If Mauchly’s sphericity test was violated, the Huyn-Feldt adjusted P value was used. P ⬍ 0.05 and P ⬍ 0.01 were noted where appropriate. Statistical analyses were performed with Statistica 9.0 (StatSoft, Tulsa, OK).
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Fig. 2. Cardiovascular measurements during different stages of the experiment on Control and LBNP days. Filled symbols indicate when sympathetic nerve activity was heightened (Resting and Active stages of the LBNP day only). HRVLF, low-frequency (0.05– 0.15 Hz) power of heart rate variability expressed relative to the power in total frequencies (0.05– 0.50 Hz); Base, Baseline stage; Rest, Resting stage; Inter., Intermission stage. **P ⬍ 0.01 between stages during LBNP day, as tested with Bonferroni post hoc test of significant day ⫻ stage interaction.
ferent between days (LBNP: 4.17 ⫾ 0.61 V, Control: 3.80 ⫾ 0.29 V). The stimulation intensity needed to elicit 1-mV PPamp MEP in the resting muscle did not vary significantly between days (Table 1). MEP PPamp and MEP area during SICI, ICF, and SICF paired-pulse protocols were expressed relative to the MEP PPamp and MEP area during the single-pulse test stimulation, respectively (Fig. 3). During the SICI protocol, there was an
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Fig. 3. Intracortical excitability in the first dorsal interosseus muscle during the Resting stage on Control and LBNP days. y-Axis shows the MEP peak-to-peak amplitude (top) and MEP area (bottom), in response to paired-pulse stimulation, normalized to the corresponding value during single-pulse test stimulation. Unloading of baroreceptors was performed on the LBNP day. *P ⬍ 0.05 compared with Control in the respective measure.
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**
average inhibition of 54% and 53% of MEP PPamp and MEP area compared with the test stimulation, respectively, on the Control day. This inhibition was reduced with LBNP, resulting in 63% greater MEP PPamp (P ⬍ 0.05) and 60% greater MEP area (P ⬍ 0.05) on the LBNP day compared with the Control day. During the ICF protocol, subjects showed an average facilitation of 15% and 16% of MEP PPamp and MEP area compared with the values for test stimulation, respectively, when collapsed across days. No significant effect of LBNP on these ICF measures was observed. For the SICF protocol, MEP PPamp and MEP area were approximately twofold compared with the values for test stimulation. There was no significant difference in these SICF measures between days. Intracortical excitability in the Active stage. There were no significant effects of day order for the variables in the Active stage. The amplitude of prestimulus EMG did not differ significantly between days (LBNP: 123 ⫾ 44.1 V, Control: 152 ⫾ 71.4 V for 10% EMGmax contraction; LBNP: 474 ⫾ 129.5 V, Control: 473 ⫾ 207.4 V for 50% EMGmax contraction). There was no significant difference in the duration of the CSP for either low- or high-intensity TMS when measured in milliseconds, or when normalized to the corresponding MEP peak or area, between days (Table 2). On
INTRACORTICAL EXCITABILITY AND BAROREFLEX
Control
n
5.77 ⫾ 2.45 40.48 ⫾ 13.75 3.5 ⫾ 20.26 10.9 ⫾ 5.83 1.4 ⫾ 0.66
4.74 ⫾ 2.13 34.80 ⫾ 13.10 58.4 ⫾ 32.36 17.4 ⫾ 18.58 1.9 ⫾ 1.20
14 14 14 14 14
6.10 ⫾ 3.35 50.97 ⫾ 24.89 249.1 ⫾ 57.38 64.8 ⫾ 53.99 6.8 ⫾ 4.76
5.70 ⫾ 2.87 45.95 ⫾ 22.46 236.6 ⫾ 49.03 73.8 ⫾ 92.29 7.7 ⫾ 7.59
13 13 14 13 13
Values are means ⫾ SD for n subjects. Contraction intensity was 50% EMGmax. MEP peak, peak amplitude of average rectified MEP; MEP area, area of average rectified MEP; CSP, cortical silent period. No significant differences observed between days.
average, CSPs with low- and high-intensity TMS were 55.9 ⫾ 26.6 ms and 242.9 ⫾ 52.8 ms, respectively, when collapsed across days. Removal of data of four subjects with high resting motor threshold from the analysis did not influence the statistical results. For the paired-pulse response in the Active stage, there was no significant effect of Day on any tested measures (Fig. 4). When collapsed across days, MEP PPamp and MEP area during the SICF protocol were 2.26 ⫾ 2.03 and 2.05 ⫾ 1.47, respectively. MEP PPamp and MEP area during the SICI protocol were 1.16 ⫾ 1.06 and 1.12 ⫾ 0.78, respectively. To confirm that subthreshold conditioning TMS at 0.9 ⫻ active motor threshold did not produce measurable motor activity, the PPamp of the potential response to the subthreshold TMS was compared with the PPamp of unrectified EMG during the background contraction. The PPamp after the subthreshold TMS (304 ⫾ 143 V) was not significantly different from the PPamp before the subthreshold TMS (338 ⫾ 206 V) during contraction.
5 4
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DISCUSSION 0 5 4
MEP area
The major finding is that SICI in the Resting stage was decreased on the LBNP day when LBNP of 40 mmHg was applied compared with the Control day. No significant differences between days were found in other measures in the Resting stage (SICF and ICF) or in any measures (CSP, SICI, and SICF) in the Active stage. LBNP of 40 mmHg unloads the baroreceptors, thereby heightening sympathetic nerve activity, as evidenced by increases of heart rate (Buharin et al. 2013; Davy et al. 1998) and HRVLF (Lee et al. 2004). In the present study, heart rate and HRVLF were significantly greater on the LBNP day during application of LBNP of 40 mmHg, while mean arterial blood pressure was maintained. The 11–12 bpm increase in heart rate with minimal change in blood pressure due to LBNP of 40 mmHg is comparable to previous studies (Buharin et al. 2013; Davy et al. 1998). These findings support that the present orthostatic stress protocol using LBNP was effective for unloading the baroreceptors and heightening sympathetic nerve activity (Cooke et al. 2004).
3 2 1 0 SICF
SICI
Fig. 4. Intracortical excitability in the first dorsal interosseus muscle during the Active stage on the Control and LBNP days. y-Axis shows the MEP peak-topeak amplitude (top) and MEP area (bottom), in response to paired-pulse stimulation, normalized to the corresponding value during single-pulse test stimulation. Unloading of baroreceptors was performed on the LBNP day. No significant differences were observed between days.
