Abstract Lundbye-Jensen J, Nielsen JB. Central nervous adaptations following 1 wk of wrist and hand immobilization. J Appl Physiol 105: 139-151, 2008. First published May 1, 2008 doi:10.1152/japplphysiol.00687.2007.-Plastic neural changes have been documented in relation to different types of physical activity, but little is known about central nervous system plasticity accompanying reduced physical activity and immobilization. In the present study we investigated whether plastic neural changes occur in relation to 1 wk of immobilization of the nondominant wrist and hand and a corresponding period of recovery in 10 able-bodied volunteers. After immobilization, maximal voluntary contraction torque decreased and the variability of submaximal static contractions increased significantly without evidence of changes in muscle contractile properties. Hoffmann (H)-reflex amplitudes and the ratios of H-slope to M-slope increased significantly in flexor carpi radialis and abductor pollicis brevis at rest and during contraction without changes in corticospinal excitability, estimated from motorevoked potentials (MEPs) elicited by transcranial magnetic stimulation. Corticomuscular coherence measures were derived from EEG and EMG obtained during static contractions. After immobilization, corticomuscular coherence in the 15-to 35-Hz range associated with maximum negative cumulant values at lags corresponding to MEP latencies decreased. One week after cast removal, all measurements returned to preimmobilization levels. The increased H-reflex amplitudes without changes in MEPs may suggest that presynaptic inhibition or postactivation depression of Ia afferents is reduced following immobilization. Reduced corticomuscular coherence may be caused by changes in afferent input at spinal and cortical levels or by changes in the descending drive from motor cortex. Further studies are needed to elucidate the mechanisms underlying the observed increased spinal excitability and reduced coupling between motor cortex and spinal motoneuronal activity following immobilization. plasticity; motor control; disuse NUMEROUS STUDIES HAVE DOCUMENTED that periods of bed rest, limb unloading, or joint immobilization all result in significant loss of muscle strength. Some of these studies have associated the observed strength decrements with whole muscle atrophy (1, 6, 7, 24, 39, 54, 59, 65, 83) or changes in myofiber characteristics (7, 39, 41, 42) , indicating that loss of contractile proteins accounts for at least part of the reduction of maximal voluntary contraction strength (MVC). It also has been established that joint immobilization induces several changes in skeletal muscle properties, such as morphology (86), fatigability (77, 95), muscle fiber type (10, 57, 88), and contractility (23, 29, 83) . Abstract For most of the studies in which reduced MVC torque and atrophy have been documented, the period of immobilization has lasted for more than 4 -6 wk, but decrements in MVC also are apparent after shorter periods of immobilization, e.g., 2 wk (9, 59). In this case muscular changes are relatively minor, making it possible that part of the observed reduction in MVC is caused by reduced drive to the muscle from the central nervous system. In line with this, Veldhuizen et al. (90), Thom et al. (87), Kawakami et al. (48), and Clark et al. (17) all found marked discrepancies between reduction of strength and anatomical cross-sectional area after immobilization, with strength loss appearing to be three-to fourfold greater than muscle atrophy. Duchateau and Hainaut (23), Davies et al. (20), and White and Davies (91) also found greater reductions in MVC compared with maximal tetanic force following immobilization. On the basis o twitch interpolation technique measurements, Gondin et al. (33) concluded that immobilization resulted in impaired neural activation of the soleus muscle after 2 wk of ankle joint immobilization, and recently, Seki et al. (82) found maximal motoneuronal firing rates to be reduced following 1 wk of finger immobilization. -Plastic neural changes have been documented in relation to different types of physical activity, but little is known about central nervous system plasticity accompanying reduced physical activity and immobilization. In the present study we investigated whether plastic neural changes occur in relation to 1 wk of immobilization of the nondominant wrist and hand and a corresponding period of recovery in 10 able-bodied volunteers. After immobilization, maximal voluntary contraction torque decreased and the variability of submaximal static contractions increased significantly without evidence of changes in muscle contractile properties. Hoffmann (H)-reflex amplitudes and the ratios of H-slope to M-slope increased significantly in flexor carpi radialis and abductor pollicis brevis at rest and during contraction without changes in corticospinal excitability, estimated from motorevoked potentials (MEPs) elicited by transcranial magnetic