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Low-intensity TMS MEP peak, mV MEP area, mV·ms CSP, ms CSP/MEP peak CSP/MEP area High-intensity TMS MEP peak, mV MEP area, mV·ms CSP, ms CSP/MEP peak CSP/MEP area
LBNP
SICI in the Resting stage decreased on the LBNP day when LBNP of 40 mmHg was applied. SICI represents the inhibition of later I waves (i.e., I-2 and I-3 waves) (Di Lazzaro et al. 1998b) and, at an interstimulus interval of 2 ms, likely reflects the activity of the intracortical inhibitory pathway that modulates the excitability of corticospinal neurons through GABAA receptors (Ziemann et al. 1996b). Several technical considerations ensured the proper measurement of intracortical excitability. Care was taken to ensure that only resting data were analyzed, since SICI is influenced by muscle activation (Ortu et al. 2008; Ridding et al. 1995). The lack of difference in prestimulus EMG activity between days further supports the maintenance of resting status across days. LBNP of 40 mmHg increases corticospinal excitability in the resting muscle when stimulated only at or above 130% resting motor threshold (Buharin et al. 2013). Since the employed intensity of test stimulation was below 130% resting motor threshold and was not different between days, it is likely that the present measurements were made within the range for consistent inputoutput properties of corticospinal neurons. Since the conditioning stimulation was chosen below the active motor threshold and no discernible MEP was produced during the Active stage, the employed conditioning stimulation likely did not produce descending volleys. Additionally, the amount of SICI in the Resting stage on the Control day was comparable to previous reports (Hanajima et al. 2002; Ortu et al. 2008; Ridding et al. 1995). Hence, the decreased SICI in the Resting stage on the LBNP day indicates a decrease in SICI due to orthostatic stress and subsequent baroreceptor unloading, supporting our hypothesis. The decrease in SICI at an interstimulus interval of 2
MEP peak amplitude
Table 2. Measures of corticospinal excitability in first dorsal interosseus muscle during voluntary muscle contraction tested with low- and high-intensity TMS on LBNP and Control days
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The potential influences of baroreflex processes on cortical monoamines affecting SICI are inferred from the following evidence. Baroreceptor unloading may result in increased noradrenergic, dopaminergic, and serotonergic function within the motor cortex. The influences of these neuromodulatory monoamines on SICI are variable. Administration of pharmacological agents that facilitate noradrenergic or serotonergic functions, including noradrenergic agonists (Gilbert et al. 2006; Herwig et al. 2002; Ili´c et al. 2003) and serotonergic agonists (Werhahn et al. 1998), can decrease SICI and, similar to the application of orthostatic stress (Buharin et al. 2013), increase corticospinal excitability (Boroojerdi et al. 2001; Gerdelat-Mas et al. 2005; Ili´c et al. 2002, 2003; Plewnia et al. 2001, 2002). Conversely, the decreased SICI with orthostatic stress is in contrast with the increased SICI due to the administration of dopamine agonists (Korchounov et al. 2007; Ziemann et al. 1996a, 1997), which have been shown to decrease corticospinal excitability (Korchounov et al. 2007). Hence, although speculative, baroreceptor unloading due to orthostatic stress may inhibit intracortical inhibitory GABAAergic activity possibly because of facilitation of noradrenergic and potentially serotonergic functions overriding the dopaminergic functions on GABAAergic activity. The delineation of the actual neural mechanisms may be examined in future studies in combination with pharmacological agents for these monoamine functions. Collectively, baroreceptor unloading induced by orthostatic stress decreases the intracortical GABAAergic pathway in the resting hand muscle, probably by altering intracortical neuromodulatory activity and perhaps somatosensory afferent input. The present findings may be further integrated into exploration of the potential mechanisms for increased corticospinal excitability with baroreceptor unloading only at higher TMS intensities (130% resting motor threshold and greater) (Buharin et al. 2013). Such higher TMS intensity is necessary to produce descending volleys with 2-ms delay and longer (later I waves) (Di Lazzaro et al. 1998a). As judged from the decrease in SICI with orthostatic stress, baroreceptor unloading may decrease the activity of intracortical inhibitory GABAAergic pathway that inhibits later I waves 2 ms after excitation (Di Lazzaro et al. 1998b), and thus allow the excitatory later I wave-generating interneurons to generate greater descending corticospinal activity. The present decrease in SICI and previous increase in corticospinal excitability due to orthostatic stress have important implications for motor control and neural plasticity during situations of baroreceptor unloading (e.g., sudden change to an upright posture, increased peripheral perfusion during exercise, jet fighter pilot in combat, blood loss). Since a decrease in SICI of resting muscle has been observed to precede muscle activation and movement initiation (Reynolds and Ashby 1999), baroreceptor unloading may potentially lead to quicker movement initiation and to unintended movement. Intracortical GABAAergic inhibition contributes to performance on the no-go task (Coxon et al. 2006), decreasing corticospinal excitability (Coxon et al. 2006; Leocani et al. 2000). Decreased intracortical inhibition and increased corticospinal excitability due to baroreceptor unloading may impair volitional inhibition of prepared action in “finger on the trigger” situations, leading to execution of the prepared movement at an inappropriate time (e.g., false start during a game of American football, premature gun firing during combat). Since intracortical GABAergic
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ms suggests that baroreceptor unloading due to orthostatic stress likely resulted in decreased excitability of the intracortical inhibitory GABAAergic pathway (Ziemann et al. 1996b), which would lead to less inhibition of later I waves (Di Lazzaro et al. 1998b). In contrast to the resting muscle, there was no significant effect of Day on the measures of intracortical inhibitory pathways in the Active stage. SICI was used to assess GABAAergic activity, and CSP with low- and high-intensity TMS was used to assess GABAAergic and GABABergic activities, respectively (Kimiskidis et al. 2006). The absence of a significant effect of Day on CSP indicates that baroreceptor unloading induced by orthostatic stress did not influence GABAAergic or GABABergic activity during muscle contraction. While the SICI protocol that previously resulted in ⬃38% inhibition during 10% EMGmax contraction (Ortu et al. 2008) was followed, we did not observe inhibition during the Active stage. It is possible that peculiarities of the experimental setup, such as lack of vision of the hand that influences somatosensory intracortical inhibition (Cardini et al. 2011), may have influenced these measures of intracortical inhibition in the contracting muscle. For the measures of excitatory pathways, ICF was used to assess the intracortical excitatory NMDAergic pathway that is stimulated by glutamate (Schwenkreis et al. 1999; Ziemann et al. 1998), and SICF in the present protocol tested the activity of a portion of the intracortical pathway implicated in generating later I waves (Hanajima et al. 2002). The absence of a significant effect of Day on ICF and SICF suggests that baroreceptor unloading induced by orthostatic stress did not influence the intracortical excitatory NMDAergic and later I wave-generating pathways. It is difficult to contemplate the mechanisms responsible for the reduction in SICI during baroreceptor unloading for a resting hand muscle. Based on the literature, the decreased SICI due to orthostatic stress may involve the potential influences of baroreflex processes on 1) somatosensory afferent input and/or 2) cortical monoamines (i.e., norepinephrine, dopamine, serotonin). For the somatosensory afferent input, electrical stimulation over motor axons (Devanne et al. 2009; Stefan et al. 2002) and electrical stimulation of digits (McDonnell et al. 2007; Ridding et al. 2005; Ridding and Rothwell 1999) can decrease SICI. However, the decrease in SICI is abolished if the interval between digit stimulation and TMS is altered (Kobayashi et al. 2003). Hence, the potential effect of somatosensory afferent input on SICI is not robust. Furthermore, there is controversy over whether physiologically heightened sympathetic nerve activity can modulate somatosensory afferent input. While no influence of sympathetic nerve activity on muscle spindle discharge is observed in humans (Macefield et al. 2003), an increased stretch reflex (Hjortskov et al. 2005; Kamibayashi et al. 2009) in the presence of an unaltered H reflex (Kamibayashi et al. 2009) due to physiological heightening of sympathetic nerve activity suggests an increase in gain of the somatosensory afferent input due to sympathetic nerve activity. However, the impact of this possible increase in gain of the somatosensory afferent input in the resting first dorsal interosseus is questionable considering that the corticospinal excitability is not increased with LBNP through TMS intensity of 120% resting motor threshold in the resting first dorsal interosseus (Buharin et al. 2013).