stimulation. Corticomuscular coherence measures were derived from EEG and EMG obtained during static contractions. After immobilization, corticomuscular coherence in the 15-to 35-Hz range associated with maximum negative cumulant values at lags corresponding to MEP latencies decreased. One week after cast removal, all measurements returned to preimmobilization levels. The increased H-reflex amplitudes without changes in MEPs may suggest that presynaptic inhibition or postactivation depression of Ia afferents is reduced following immobilization. Reduced corticomuscular coherence may be caused by changes in afferent input at spinal and cortical levels or by changes in the descending drive from motor cortex. Further studies are needed to elucidate the mechanisms underlying the observed increased spinal excitability and reduced coupling between motor cortex and spinal motoneuronal activity following immobilization. plasticity; motor control; disuse NUMEROUS STUDIES HAVE DOCUMENTED that periods of bed rest, limb unloading, or joint immobilization all result in significant loss of muscle strength. Some of these studies have associated the observed strength decrements with whole muscle atrophy (1, 6, 7, 24, 39, 54, 59, 65, 83) or changes in myofiber characteristics (7, 39, 41, 42) , indicating that loss of contractile proteins accounts for at least part of the reduction of maximal voluntary contraction strength (MVC). It also has been established that joint immobilization induces several changes in skeletal muscle properties, such as morphology (86) , fatigability (77, 95) , muscle fiber type (10, 57, 88) , and contractility (23, 29, 83) . For most of the studies in which reduced MVC torque and atrophy have been documented, the period of immobilization has lasted for more than 4 -6 wk, but decrements in MVC also are apparent after shorter periods of immobilization, e.g., 2 wk (9, 59) . In this case muscular changes are relatively minor, making it possible that part of the observed reduction in MVC is caused by reduced drive to the muscle from the central nervous system. In line with this, Veldhuizen et al. (90) , Thom et al. (87) , Kawakami et al. (48) , and Clark et al. (17) all found marked discrepancies between reduction of strength and anatomical cross-sectional area after immobilization, with strength loss appearing to be three-to fourfold greater than muscle atrophy. Duchateau and Hainaut (23) , Davies et al. (20) , and White and Davies (91) also found greater reductions in MVC compared with maximal tetanic force following immobilization. On the basis o twitch interpolation technique measurements, Gondin et al. (33) concluded that immobilization resulted in impaired neural activation of the soleus muscle after 2 wk of ankle joint immobilization, and recently, Seki et al. (82) found maximal motoneuronal firing rates to be reduced following 1 wk of finger immobilization. A great deal of experimental evidence has been gathered on functional and structural neuroplasticity following motor learning, documenting that training is capable of inducing widespread changes in the functional properties of the nervous system at many levels (for reviews, see Refs. 12, 66, and 92) . It is the general notion that plasticity is an intrinsic property that serves to continuously modify the properties of the central nervous system. Neuroplasticity takes place in response to changes in afferent input and/or efferent demand and thereby enables the nervous system to adapt to physiological changes and experience. Plastic changes within the nervous system have been documented to occur rapidly with motor skill training (70 -72) and progress during longer periods of training (see e.g., Ref. 46) . Although decreases in maximal voluntary strength are frequently observed behavioral consequences of immobilization, previous studies focusing on plastic changes in the central nervous system accompanying physical inactivity or immobilization have led to somewhat ambiguous findings. Anderson et al. (2) reported increases in the H-reflex gain following 3 wk of hindlimb unloading in the rat. In humans, Kaneko et al. (47) observed no differences in Hoffmann (H)-reflex parameters following 3-6 wk of arm immobilization, whereas Clark et al. (18) observed increased H-reflex amplitudes at rest following 4 wk of lower limb suspension. In contrast, Yamanaka et al. (93) reported reduced H-reflex amplitudes after 20 days of bed rest, but these measurements were made during standing and thus may be difficult to compare with those from the other studies. Using transcranial magnetic stimulation (TMS), Liepert et al. (55) found a reduced size of motor cortex representational maps following ankle immobilization for 16 wk, and Facchini et al. (26) reported reduced motor-evoked potentials (MEPs) evoked by TMS following finger immobilization for 4 days. In addition, Kaneko et al. (47) found reduced MEP amplitudes during motor imagery following arm immobilization. Contrary to this, Zanette et al. (96, 97) found