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activity appears to exert inhibitory control over practice-dependent plasticity (Butefisch et al. 2000; Teo et al. 2009; Ziemann et al. 2001), decreased intracortical inhibition during orthostatic stress may promote practice-dependent plasticity. In conclusion, baroreceptor unloading induced by orthostatic stress decreased SICI, and did not affect SICF or ICF, in a resting hand muscle. In an active muscle, orthostatic stress had no effect on CSP, SICF, or SICI. These findings suggest that baroreceptor unloading due to orthostatic stress diminishes intracortical inhibition, at least in the resting muscle. ACKNOWLEDGMENTS
GRANTS The study was supported, in part, by a U.S. Department of Veterans Affairs (VA) Pilot Grant (1 I01 RX000421-01). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: V.E.B., A.J.B., and M.S. conception and design of research; V.E.B., A.J.B., and M.S. performed experiments; V.E.B. analyzed data; V.E.B., A.J.B., and M.S. interpreted results of experiments; V.E.B. prepared figures; V.E.B. drafted manuscript; V.E.B., A.J.B., and M.S. edited and revised manuscript; V.E.B., A.J.B., and M.S. approved final version of manuscript. REFERENCES Berne C, Fagius J, Niklasson F. Sympathetic response to oral carbohydrate administration. Evidence from microelectrode nerve recordings. J Clin Invest 84: 1403–1409, 1989. Boroojerdi B, Battaglia F, Muellbacher W, Cohen LG. Mechanisms influencing stimulus-response properties of the human corticospinal system. Clin Neurophysiol 112: 931–937, 2001. Brasil-Neto JP, Cohen LG, Panizza M, Nilsson J, Roth BJ, Hallett M. Optimal focal transcranial magnetic activation of the human motor cortex: effects of coil orientation, shape of the induced current pulse, and stimulus intensity. J Clin Neurophysiol 9: 132–136, 1992. Buharin VE, Butler AJ, Rajendra JK, Shinohara M. Enhanced corticospinal excitability with physiologically heightened sympathetic nerve activity. J Appl Physiol 114: 429 – 435, 2013. Butefisch CM, Davis BC, Wise SP, Sawaki L, Kopylev L, Classen J, Cohen LG. Mechanisms of use-dependent plasticity in the human motor cortex. Proc Natl Acad Sci USA 97: 3661–3665, 2000. Cardini F, Longo MR, Haggard P. Vision of the body modulates somatosensory intracortical inhibition. Cereb Cortex 21: 2014 –2022, 2011. Clement HW, Gemsa D, Wesemann W. The effect of adrenergic drugs on serotonin metabolism in the nucleus raphe dorsalis of the rat, studied by in vivo voltammetry. Eur J Pharmacol 217: 43– 48, 1992. Cooke WH, Ryan KL, Convertino VA. Lower body negative pressure as a model to study progression to acute hemorrhagic shock in humans. J Appl Physiol 96: 1249 –1261, 2004. Coxon JP, Stinear CM, Byblow WD. Intracortical inhibition during volitional inhibition of prepared action. J Neurophysiol 95: 3371–3383, 2006. Darling WG, Wolf SL, Butler AJ. Variability of motor potentials evoked by transcranial magnetic stimulation depends on muscle activation. Exp Brain Res 174: 376 –385, 2006. Davy KP, Seals DR, Tanaka H. Augmented cardiopulmonary and integrative sympathetic baroreflexes but attenuated peripheral vasoconstriction with age. Hypertension 32: 298 –304, 1998.
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The authors thank Dr. Douglas R. Seals (University of Colorado at Boulder) for providing the LBNP equipment, Drs. Paul Fadel (University of Missouri) and Shigehiko Ogoh (Toyo University) for advice on the LBNP procedure, Justin K. Rajendra for advice on automating the TMS procedure, and Dr. Teresa Snow for statistical advice.
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Motor cortical disinhibition with baroreceptor unloading induced by orthostatic stress Vasiliy E. Buharin,1 Andrew J. Butler,1,2,3 and Minoru Shinohara1,3 1 School of Applied Physiology, Georgia Institute of Technology, Atlanta, Georgia; 2Department of Physical Therapy, Georgia State University, Atlanta, Georgia; and 3Rehabilitation R&D Center of Excellence, Atlanta Department of Veterans Affairs Medical Center, Decatur, Georgia
Submitted 30 October 2013; accepted in final form 21 March 2014
transcranial magnetic stimulation; motor cortex; autonomic nervous system
is a measure of the excitability of the excitatory and inhibitory interneurons within the motor cortex, which influence fine motor skill and neural plasticity. Interventions that can alter intracortical excitability are useful for neurophysiological research on motor control and for neurological treatment of individuals with movement disorders. Aside from being altered artificially by applications of exogenous neuromodulatory agents (e.g., for dopamine and norepinephrine; Ziemann 2003), intracortical excitability is physiologically modulated with hypoxia (Szubski et al. 2006), high altitude exposure (Miscio et al. 2009), and circadian rhythm (Lang et al. 2011). Since these physiological conditions commonly influence autonomic nervous activity, it is possible that intracortical excitability is modified with the neurophysiological processes involved in the control of autonomic nervous activity. In human studies, orthostatic stress by the lower body negative pressure (LBNP) procedure has been applied for enhancing sympathetic nerve activity through baroreceptor unloading, as a human model of acute central hypovolemia
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Address for reprint requests and other correspondence: M. Shinohara, School of Applied Physiology, Georgia Inst. of Technology, 555 14th St. NW, Atlanta, GA 30332-0356 (e-mail: [email protected]). 2656
(Cooke et al. 2004; Davy et al. 1998). Our recent data show that corticospinal excitability for a resting muscle increases with LBNP (Buharin et al. 2013). However, the effects of orthostatic stress and subsequent baroreceptor unloading on intracortical excitatory and inhibitory pathways within the motor cortex in resting or contracting muscle are unknown. There is substantial indirect evidence that suggests that orthostatic stress and subsequent baroreceptor unloading may influence intracortical excitability because of potential increases in the function of neuromodulatory monoamines in the motor cortex (Clement et al. 1992; Devoto et al. 2005; Foote and Morrison 1987; Kaehler et al. 2000; Singewald and Philippu 1993) and the amount of somatosensory afferent input (Hjortskov et al. 2005; Kamibayashi et al. 2009). The purpose of this study was to examine the effects of orthostatic stress on intracortical excitatory and inhibitory pathways in the motor cortex for resting and contracting muscle. The potential intracortical pathways influenced by orthostatic stress may be inferred from previous findings demonstrating that increases in corticospinal excitability with orthostatic stress are observed only at higher intensities of transcranial magnetic stimulation (TMS) (Buharin et al. 2013). Since TMS of higher intensity recruits interneurons that generate later I waves (i.e., I-2 and I-3 waves) in the motor cortex (Di Lazzaro et al. 1998a), greater contribution of later I waves may lead to increased corticospinal excitability with orthostatic stress. Contribution of later I waves to corticospinal excitability can be increased by 1) increasing the activity of the intracortical excitatory glutamatergic pathway responsible for later I-wave generation or by 2) decreasing the activity of the intracortical inhibitory GABAAergic pathway that inhibits later I waves (Di Lazzaro et al. 1998b; Hanajima et al. 2002). In the present study, it was hypothesized that orthostatic stress for baroreceptor unloading would increase the activity of the intracortical excitatory glutamatergic pathway and/or decrease the activity of the intracortical inhibitory GABAAergic pathway in resting and contracting muscle. MATERIALS AND METHODS
Subjects. Sixteen healthy adults (19.8 ⫾ 1.5 yr of age; 6 women, 10 men) participated in the study on 2 days separated by ⬃4 wk. Women were tested during their follicular phase to avoid potential confounding effects of estrogen and progesterone (Minson et al. 2000). All subjects were right-handed, as confirmed with the Edinburgh handedness inventory (laterality quotient: 0.82 ⫾ 0.155) (Oldfield 1971). Volunteers reported no symptoms of chronically altered sympathetic nerve activity: no history of diabetes, cardiovascular problems, brain or nerve disorder, obesity, hypertension, or hypotension (Landsberg www.jn.org
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Buharin VE, Butler AJ, Shinohara M. Motor cortical disinhibition with baroreceptor unloading induced by orthostatic stress. J Neurophysiol 111: 2656 –2664, 2014. First published March 26, 2014; doi:10.1152/jn.00778.2013.—Unloading of the baroreceptors due to orthostatic stress increases corticospinal excitability. The purpose of this study was to examine the effects of baroreceptor unloading due to orthostatic stress on intracortical excitatory and inhibitory pathways in the motor cortex. With transcranial magnetic stimulation, measures of intracortical excitability for a hand muscle were tested on 2 days in healthy young adults. Lower body negative pressure (LBNP) of 40 mmHg was applied during one of the days and not during the Control day. During application of LBNP heart rate and the low-frequency component of heart rate variability increased, while mean arterial blood pressure was maintained. In the resting state, LBNP decreased short-interval intracortical inhibition (SICI) and had no effect on intracortical facilitation (ICF) or short-interval intracortical facilitation (SICF) compared with the Control day. During isometric contraction, no effects of LBNP were observed on tested measures of intracortical excitability including SICI, SICF, and cortical silent period. It was concluded that baroreceptor unloading due to orthostatic stress results in diminished intracortical inhibition, at least in the resting muscle.
INTRACORTICAL EXCITABILITY AND BAROREFLEX
Subjects did not have vision of their hands because of obstruction by the LBNP chamber. The experiment was conducted in an electrically shielded room. Surface electromyogram (EMG) was recorded with Ag-AgCl electrodes (E224A, IVM, Healdsburg, CA) placed on the skin overlying the right first dorsal interosseus in a belly-tendon montage (Buharin et al. 2013). One electrode was placed over the belly of the muscle, and the other was attached to the skin over the distal tendon after abrasion of the skin. A wet circumferential strap electrode (F-E10SG1, Grass Technologies, West Warwick, RI) was placed around the right wrist for a reference. EMG was differentially amplified 300 times and band-pass filtered between 15 and 2,000 Hz (Y03-000, MotionLabs). EMG data were sampled at 5,000 samples/s with an analog-to-digital converter (Power 1401, Cambridge Electronic Design). Contraction intensity was determined based on the EMG amplitude of the first dorsal interosseus during maximal voluntary isometric contraction. A rectified running average EMG with an averaging window of 0.175 s was used to provide visual feedback to subjects and to calculate the maximal EMG (EMGmax) of the muscle. With their right hand secured in the hand brace, subjects increased their EMG amplitude to maximum in a ramp fashion over 3 s and maintained it at maximum for 2 s before relaxing. Verbal instruction and encouragement were provided while the right hand of subjects was visually monitored. TMS intensity was adjusted based on MEP in the first dorsal interosseus. A figure-of-eight coil (Magstim second-generation double 70-mm remote coil, Magstim) was held over the left primary motor cortex at the optimum position (i.e., hot spot) for eliciting an MEP in the resting muscle of the right hand (Buharin et al. 2013). The coil was held with the handle pointing posteriorly at an angle of ⬃45° to the sagittal plane, yielding an E field perpendicular to the central sulcus (Brasil-Neto et al. 1992). A TMS coil navigation system (NDI TMS Manager, Northern Digital, Waterloo, ON, Canada) was used to maintain the coil position in three-dimensional space relative to the head. Continuous running visual feedback of the EMG was provided to the subject and experimenter to ensure relaxation or appropriate activation of the muscle when necessary. The resting motor threshold was determined as the smallest TMS intensity needed to elicit an MEP with peak-to-peak amplitude (PPamp) ⬎ 50 V in 5 of 10 consecutive stimulations in the relaxed muscle (Darling et al. 2006). Active motor threshold was assessed during the isometric contraction at 10% EMGmax (Ortu et al. 2008). Because of the difficulty in differentiating between MEP and background contraction EMG in single stimulation traces, the active motor threshold was defined as the largest TMS intensity that produced an EMG response ⬍50 V above background EMG activity in the triggered average of 10 consecutive rectified stimulation traces (Ortu et al. 2008). TMS paradigms were chosen to test whether orthostatic stress would increase the activity of the intracortical excitatory glutamatergic pathway and/or decrease the activity of the intracortical inhibitory GABAAergic pathway. During the Resting stage, activity of the intracortical later I-wave, GABAAergic, and NMDAergic pathways in the resting muscle were assessed with the paired-pulse TMS paradigms for short-interval intracortical facilitation (SICF) (Hanajima et al. 2002), short-interval intracortical inhibition (SICI) (Ziemann et al. 1996b), and intracortical facilitation (ICF) (Schwenkreis et al. 1999; Ziemann et al. 1998), respectively (Fig. 1). The test stimulus for the paired-pulse stimulation in the Resting stage was determined as the TMS intensity that produced an average MEP with PPamp of 1 mV in 10 consecutive stimulations in the relaxed muscle (Ortu et al. 2008). SICF was measured by delivering the TMS pulse at 0.9 ⫻ active motor threshold 1.5 ms after the suprathreshold test stimulus pulse (Ortu et al. 2008). For SICI and ICF, a subthreshold TMS pulse at 0.9 ⫻ active motor threshold was followed by the suprathreshold test stimulus pulse with an interval of 2 and 10 ms, respectively (Kujirai et al. 1993; Ortu et al. 2008). An interstimulus interval of 2 ms was
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1986). On the testing days, heart rate and blood pressure in the resting state were confirmed to be normal. They did not perform extensive hand grip activity, exhibit skilled use of hands, report arthritis of the hands, or take any medication that may affect motor control or brain and nerve function. In addition, subjects were excluded if they had a family history of seizure or epilepsy, had skin allergies, were pregnant, were prone to severe headaches, or had metal in their head, excluding dental fillings (Rossi et al. 2011). To minimize the variability in the basal physiological level and responsiveness of sympathetic nerve activity across subjects, all experiments were conducted at 8 AM; participants abstained from food and drink, with the exception of water, for 10 h prior to the experiment (Berne et al. 1989). All subjects gave written informed consent. This study was approved by the Georgia Institute of Technology and Emory University Institutional Review Boards. Experimental approach. Intracortical excitability was assessed with TMS delivered over the motor representation area of the first dorsal interosseus muscle. Motor evoked potentials (MEPs) were recorded from the muscle in the right hand with orthostatic stress on the LBNP day and without orthostatic stress on the Control day. The experimental protocol of each subject was temporally comparable between the two days. On a given day, intracortical excitability was assessed at rest (Resting stage), followed by contraction of the first dorsal interosseus (Active stage) with a 10-min intermission (Intermission stage) in between. The LBNP vacuum was turned off during the Intermission stage. Effects of LBNP on intracortical excitability were interpreted from the differences of corresponding measures between the Control day and the LBNP day. The order of the two days was randomized across subjects. LBNP. The LBNP procedure in our previous study (Buharin et al. 2013) was employed to apply orthostatic stress and unload the baroreceptors. The participants lay supine with their lower body inside the LBNP chamber (1.2 m ⫻ 0.6 m ⫻ 0.5 m). Subjects wore a neoprene belt about their hips at the level of the iliac crest. An airtight flexible nylon cover was fit over the opening of the LBNP chamber to form a seal between the chamber and the belt. A commercial vacuum (model 3Z708B, Dayton Industrial, Dayton, OH) attached to the chamber was used to lower the pressure inside the chamber. This setup has been used repeatedly in cardiovascular studies by others (e.g., Davy et al. 1998) and in our previous study (Buharin et al. 2013). On the LBNP day, orthostatic stress was applied to unload aortic and carotid baroreceptors by gradually reducing the pressure in the chamber to ⫺40 mmHg relative to ambient pressure in 20 s. MEP testing commenced 30 s after application of LBNP. On the Control day, the vacuum remained on and the pressure was set to 0 mmHg during the recording of MEPs. LBNP of 40 mmHg is known to physiologically unload the baroreceptors, heighten sympathetic nerve activity, and increase heart rate and low-frequency content of heart rate variability (HRVLF), with little change in blood pressure (Buharin et al. 2013; Lee et al. 2004). Blood pressure at the brachial artery in the left arm and heart rate at the fingertip were monitored noninvasively (Cardiocap/5, GE Healthcare, Chalfont St. Giles, UK), and mean arterial blood pressure was recorded in each TMS block. Electrocardiogram (ECG; Cardiocap/5, GE Healthcare) was sampled at 10,000 samples/s with an analog-to-digital converter (Power 1401, Cambridge Electronic Design, Cambridge, UK) and data acquisition software (Spike2 v7.0, Cambridge Electronic Design). ECG was not obtained in one subject because of technical issues. The cardiovascular data were recorded during TMS as well as before delivery of TMS (Baseline stage) and during the Intermission stage. Intracortical excitability. Intracortical excitability was assessed with single- and paired-pulse TMS (Magstim 2002, Magstim, Whitland, UK) of the left primary motor cortex. The head was oriented in neutral position on a pillow. The arms of each subject lay at his/her sides with the right hand in pronation, in a wooden brace when producing isometric contractions or resting on the bed when not.