an increased representation of muscles in the motor cortex following wrist immobilization for 30 -45 days, and recently, Roberts et al. (76) reported increased corticospinal excitability following 10 days of leg immobilization. These differences may be explained by different duration of the immobilization period, differences in immobilization paradigm, differences between the upper and lower limb, and differences in the conditions under which the measurements were obtained. The aim of the present study was to investigate which changes in the central nervous system accompany immobilization in able-bodied subjects. MEPs and H-reflexes were measured to obtain information about the excitability of the corticospinal and monosynaptic Ia-afferent pathways, respectively. In contrast to previous studies, in which measurements were only obtained at rest, we obtained measurements at rest and during submaximal static contractions. Furthermore, corticomuscular coherence was also extracted from electroencephalographic (EEG) and electromyographic (EMG) measurements (19, 27, 81) during submaximal static contractions to obtain information about the coupling between the firing of corticospinal cells and the spinal motoneurons. Corticomuscular coherence is commonly regarded as a measure of the coupling between the frequency content of the corticospinal drive and the firing pattern of the spinal motoneurons (37, 45) , but it also has been shown that corticomuscular coherence may be affected by changes in afferent feedback (75) . Recently, it was demonstrated that measures of corticomuscular coherence increased following visuomotor learning (70) . It is, however, unknown whether corticomuscular coherence changes following a period of limb immobilization in healthy humans. MATERIALS AND METHODS In 10 healthy volunteers (6 male and 4 female; ages 24 Ϯ 6 yr), the nondominant (left) forearm, hand, and fingers were immobilized by application of a cast for 1 wk. All subjects were right-handed according to the Edinburgh Handedness Inventory (64), and no volunteers had any history of neurological disease. Subjects gave informed, written consent to the experiments, which were approved by the local ethics committee (J. No. KF 100.1969/1991). The experiments were performed in accordance with the Helsinki Declaration. Experimental Protocol All subjects participated in four identical experimental sessions on four separate days. To assess day-to-day variability, we obtained measurements on two different days before immobilization (pretests). Experimental Protocol In addition, subjects were tested after 1 wk of immobilization immediately after cast removal (posttest) and once again following 1 wk of recovery after cast removal (retest). Experimental Protocol During all experimental sessions, subjects were seated in a comfortable armchair. Subjects were positioned with the head, torso, and arms supported. The nondominant arm was placed in a molded rigid arm support with the shoulder joint in a neutral position, 90°elbow joint angle, and the forearm was placed in a neutral semiprone position. The forearm was firmly strapped to the arm support proximally and distally (Fig. 1) . The distal part of the arm support consisted of a handle with a rotational axis located coaxially with the axis of rotation of the wrist. The handle had a built-in potentiometer and was connected to a strain gauge transducer providing information on position and torque applied to the handle. The position of each individual was recorded and reestablished in all tests. Maximal Voluntary Contraction In each test, maximal voluntary isometric contraction torque (MVC) was measured for the wrist flexors and extensors, respectively, to evaluate alterations in the maximal strength of the subjects before and after immobilization. Subjects were instructed to perform a maximal contraction of the left arm wrist flexors or extensors by increasing the torque to maximum within a few seconds and to exert maximal torque for 2 s while maintaining the standardized position. Verbal encouragement and visual feedback of the torque exerted were provided. Typically, four or five successive trials were performed until the peak torque did not increase any further. The peak torque recorded in either of the trials was taken as the MVC. During these MVC tests, both torque and EMG measurements from the flexor carpi radialis (FCR), extensor carpi radialis longus (ECR), and abductor pollicis brevis (APB) muscles were obtained. In separate trials, subjects also performed MVCs by exerting maximal thumb abduction torque against the handle. During these measurements, APB EMG was obtained to enable quantification of APB contraction levels during the following electrophysiological testing procedures. After completion of the strength tests, electrophysiological testing procedures involving peripheral electrical nerve stimulation, H-reflexes, TMS, and EEG recordings were performed. Stimulation and Recording EMG recording. Electromyographic activity