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SICF
SICI
ICF
1 mV
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50 ms
used for SICI assessment to avoid contamination of SICI measurement by SICF peaks (Peurala et al. 2008). With this set of stimulation intensity and interval combinations, MEPs in response to test stimulation, SICF, SICI, and ICF were collected. TMS was delivered every 6 s. Seventeen MEPs were collected for each TMS paradigm in the Resting stage. Because of technical issues, SICF was not measured in one subject. During the Active stage, the primary variables of interest were activity of the intracortical GABAAergic and GABABergic pathways, assessed with cortical silent period (CSP) in the contracting muscle. CSP was measured during isometric contraction at 50% EMGmax to minimize variability of CSP due to the contraction strategy of subjects (Mathis et al. 1998) and to delineate potential effects of orthostatic stress on intracortical GABAAergic and GABABergic pathways (Kimiskidis et al. 2006). Subjects were instructed to ramp up to the 50% EMGmax contraction, to maintain the contraction through the ensuing stimulation, and to relax only when told to do so, at least 1 s after TMS (Mathis et al. 1998). Data from subjects who failed to follow this instruction were removed from analysis of 50% EMGmax data (2 subjects). CSP was measured in response to TMS at resting motor threshold (low-intensity TMS) and TMS at 2 ⫻ resting motor threshold (high-intensity TMS), separately, because the response to lowand high-intensity TMS is indicative of GABAAergic and GABABergic activity, respectively (Kimiskidis et al. 2006). For those subjects whose 2 ⫻ resting motor threshold was greater than the maximal stimulator output (n ⫽ 4), TMS at the maximal stimulator output was used to assess CSP with high-intensity TMS. TMS was delivered every 15 s. CSPs with low- and high-intensity TMS were collected in this order, and 12 responses were collected for each. Additionally, activity of the later I-wave and GABAAergic pathways of the active first dorsal interosseus were investigated with the paired-pulse protocols for SICF and SICI during isometric contraction at 10% EMGmax. While there was a concern that SICI may not be induced in contracting muscles (Ridding et al. 1995), SICI was
measured following the protocol of Ortu et al. (2008) that has observed SICI of 38% during isometric contraction with the first dorsal interosseus at 10% EMGmax. Note that activity of the intracortical NMDAergic pathway cannot be measured during muscle contraction because of the disappearance of ICF (Ortu et al. 2008). The test stimulus for paired-pulse stimulation in the Active stage was determined as the TMS intensity that produced a peak amplitude between 0.5 mV and 1 mV in the average of 10 consecutive rectified stimulation traces during 10% EMGmax contraction (Ortu et al. 2008). SICF was measured by delivering the TMS pulse at 0.9 ⫻ active motor threshold 1.5 ms after the suprathreshold test pulse (Ortu et al. 2008). For SICI, a subthreshold TMS pulse at 0.7 ⫻ active motor threshold was followed by a suprathreshold test stimulus pulse at 2 ms. The 2-ms interstimulus interval and conditioning stimulation were chosen to best analyze SICI without interference from SICF during voluntary contraction (Ortu et al. 2008; Peurala et al. 2008). Response to single-pulse TMS at 0.9 ⫻ active motor threshold was also measured to check whether the conditioning stimulations produced an MEP. MEPs in response to test stimulation were collected first, followed by SICF, SICI, and single-pulse TMS at 0.9 ⫻ active motor threshold, in random order, during 10% EMGmax contraction. TMS was delivered every 6 s, and subjects maintained the contraction level for the duration of each TMS paradigm block. Twelve MEP responses were collected for each TMS paradigm. Data reduction. The first two recordings in each TMS paradigm were discarded to control for possible startle responses. In the Resting stage, EMG recordings that showed prestimulus EMG activity above baseline 100 ms preceding TMS were discarded. The root mean square amplitude of the prestimulus EMG, the MEP PPamp, and the MEP area bounded by the MEP and the 0-mV axis (MEP area) were calculated for each response. The amplitude of the prestimulus EMG was calculated from data 100 ms preceding the application of TMS. The EMG in the period between 20 and 50 ms after application of TMS was used to measure MEP PPamp and MEP area. MEP PPamp
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Fig. 1. Representative recordings of motor evoked potentials (MEPs) in response to transcranial magnetic stimulation (TMS) recorded from the first dorsal interosseus muscle in 1 subject on the Control day. Traces represent different TMS protocols (top), a single MEP in the Resting stage (middle), and an average of 10 MEPs in the Active stage (bottom) without lower body negative pressure (LBNP). Test, single-pulse test stimulation; SICF, short-interval intracortical facilitation; SICI, short-interval intracortical inhibition; ICF, intracortical facilitation. Vertical scale is for middle and bottom rows.
Resting
TMS
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INTRACORTICAL EXCITABILITY AND BAROREFLEX
Unless otherwise stated, data are presented as means ⫾ SD in the text and tables and as means ⫾ SE in the figures. RESULTS
Subject characteristics. Basic subject characteristics, including EMGmax, resting motor threshold, active motor threshold, and test stimulation MEP and intensity for Resting and Active stages were not different between the LBNP and Control days (Table 1). No effects of day order were observed for these variables. Cardiovascular measurements. No effects of day order were observed for heart rate, HRVLF, or mean arterial blood pressure. With the application of 40 mmHg LBNP on the LBNP day, both heart rate and HRVLF were increased while mean arterial blood pressure was not significantly different compared with the Control day (Fig. 2). There were main effects of Day (P ⬍ 0.01), Stage (P ⬍ 0.01), and their interaction (P ⬍ 0.01) on heart rate. Heart rate was greater on the LBNP day when 40 mmHg LBNP was applied in Resting [by 11 beats per min (bpm), P ⬍ 0.01] and Active (by 12 bpm, P ⬍ 0.01) stages, respectively, compared with the Baseline and Intermission stages of the same day. Main effects of Day (P ⬍ 0.05), Stage (P ⬍ 0.05), and their interaction (P ⬍ 0.01) were detected for HRVLF. HRVLF was greater with the application of LBNP on the LBNP day by 48% in the Resting stage (P ⬍ 0.01) and by 40% in the Active stage (P ⬍ 0.01) compared with the Baseline and Intermission stages of the same day. There was no significant difference in heart rate or HRVLF between days during the Baseline or Intermission stage. There was only a main effect of Stage (P ⬍ 0.05) on mean arterial blood pressure. Mean arterial blood pressure was slightly greater at the end of data collection (Active stage, 88.6 ⫾ 7.3 mmHg when collapsed across days) compared with the beginning of data collection (Baseline stage, 86.1 ⫾ 6.8 mmHg, P ⬍ 0.05). Intracortical excitability in the Resting stage. No significant effects of day order were observed for any of the intracortical excitability measures in the Resting stage. Root mean square amplitude of the prestimulus EMG was not significantly difTable 1. Neuromuscular characteristics in first dorsal interosseus muscle used for TMS protocol on LBNP and Control days LBNP EMGmax, mV 0.95 ⫾ 0.32 RMT, %* 44.3 ⫾ 7.61 AMT, %* 32.7 ⫾ 8.09 Test stimulation during Resting stage TMS intensity, %* 55.6 ⫾ 12.52 TMS intensity, % RMT 122.5 ⫾ 11.23 MEP PPamp, mV 1.0 ⫾ 0.36 MEP area, mV·ms 3.4 ⫾ 1.31 Test stimulation during Active stage TMS intensity, %* 39.8 ⫾ 8.64 MEP peak, mV 0.9 ⫾ 0.26 MEP area, mV·ms 7.2 ⫾ 2.30
Control 0.95 ⫾ 0.29 44.7 ⫾ 7.15 32.4 ⫾ 5.75 53.9 ⫾ 10.04 118.7 ⫾ 9.44 1.2 ⫾ 0.37 4.7 ⫾ 2.00 38.5 ⫾ 7.93 0.8 ⫾ 0.31 7.2 ⫾ 2.69
Values are means ⫾ SD. TMS, transcranial magnetic stimulation; LBNP, lower body negative pressure; EMGmax, electromyogram amplitude during maximal voluntary contraction; RMT, resting motor threshold; AMT, active motor threshold; MEP, motor evoked potential; PPamp, peak-to-peak amplitude. *Expressed relative to maximal stimulator output. No significant difference observed between LBNP and Control days.