was recorded from the FCR, ECR, and APB muscles using bipolar Ag-AgCl surface electrodes (0.5-cm-diameter electrodes; Blue Sensor; Ambu, Ølstykke, Denmark) with an interelectrode distance of 2 cm. The amplified EMG signals were filtered (band pass, 25 Hz to 1 kHz), sampled at 2 kHz, and stored on a personal computer (PC) for off-line analysis (CED 1401ϩ with Signal & Spike 2.5 software; Cambridge Electronic Design, Cambridge, UK). Stimulation and Recording Contractile properties. To test changes in muscular contractile properties, we delivered supramaximal electrical stimulation to the median nerve to elicit maximal twitch contractions. Stimulation consisted of a 1-ms rectangular pulse delivered by a constant-current stimulator (model DS7A; Digitimer). The elicited twitch torque was registered, and peak torque, time to peak torque, and half relaxation time were calculated during off-line analysis. Stimulation and Recording H-reflex testing. FCR H-reflexes were evoked by stimulating the median nerve at the elbow through a bipolar electrode placed on the medial aspect of the upper arm just above the elbow joint. The two legs of the electrode had plates that were 0.5 cm in diameter and separated by 2 cm. The cathode was placed proximally to the anode. Stimulation consisted of a 1-ms rectangular pulse delivered by a constant-current stimulator (model DS7A; Digitimer). The interstimulus interval (ISI) was 4 s. Although postactivation (homosynaptic) depression affects stimulus responses at rest at an ISI of 4 s, this ISI was chosen for several reasons. First, postactivation depression is assumed to be relatively constant during this constant ISI. Second, postactivation depression may have an effect lasting up to 15 s. If we were to use ISI Ͼ15 s, the duration of the experiment alone would induce several additional problems. Stimulation and Recording The reflex was measured as peak-to-peak amplitude of the nonrectified reflex response. Stimulus intensities were randomly increased in steps of 0.05 mA, starting below H-reflex threshold (H threshold) and increasing up to supramaximal intensity, at which the maximal compound motor response (Mmax) was measured. Stimulus intensities ranged from 0 to 100 mA. H-reflex recruitment curves were assessed by averaging five responses at each stimulus intensity. This procedure was performed at rest and during contraction. During voluntary static contractions, the torque and full-wave rectified and integrated EMG (iEMG) was displayed as a line on a PC screen in front of the subject. The iEMG was used as visual feedback of the level of the performed contraction during the measurements. Subjects were instructed to match and maintain a contraction (iEMG amplitude) as precisely as possible to a displayed target iEMG amplitude corresponding to 10% of MVC on the day of the test. In four of the subjects, it was possible to elicit H-reflexes in APB by stimulating the median nerve with an identical electrode placed at the wrist with the anode on the dorsum of the wrist and the cathode proximally. In these subjects, APB recruitment curves were generated by applying identical procedures. During static contraction, subjects were asked to match iEMG as precisely as possible to a target level of 10% MVC iEMG on the day of the test. To enable comparison between recruitment curves obtained in different sessions, H-reflex amplitudes were normalized to the corresponding M max and stimulation intensities were normalized to motor threshold (Mthreshold) in pretest 1. Stimulation and Recording Transcranial magnetic stimulation. MEPs were evoked by TMS over the arm area of the right motor cortex at the hot spot for activation of FCR and APB, respectively, by using a magnetic stimulator (Magstim rapid; Magstim Whitland, Dyfed, UK) with the capability to deliver a magnetic field of 2 T for 100 s through the figure-of-eight coil (loop diameter, 9 cm; type no. 8106). The MEPs were recorded from FCR and APB. Cz (vertex) was identified, and a cap with a coordinate system marked on it was placed on the subject's head. The stimulating coil was oriented and positioned with the handle of the coil pointing backwards and with the axis of intersection between the two loops oriented at ϳ45°to the sagittal plane. The hot spots were identified as the coordinates in which TMS at the lowest possible intensity evoked an MEP of 50-V peak-to-peak amplitude in at least three of five consecutive trials (79) . After the hot spot coordinates were established, the coil was secured to ensure that the same area of the cortex was stimulated throughout the experiment. Stimulation and Recording Single pulse stimuli were delivered at an ISI of 4 s. During the experiment, MEPs were displayed and averaged online for visual inspection as well as stored on a computer for off-line analysis. At first, TMS was ronaldramos@example.com. Magnetic stimuli were applied at 10 -15 different stimulation intensities from 0.6 to 2.0 times the minimal stimulation intensity required to elicit MEPs (MEP threshold) with 10 stimulations at each intensity. The sequence of intensities was randomly varied. Responses were measured as the peak-to-peak amplitude and expressed as a percentage of the corresponding M max. For each stimulation intensity, responses were averaged, and the peak-topeak amplitude was plotted until a stimulus response curve with a well-defined MEP threshold, slope, and maximal level (MEPmax) had been obtained. This procedure was performed for both FCR and APB at rest and during static contractions corresponding to 10% of MVC as described for the H-reflex measurements. Stimulation and Recording EEG recordings. Electroencephalographic activity was recorded by a pair of bipolar 5-mm diameter silver cup electrodes. One electrode was placed at the vertex (Cz) and the other 5 cm lateral to and 2 cm in front of Cz. EEG signals were amplified (50,000 times), filtered (1-1,000 Hz), and stored for later analysis. EEG and corresponding EMG were obtained in periods of 2 min during two different tasks: static wrist flexion (FCR) and thumb abduction (APB) corresponding to 10% of MVC as described for the H-reflex and TMS measurements. Again, subjects were instructed to align the EMG signal and the target Fig. 1 . Experimental setup. During experiments, subjects were seated in an armchair with the left arm fixated in an arm support. The manipulandum at the end of the arm support made it possible to register wrist joint position and torque. Transcranial magnetic stimulation (TMS) was delivered to the right hemisphere primary motor cortex, and electroencephalographic (EEG) signals were recorded from electrodes placed on the scalp above the primary motor cortex. Peripheral nerve electrical stimulation was delivered to the median nerve proximal to the elbow and to the wrist, respectively. Electromyographic (EMG) signal was obtained from flexor carpi radials (FCR), extensor carpi radialis (ECR) and abductor pollicis brevis (APB). Stimulation and Recording level as precisely as possible. The subjects' motor performance during the EEG measurements was calculated off-line as the variability (SD) of the displayed and obtained iEMG during the 2-min static contractions (see further details in Data Analysis and Statistics). Immobilization At the end of pretest 2, a custom-molded circular arm cast (X-lite; Camp Scandinavia) was configured for each subject. The subject's arm was covered by a stockinette and foam padding, and the cast was positioned from the proximal point of the forearm, continued along the forearm, and wrapped around the hand and all digits, maintaining the wrist in a neutral slightly extended position and restricting finger movements. Subjects were not able to remove the cast themselves, ensuring that joint immobilization was effective for the full period. Subjects were instructed not to attempt movements of the immobilized joints throughout the immobilization period. After 1 wk of immobilization, the cast was removed by the experimenter immediately before the posttest procedures were begun. Data Analysis and Statistics The data were calculated as means Ϯ SE for all parameters and conditions. Data Analysis and Statistics H-reflex and TMS measurements. H-reflex, M-wave, and TMS recruitment curves were analyzed following similar procedures. The mean amplitude was determined for the responses obtained at each stimulus intensity. All response amplitudes were normalized to the corresponding M max, and all stimulation intensities were normalized to the first Mthreshold pretest for H-reflexes and the first MEPthreshold pretest for MEP data. Stimulus intensity was plotted against response magnitude, and the data of each individual curve were fitted with the following three-parameter sigmoid function: Data Analysis and Statistics where S is stimulus intensity, MEPmax represents the maximum MEP defined by the function, and m is the slope parameter of the function. S 50 is the stimulus intensity at which the MEP amplitude size is 50% of MEPmax. This equation has previously been used to fit TMS recruitment curve data. It is referred to as an analog of the Boltzmann equation with the exception that the slope parameter m is the inverse of the Boltzmann slope parameter k. As such, an increase in slope will represent a larger increment in MEP size per unit of TMS intensity, whereas the opposite is true for the Boltzmann slope parameter (15, 16, 22, 74) . Klimstra and Zehr (52) recently demonstrated that a sigmoid function is the best fit for the ascending limb of the H-reflex recruitment curve, and an identical curve-fitting procedure was performed for the H-reflex and M-wave recruitment curves. Data Analysis and Statistics For all H-reflex, M-wave, and MEP recruitment curves, maximal values were quantified based on the obtained data. Furthermore, every curve was characterized by the slope parameter of the function and the MEP threshold. Based on the sigmoidal fit, the slope was calculated for the steepest part of the curve (i.e., at