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and area of each response were averaged together within each paradigm block and expressed relative to the corresponding values for test stimulation. Effects of orthostatic stress on intracortical excitability were judged from differences in MEP ratios that were calculated from the measurements made on the same date. During the Active stage, collected recordings were inspected for comparable muscle activation. At each contraction level, those recordings whose amplitude of the prestimulus EMG fell 2 standard deviations outside of the mean were discarded. The recordings collected during subthreshold TMS at 0.9 ⫻ active motor threshold were averaged together. To observe whether subthreshold TMS produced an MEP during 10% EMGmax contraction, the PPamp of the prestimulus EMG and the PPamp between 20 and 50 ms after TMS were calculated. PPamp was chosen 1) to take into account possible fluctuations in the prestimulus EMG and 2) to not underestimate the MEP response. All other recordings were rectified and averaged together within each paradigm block. The automated cumulative sum method was used to measure the CSP objectively (King et al. 2006). The start and end of the CSP were defined as the times, after the MEP, when the average rectified EMG fell below and increased back up to the prestimulus EMG level, respectively. Each CSP was expressed in milliseconds and, in addition, normalized to the peak amplitude and area of the accompanying MEP (Orth and Rothwell 2004). The normalized value for CSP with high-intensity TMS was not available in one subject because of a technical issue in the EMG gain for measuring MEP size. The MEP peak amplitude and MEP area of paired-pulse stimulations were calculated and expressed relative to the corresponding values for test stimulation. As measures of sympathetic nerve activity, heart rate and heart rate variability were assessed from the ECG recordings taken during Baseline, Resting, Intermission, and Active stages. All ECG recordings used in calculation of heart rate variability were ⬎2 min in duration. From the ECG recording, all R-wave peaks were identified, marked, and visually inspected to rule out artifacts. Then the power spectrum of the R-to-R interval was calculated. HRVLF was defined as low-frequency (0.05– 0.15 Hz) power expressed relative to the power in total frequencies (0.05– 0.50 Hz) and was used as a measure of sympathetic nerve activity (Task Force of European Society of Cardiology and North American Society of Pacing and Electrophysiology 1996). Statistical analysis. In this within-subject study design, the main independent variable was Day (Control vs. LBNP). The main dependent variables for intracortical excitability were the appropriate ratios of MEP PPamp, MEP area, and MEP peak amplitude of SICI, ICF, and SICF to the corresponding values for test stimulation and the absolute and normalized durations of CSP. EMGmax, resting motor threshold, active motor threshold, test stimulation intensity for Resting and Active stages, prestimulus EMG from the Resting stage, and the main dependent variables were individually tested for difference between the two days with a two-tailed paired-samples t-test. To test whether the subthreshold stimulation produced motor activity, PPamp of the unrectified prestimulus EMG was tested against the PPamp within the MEP period of the unrectified response to the subthreshold TMS at 0.9 ⫻ active motor threshold during 10% EMGmax contraction, with a two-tailed paired-samples t-test. Effects of LBNP on heart rate, mean arterial blood pressure, and HRVLF were each tested with a two-factor (day, stage) analysis of variance (ANOVA) with repeated measures, where factor stage had four levels: Baseline, Resting, Intermission, and Active. Significant main effects and interaction of all ANOVAs were further tested with the Bonferroni post hoc test. To test the effects of experiment day order, the data were reorganized such that the independent variable Day reflected either day 1 or day 2 of the experiment, and the same statistical analyses were repeated. An ␣ level of 0.05 was used for all significance testing. If Mauchly’s sphericity test was violated, the Huyn-Feldt adjusted P value was used. P ⬍ 0.05 and P ⬍ 0.01 were noted where appropriate. Statistical analyses were performed with Statistica 9.0 (StatSoft, Tulsa, OK).
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Heart rate (beats min-1)
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**
**
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Fig. 2. Cardiovascular measurements during different stages of the experiment on Control and LBNP days. Filled symbols indicate when sympathetic nerve activity was heightened (Resting and Active stages of the LBNP day only). HRVLF, low-frequency (0.05– 0.15 Hz) power of heart rate variability expressed relative to the power in total frequencies (0.05– 0.50 Hz); Base, Baseline stage; Rest, Resting stage; Inter., Intermission stage. **P ⬍ 0.01 between stages during LBNP day, as tested with Bonferroni post hoc test of significant day ⫻ stage interaction.
ferent between days (LBNP: 4.17 ⫾ 0.61 V, Control: 3.80 ⫾ 0.29 V). The stimulation intensity needed to elicit 1-mV PPamp MEP in the resting muscle did not vary significantly between days (Table 1). MEP PPamp and MEP area during SICI, ICF, and SICF paired-pulse protocols were expressed relative to the MEP PPamp and MEP area during the single-pulse test stimulation, respectively (Fig. 3). During the SICI protocol, there was an
MEP area
Stage 2
*
1
0
SICF
SICI
ICF
Fig. 3. Intracortical excitability in the first dorsal interosseus muscle during the Resting stage on Control and LBNP days. y-Axis shows the MEP peak-to-peak amplitude (top) and MEP area (bottom), in response to paired-pulse stimulation, normalized to the corresponding value during single-pulse test stimulation. Unloading of baroreceptors was performed on the LBNP day. *P ⬍ 0.05 compared with Control in the respective measure.
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**
average inhibition of 54% and 53% of MEP PPamp and MEP area compared with the test stimulation, respectively, on the Control day. This inhibition was reduced with LBNP, resulting in 63% greater MEP PPamp (P ⬍ 0.05) and 60% greater MEP area (P ⬍ 0.05) on the LBNP day compared with the Control day. During the ICF protocol, subjects showed an average facilitation of 15% and 16% of MEP PPamp and MEP area compared with the values for test stimulation, respectively, when collapsed across days. No significant effect of LBNP on these ICF measures was observed. For the SICF protocol, MEP PPamp and MEP area were approximately twofold compared with the values for test stimulation. There was no significant difference in these SICF measures between days. Intracortical excitability in the Active stage. There were no significant effects of day order for the variables in the Active stage. The amplitude of prestimulus EMG did not differ significantly between days (LBNP: 123 ⫾ 44.1 V, Control: 152 ⫾ 71.4 V for 10% EMGmax contraction; LBNP: 474 ⫾ 129.5 V, Control: 473 ⫾ 207.4 V for 50% EMGmax contraction). There was no significant difference in the duration of the CSP for either low- or high-intensity TMS when measured in milliseconds, or when normalized to the corresponding MEP peak or area, between days (Table 2). On
INTRACORTICAL EXCITABILITY AND BAROREFLEX
Control
n
5.77 ⫾ 2.45 40.48 ⫾ 13.75 3.5 ⫾ 20.26 10.9 ⫾ 5.83 1.4 ⫾ 0.66
4.74 ⫾ 2.13 34.80 ⫾ 13.10 58.4 ⫾ 32.36 17.4 ⫾ 18.58 1.9 ⫾ 1.20
14 14 14 14 14
6.10 ⫾ 3.35 50.97 ⫾ 24.89 249.1 ⫾ 57.38 64.8 ⫾ 53.99 6.8 ⫾ 4.76
5.70 ⫾ 2.87 45.95 ⫾ 22.46 236.6 ⫾ 49.03 73.8 ⫾ 92.29 7.7 ⫾ 7.59
13 13 14 13 13
Values are means ⫾ SD for n subjects. Contraction intensity was 50% EMGmax. MEP peak, peak amplitude of average rectified MEP; MEP area, area of average rectified MEP; CSP, cortical silent period. No significant differences observed between days.