S50), indicating the maximal increase of MEP, H-reflex, or M-wave amplitude with increasing stimulus intensity. Because the stimulus intensity necessary to elicit a threshold response is not an explicit parameter of the equation and cannot be directly derived, it was quantified as the x-intercept between the baseline activity (SD; linear regression) and the tangent to the function at the point of maximal slope (i.e., at S 50) and the mean baseline (SD) (16) . It has previously been demonstrated for MEP recruitment curves that the sigmoid function parameters can be obtained reliably in testing sessions conducted on different days (15) . In addition to the slope of individual curves, the ratio H slope/Mslope was calculated for each pair of curves to obtain additional information on spinal reflex gain (31). Data Analysis and Statistics Motor performance: variability of static contractions. During static contractions, the ability of the subjects to accurately match the specified target level was quantified as the mean amplitude and variability (SD) of the iEMG signal obtained during the 2-min EEG measurements relative to the target level of 10% MVC iEMG. Analysis of motor performance was performed on the EMG signal displayed to the subjects. The EMG was amplified, filtered (band pass, 25 Hz to 1 kHz), and full-wave rectified. After averaging with a 3rd-order low-pass averaging filter with a 200-ms averaging period (32), the signal was displayed to the subjects as a line on a PC screen. The signal was sampled at 2 kHz and stored on a PC for off-line analysis (CED 1401ϩ with Spike 2.5 software; Cambridge Electronic Design). Data Analysis and Statistics A one-way repeated-measures ANOVA was used to determine the effect of immobilization on MVC torque, MVC EMG, twitch torque, half relaxation time, static contraction variability, M max, Mslope, Hthreshold, Hslope, Hmax, Hslope/Mslope, MEPthreshold, MEPslope, MEPmax, and MEP latencies with time of measurements as a factor. Furthermore, a two-way repeated-measures ANOVA was performed on recruitment curves with stimulation intensity and time of measurement as factors. Tukey's post hoc test was performed on significant comparisons. All values are reported as means (SD) unless stated otherwise. In all tests, statistical significance was assumed if P Ͻ 0.05. Data Analysis and Statistics Coherence. EEG and EMG recordings were analyzed with respect to coherence and cumulant density function. Corticomuscular coherence was analyzed for EEG and FCR and APB EMG, respectively, during isolated static contraction. Data Analysis and Statistics The procedures for calculation of coherence and cumulant density functions between two signals have been described in detail in previous publications (27, 36) and are described only briefly below. Data Analysis and Statistics Briefly, f xx() and fyy() represent the power spectra of processes x and y, respectively. In the frequency domain, the correlation between the EEG and EMG signals is assessed through coherence functions (36) . The coherence function between the two signals is defined at frequency as Data Analysis and Statistics Coherence functions provide normative measures of linear association on a scale from 0 to 1. For the present data, the coherence provides a measure, at each Fourier frequency , of the fraction of the activity in one surface EMG signal that can be predicted by the activity in the EEG signal. In this way, the coherence is used to quantify the strength and frequency of rhythmic synaptic inputs that are distributed across the motoneuron pool (27) . Cumulant density function. In the time domain, the cumulant density function, denoted by q xy(u), is defined as the inverse Fourier transform of the cross spectrum. Estimates of the cumulant density function are used to characterize the correlation between the two signals. Data Analysis and Statistics For two uncorrelated signals, the cumulant has an expected value of zero; deviations from this indicate a correlation between the EEG and EMG signals at a particular time lag u. To summarize the correlation structure across subjects, we added the values for coherence and cumulant density functions for all subjects displaying significant coherence (Ͼ95% confidence interval) between the EEG and EMG recordings around 15-to 35-Hz frequency during tonic contraction. The spectra and cumulant density functions were compared before and after immobilization and recovery. EEG-EMG coherence estimates were compared for statistically significant differences before and after immobilization and recovery using the difference of coherence test described by Rosenberg et al. (78) . This test is applied to the coherency estimates (where the coherence is the magnitude squared of the coherency), which are first transformed using Tanh-1 to stabilize the variance. The difference of these transformed coherency estimates were tested for significance using a standardized normal variate, with variance 1/L, at a 5% significance level (see Ref. 78 for further details). In addition to this individual statistical analysis, a group-level one-way repeated-measures ANOVA test was used to determine the effect of the immobilization and recovery on EEG-EMG coherence (peak coherence between 15 and 35 Hz) and cumulant density function parameters with time of measurement as a factor (pretest 1, pretest 2, posttest, and retest). A post hoc Tukey's test was done on significant comparisons. RESULTS After immobilization, maximal voluntary wrist flexion and extension torque decreased significantly (Fig. 2) . RESULTS and 0.013, respectively). After recovery, MVC EMG returned to 1.67 Ϯ 0.32% M max (348 Ϯ 66 V) (P ϭ 0.2 and 0.3 compared with pretests and P ϭ 0.03 compared with posttest, respectively). For ECR, M max was not elicited, but nonnormalized MVC EMG amplitudes decreased from 297 Ϯ 43 and 294 Ϯ 46 V in pretests to 239 Ϯ 51 V following immobilization (P ϭ 0.032 and 0.038, respectively) and returned to 285 Ϯ 52 V after recovery (P ϭ 0.6 compared with pretests and P ϭ 0.05 compared with posttest). There were no significant changes in the obtained M max values following immobilization and recovery. RESULTS After supramaximal median nerve stimulation, three parameters were quantified from the elicited twitch contraction to assess changes in contractile properties (Fig. 3) . These three parameters were twitch contraction torque, contraction time (time to peak torque), and half relaxation time. None of the parameters changed significantly following the immobilization period. Twitch contraction torque was 0.79 Ϯ 0.49 and 0.80 Ϯ 0.11 Nm in pretests 1 and 2, 0.77 Ϯ 0.17 Nm following immobilization, and 0.82 Ϯ 0.12 Nm after 1 wk of recovery. Contraction time was 65.2 Ϯ 7 and 66.1 Ϯ 8 ms in pretests 1 and 2, 65.1 Ϯ 7 ms following immobilization, and 63.9 Ϯ 6 ms after 1 wk of recovery. There also was no significant change in half relaxation time between tests: 57.2 Ϯ 4 and 59.4 Ϯ 8 ms in pretests 1 and 2, 59.2 Ϯ 6 ms after immobilization, and 60.3 Ϯ 7 ms after 1 wk of recovery. H-Reflexes Both at rest and during static contraction, the H-reflex amplitudes increased significantly following immobilization. The FCR H-reflexes measured at rest (Fig. 4, A and B) increased significantly following immobilization at all stimulation intensities, ranging from 0.7 to 1.3 times M threshold pretest 1 (P ϭ 0.014 -0.042). H max /M max increased from 21.5 Ϯ 5.5 and 19.7 Ϯ 6.6% of M max in pretests to 36.3 Ϯ 7.2% of M max after immobilization. After recovery, H max returned to 23 Ϯ 6% of M max , which was a statistically significant difference from the posttest value (P ϭ 0.001). H-Reflexes FCR H-reflexes obtained during static contraction (Fig. 4 , C and D) also increased significantly at all stimulation intensities, ranging from 1.1 to 1.7 times M threshold pretest 1 (P ϭ 0.007-0.049). H max /M max increased from 32.8 Ϯ 8 and 32.7 Ϯ 5.4% of M max in pretests to 44 Ϯ 7% of M max after immobilization. H-reflex amplitudes returned to preimmobilization levels after 1 wk of recovery (29.6 Ϯ 3.8% of M max ), which was a statistically significant difference from the posttest values (P ϭ 0.001). H-Reflexes Sigmoid curve-fitting analysis of the recruitment curves did not show any significant changes in M-wave and H-reflex threshold and slope. There was, however, a tendency toward an increased H slope of the static contraction recruitment curve after immobilization (P ϭ 0.059) and a decrease in H threshold at rest (P ϭ 0.07). H slope /M slope increased significantly following immobilization both at rest (P ϭ 0.028 and 0.031 compared with pretests) and during static contraction (P ϭ 0.032 and 0.036 compared with pretests). After 1 wk of recovery, H slope / M slope decreased to preimmobilization levels at rest (P ϭ 0.7 compared with pretests) and during static contraction (P ϭ 0.63 compared with pretests). There were no significant changes in the peak-to-peak amplitude of the FCR maximal compound action potential (M max ) obtained at rest (P ϭ 0.48) or during tonic contraction (P ϭ 0.42) following immobilization. H-Reflexes In four subjects it was possible to evoke H-reflexes in APB during tonic contraction (Fig. 4, E and F) . The same tendency, although nonsignificant, for an increase in H max without changes in M max amplitude, as reported for the FCR was observed. Also for APB, H slope /M slope tended to increase in all four subjects (P ϭ 0.17). MEPs TMS stimulus-response curves were generated at rest and during static contraction corresponding to 10% MVC for both FCR and APB. There were no significant changes in the stimulusresponse curve parameters (MEP threshold , slope, and MEP max ) after immobilization for either FCR or APB (Fig. 5) . At rest, FCR MEP max increased insignificantly following immobilization from 8.7 Ϯ 1.8%M max before to 11.9 Ϯ 2.6%M max following immobilization and 9.7 Ϯ 1%M max after recovery (P ϭ 0.25) and during contraction from 42.6 Ϯ 5.2 to 51.4 Ϯ 4.6%M max following immobilization and 46.6 Ϯ 