average, CSPs with low- and high-intensity TMS were 55.9 ⫾ 26.6 ms and 242.9 ⫾ 52.8 ms, respectively, when collapsed across days. Removal of data of four subjects with high resting motor threshold from the analysis did not influence the statistical results. For the paired-pulse response in the Active stage, there was no significant effect of Day on any tested measures (Fig. 4). When collapsed across days, MEP PPamp and MEP area during the SICF protocol were 2.26 ⫾ 2.03 and 2.05 ⫾ 1.47, respectively. MEP PPamp and MEP area during the SICI protocol were 1.16 ⫾ 1.06 and 1.12 ⫾ 0.78, respectively. To confirm that subthreshold conditioning TMS at 0.9 ⫻ active motor threshold did not produce measurable motor activity, the PPamp of the potential response to the subthreshold TMS was compared with the PPamp of unrectified EMG during the background contraction. The PPamp after the subthreshold TMS (304 ⫾ 143 V) was not significantly different from the PPamp before the subthreshold TMS (338 ⫾ 206 V) during contraction.
5 4
Control LBNP
3 2 1
DISCUSSION 0 5 4
MEP area
The major finding is that SICI in the Resting stage was decreased on the LBNP day when LBNP of 40 mmHg was applied compared with the Control day. No significant differences between days were found in other measures in the Resting stage (SICF and ICF) or in any measures (CSP, SICI, and SICF) in the Active stage. LBNP of 40 mmHg unloads the baroreceptors, thereby heightening sympathetic nerve activity, as evidenced by increases of heart rate (Buharin et al. 2013; Davy et al. 1998) and HRVLF (Lee et al. 2004). In the present study, heart rate and HRVLF were significantly greater on the LBNP day during application of LBNP of 40 mmHg, while mean arterial blood pressure was maintained. The 11–12 bpm increase in heart rate with minimal change in blood pressure due to LBNP of 40 mmHg is comparable to previous studies (Buharin et al. 2013; Davy et al. 1998). These findings support that the present orthostatic stress protocol using LBNP was effective for unloading the baroreceptors and heightening sympathetic nerve activity (Cooke et al. 2004).
3 2 1 0 SICF
SICI
Fig. 4. Intracortical excitability in the first dorsal interosseus muscle during the Active stage on the Control and LBNP days. y-Axis shows the MEP peak-topeak amplitude (top) and MEP area (bottom), in response to paired-pulse stimulation, normalized to the corresponding value during single-pulse test stimulation. Unloading of baroreceptors was performed on the LBNP day. No significant differences were observed between days.
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Low-intensity TMS MEP peak, mV MEP area, mV·ms CSP, ms CSP/MEP peak CSP/MEP area High-intensity TMS MEP peak, mV MEP area, mV·ms CSP, ms CSP/MEP peak CSP/MEP area
LBNP
SICI in the Resting stage decreased on the LBNP day when LBNP of 40 mmHg was applied. SICI represents the inhibition of later I waves (i.e., I-2 and I-3 waves) (Di Lazzaro et al. 1998b) and, at an interstimulus interval of 2 ms, likely reflects the activity of the intracortical inhibitory pathway that modulates the excitability of corticospinal neurons through GABAA receptors (Ziemann et al. 1996b). Several technical considerations ensured the proper measurement of intracortical excitability. Care was taken to ensure that only resting data were analyzed, since SICI is influenced by muscle activation (Ortu et al. 2008; Ridding et al. 1995). The lack of difference in prestimulus EMG activity between days further supports the maintenance of resting status across days. LBNP of 40 mmHg increases corticospinal excitability in the resting muscle when stimulated only at or above 130% resting motor threshold (Buharin et al. 2013). Since the employed intensity of test stimulation was below 130% resting motor threshold and was not different between days, it is likely that the present measurements were made within the range for consistent inputoutput properties of corticospinal neurons. Since the conditioning stimulation was chosen below the active motor threshold and no discernible MEP was produced during the Active stage, the employed conditioning stimulation likely did not produce descending volleys. Additionally, the amount of SICI in the Resting stage on the Control day was comparable to previous reports (Hanajima et al. 2002; Ortu et al. 2008; Ridding et al. 1995). Hence, the decreased SICI in the Resting stage on the LBNP day indicates a decrease in SICI due to orthostatic stress and subsequent baroreceptor unloading, supporting our hypothesis. The decrease in SICI at an interstimulus interval of 2
MEP peak amplitude
Table 2. Measures of corticospinal excitability in first dorsal interosseus muscle during voluntary muscle contraction tested with low- and high-intensity TMS on LBNP and Control days
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The potential influences of baroreflex processes on cortical monoamines affecting SICI are inferred from the following evidence. Baroreceptor unloading may result in increased noradrenergic, dopaminergic, and serotonergic function within the motor cortex. The influences of these neuromodulatory monoamines on SICI are variable. Administration of pharmacological agents that facilitate noradrenergic or serotonergic functions, including noradrenergic agonists (Gilbert et al. 2006; Herwig et al. 2002; Ili´c et al. 2003) and serotonergic agonists (Werhahn et al. 1998), can decrease SICI and, similar to the application of orthostatic stress (Buharin et al. 2013), increase corticospinal excitability (Boroojerdi et al. 2001; Gerdelat-Mas et al. 2005; Ili´c et al. 2002, 2003; Plewnia et al. 2001, 2002). Conversely, the decreased SICI with orthostatic stress is in contrast with the increased SICI due to the administration of dopamine agonists (Korchounov et al. 2007; Ziemann et al. 1996a, 1997), which have been shown to decrease corticospinal excitability (Korchounov et al. 2007). Hence, although speculative, baroreceptor unloading due to orthostatic stress may inhibit intracortical inhibitory GABAAergic activity possibly because of facilitation of noradrenergic and potentially serotonergic functions overriding the dopaminergic functions on GABAAergic activity. The delineation of the actual neural mechanisms may be examined in future studies in combination with pharmacological agents for these monoamine functions. Collectively, baroreceptor unloading induced by orthostatic stress decreases the intracortical GABAAergic pathway in the resting hand muscle, probably by altering intracortical neuromodulatory activity and perhaps somatosensory afferent input. The present findings may be further integrated into exploration of the potential mechanisms for increased corticospinal excitability with baroreceptor unloading only at higher TMS intensities (130% resting motor threshold and greater) (Buharin et al. 2013). Such higher TMS intensity is necessary to produce descending volleys with 2-ms delay and longer (later I waves) (Di Lazzaro et al. 1998a). As judged from the decrease in SICI with orthostatic stress, baroreceptor unloading may decrease the activity of intracortical inhibitory GABAAergic pathway that inhibits later I waves 2 ms after excitation (Di Lazzaro et al. 1998b), and thus allow the excitatory later I wave-generating interneurons to generate greater descending corticospinal activity. The present decrease in SICI and previous increase in corticospinal excitability due to orthostatic stress have important implications for motor control and neural plasticity during situations of baroreceptor unloading (e.g., sudden change to an upright posture, increased peripheral perfusion during exercise, jet fighter pilot in combat, blood loss). Since a decrease in SICI of resting muscle has been observed to precede muscle activation and movement initiation (Reynolds and Ashby 1999), baroreceptor unloading may potentially lead to quicker movement initiation and to unintended movement. Intracortical GABAAergic inhibition contributes to performance on the no-go task (Coxon et al. 2006), decreasing corticospinal excitability (Coxon et al. 2006; Leocani et al. 2000). Decreased intracortical inhibition and increased corticospinal excitability due to baroreceptor unloading may impair volitional inhibition of prepared action in “finger on the trigger” situations, leading to execution of the prepared movement at an inappropriate time (e.g., false start during a game of American football, premature gun firing during combat). Since intracortical GABAergic
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ms suggests that baroreceptor unloading due to orthostatic stress likely resulted in decreased excitability of the intracortical inhibitory GABAAergic pathway (Ziemann et al. 1996b), which would lead to less inhibition of later I waves (Di Lazzaro et al. 1998b). In contrast to the resting muscle, there was no significant effect of Day on the measures of intracortical inhibitory pathways in the Active stage. SICI was used to assess GABAAergic activity, and CSP with low- and high-intensity TMS was used to assess GABAAergic and GABABergic activities, respectively (Kimiskidis et al. 2006). The absence of a significant effect of Day on CSP indicates that baroreceptor unloading induced by orthostatic stress did not influence GABAAergic or GABABergic activity during muscle contraction. While the SICI protocol that previously resulted in ⬃38% inhibition during 10% EMGmax contraction (Ortu et al. 2008) was followed, we did not observe inhibition during the Active stage. It is possible that peculiarities of the experimental setup, such as lack of vision of the hand that influences somatosensory intracortical inhibition (Cardini et al. 2011), may have influenced these measures of intracortical inhibition in the contracting muscle. For the measures of excitatory pathways, ICF was used to assess the intracortical excitatory NMDAergic pathway that is stimulated by glutamate (Schwenkreis et al. 1999; Ziemann et al. 1998), and SICF in the present protocol tested the activity of a portion of the intracortical pathway implicated in generating later I waves (Hanajima et al. 2002). The absence of a significant effect of Day on ICF and SICF suggests that baroreceptor unloading induced by orthostatic stress did not influence the intracortical excitatory NMDAergic and later I wave-generating pathways. It is difficult to contemplate the mechanisms responsible for the reduction in SICI during baroreceptor unloading for a resting hand muscle. Based on the literature, the decreased SICI due to orthostatic stress may involve the potential influences of baroreflex processes on 1) somatosensory afferent input and/or 2) cortical monoamines (i.e., norepinephrine, dopamine, serotonin). For the somatosensory afferent input, electrical stimulation over motor axons (Devanne et al. 2009; Stefan et al. 2002) and electrical stimulation of digits (McDonnell et al. 2007; Ridding et al. 2005; Ridding and Rothwell 1999) can decrease SICI. However, the decrease in SICI is abolished if the interval between digit stimulation and TMS is altered (Kobayashi et al. 2003). Hence, the potential effect of somatosensory afferent input on SICI is not robust. Furthermore, there is controversy over whether physiologically heightened sympathetic nerve activity can modulate somatosensory afferent input. While no influence of sympathetic nerve activity on muscle spindle discharge is observed in humans (Macefield et al. 2003), an increased stretch reflex (Hjortskov et al. 2005; Kamibayashi et al. 2009) in the presence of an unaltered H reflex (Kamibayashi et al. 2009) due to physiological heightening of sympathetic nerve activity suggests an increase in gain of the somatosensory afferent input due to sympathetic nerve activity. However, the impact of this possible increase in gain of the somatosensory afferent input in the resting first dorsal interosseus is questionable considering that the corticospinal excitability is not increased with LBNP through TMS intensity of 120% resting motor threshold in the resting first dorsal interosseus (Buharin et al. 2013).
INTRACORTICAL EXCITABILITY AND BAROREFLEX
activity appears to exert inhibitory control over practice-dependent plasticity (Butefisch et al. 2000; Teo et al. 2009; Ziemann et al. 2001), decreased intracortical inhibition during orthostatic stress may promote practice-dependent plasticity. In conclusion, baroreceptor unloading induced by orthostatic stress decreased SICI, and did not affect SICF or ICF, in a resting hand muscle. In an active muscle, orthostatic stress had no effect on CSP, SICF, or SICI. These findings suggest that baroreceptor unloading due to orthostatic stress diminishes intracortical inhibition, at least in the resting muscle. ACKNOWLEDGMENTS
GRANTS The study was supported, in part, by a U.S. Department of Veterans Affairs (VA) Pilot Grant (1 I01 RX000421-01). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: V.E.B., A.J.B., and M.S. conception and design of research; V.E.B., A.J.B., and M.S. performed experiments; V.E.B. analyzed data; V.E.B., A.J.B., and M.S. interpreted results of experiments; V.E.B. prepared figures; V.E.B. drafted manuscript; V.E.B., A.J.B., and M.S. edited and revised manuscript; V.E.B., A.J.B., and M.S. approved final version of manuscript. REFERENCES Berne C, Fagius J, Niklasson F. Sympathetic response to oral carbohydrate administration. Evidence from microelectrode nerve recordings. J Clin Invest 84: 1403–1409, 1989. Boroojerdi B, Battaglia F, Muellbacher W, Cohen LG. Mechanisms influencing stimulus-response properties of the human corticospinal system. Clin Neurophysiol 112: 931–937, 2001. Brasil-Neto JP, Cohen LG, Panizza M, Nilsson J, Roth BJ, Hallett M. Optimal focal transcranial magnetic activation of the human motor cortex: effects of coil orientation, shape of the induced current pulse, and stimulus intensity. J Clin Neurophysiol 9: 132–136, 1992. Buharin VE, Butler AJ, Rajendra JK, Shinohara M. Enhanced corticospinal excitability with physiologically heightened sympathetic nerve activity. J Appl Physiol 114: 429 – 435, 2013. Butefisch CM, Davis BC, Wise SP, Sawaki L, Kopylev L, Classen J, Cohen LG. Mechanisms of use-dependent plasticity in the human motor cortex. Proc Natl Acad Sci USA 97: 3661–3665, 2000. Cardini F, Longo MR, Haggard P. Vision of the body modulates somatosensory intracortical inhibition. Cereb Cortex 21: 2014 –2022, 2011. Clement HW, Gemsa D, Wesemann W. The effect of adrenergic drugs on serotonin metabolism in the nucleus raphe dorsalis of the rat, studied by in vivo voltammetry. Eur J Pharmacol 217: 43– 48, 1992. Cooke WH, Ryan KL, Convertino VA. Lower body negative pressure as a model to study progression to acute hemorrhagic shock in humans. J Appl Physiol 96: 1249 –1261, 2004. Coxon JP, Stinear CM, Byblow WD. Intracortical inhibition during volitional inhibition of prepared action. J Neurophysiol 95: 3371–3383, 2006. Darling WG, Wolf SL, Butler AJ. Variability of motor potentials evoked by transcranial magnetic stimulation depends on muscle activation. Exp Brain Res 174: 376 –385, 2006. Davy KP, Seals DR, Tanaka H. Augmented cardiopulmonary and integrative sympathetic baroreflexes but attenuated peripheral vasoconstriction with age. Hypertension 32: 298 –304, 1998.
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The authors thank Dr. Douglas R. Seals (University of Colorado at Boulder) for providing the LBNP equipment, Drs. Paul Fadel (University of Missouri) and Shigehiko Ogoh (Toyo University) for advice on the LBNP procedure, Justin K. Rajendra for advice on automating the TMS procedure, and Dr. Teresa Snow for statistical advice.
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