3.5%M max after recovery (P ϭ 0.22). MEP latencies also did not change following immobilization. For FCR, MEP latencies were 16.0 Ϯ 1.5 and 16.1 Ϯ 1.5 ms in pretests and 16.4 Ϯ 1.6 ms postimmobilization (P ϭ 0.5). For APB, MEP latencies were 21.3 Ϯ 1.3 and 21,4 Ϯ 1.3 ms in pretests and 21.7 Ϯ 1.6 ms postimmobilization (P ϭ 0.54). Corticomuscular Coherence In seven subjects, we found significant corticomuscular coherence in the 15-to 35-Hz area between EEG and FCR EMG during tonic wrist flexion (Fig. 6) . In eight subjects, significant corticomuscular coherence was found between the EEG and the APB EMG signals during tonic contraction (Fig. 6 ). Corticomuscular Coherence For FCR, group average peak coherence in the 15-to 35-Hz range was 0.21 Ϯ 0.03 in pretest 1 and 0.2 Ϯ 0.03 in pretest 2. In six of seven subjects, corticomuscular coherence decreased significantly following immobilization. For the population of subjects (n ϭ 7), group average peak coherence decreased to 0.13 Ϯ 0.02 (P Ͻ 0.01) after immobilization. One week after immobilization, coherence values returned to a preimmobilization level of 0.20 Ϯ 0.02 (P Ͻ 0.01 compared with postimmobilization, P ϭ 0.68 and 0.73 compared with pretests). Corticomuscular Coherence For APB, group average peak coherence in the 15-to 35-Hz range was 0.23 Ϯ 0.02 in pretest 1 and 0.22 Ϯ 0.03 in pretest 2. In seven of eight subjects, the amount of coherence decreased significantly following immobilization. For the population of subjects (n ϭ 8), group average coherence decreased to 0.15 Ϯ 0.02 (P Ͻ 0.01) after immobilization. One week after immobilization, coherence values increased significantly to a preimmobilization level of 0.2 Ϯ 0.02 (P Ͻ 0.01 compared with postimmobilization, P ϭ 0.61 and 0.69 compared with pretests). Corticomuscular Coherence The cumulant density function was calculated between EEG and rectified EMG with EEG as the reference. The plots (Fig. 6, B and D) show a characteristic maximum negative cumulant deflection displaced to the right of time 0, indicating a delay with respect to the EEG. In all tests, the lag of the most negative cumulant for the different subjects was found to be 13-20 ms for FCR and 16 -24 ms for APB; i.e., within the range of the measured MEP latencies and consistent with transmission in corticospinal pathways (28) . Corticomuscular Coherence In five of seven subjects for FCR and five of eight subjects for APB, the maximum negative cumulant value decreased significantly following immobilization. However, despite these changes in individual subjects, group average maximum negative cumulant did not change significantly (P ϭ 0.11 for FCR and P ϭ 0.14 for APB). Motor Performance: Variability of Static Contractions During static contractions, the ability of the subjects to accurately match the specified target level was quantified as the mean amplitude and variability (SD) of the iEMG signal obtained during the EEG measurements relative to the target level of 10% MVC iEMG for FCR and APB, respectively. In all tests, the group average mean contraction level was matched at 10% MVC (P ϭ 0.7 for FCR and P ϭ 0.61 for APB). The subjects' ability to accurately maintain the specified contraction level did, however, decrease following immobilization. This was signified by an increased variability (SD) of the FCR contraction level from Ϯ2.5 and Ϯ2.7% of MVC in Fig. 4 . M-waves and Hoffmann (H)-reflexes were elicited in FCR (n ϭ 10) and APB (n ϭ 4) before (pretests 1 and 2) and after immobilization (cast removal) and again 1 wk after cast removal (retest). A, C, and E show H-and M-wave recruitment curves in absolute values (group average Ϯ SE), whereas B, D, and F show H-reflex recruitment curves normalized to the corresponding maximal compound motor response (Mmax). A-D show data from the FCR at rest (A and B) and during contraction (C and D) ; E and F show data from APB during contraction. In all panels, the abscissa represents the stimulation intensity normalized to the M-wave threshold in pretest 1. *P Յ 0.05. pretests to Ϯ5.2% of MVC following immobilization (P ϭ 0.036 and 0.04, respectively). Similarly, the SD of the static APB contraction level also increased significantly from Ϯ3.0 and Ϯ3.2% of MVC before immobilization to Ϯ6.2% of MVC following immobilization (P ϭ 0.042 and 0.048, respectively). After 1 wk of recovery, the variability of the contraction levels (SD) returned to preimmobilization levels of 2.9% MVC for FCR (P ϭ 0.67 and 0.71 compared with pretests) and 3.3% MVC for APB (P ϭ 0.55 and 0.75 compared with pretests). DISCUSSION The major behavioral finding in this study is that 1 wk of joint immobilization was associated with significant decreases in maximal voluntary muscle strength and significantly increased variability of the submaximal static contractions with no changes in muscle contractile properties quantified as twitch torque, time to peak torque, and half relaxation time based on supramaximal electrical stimulation of the median nerve. Although we cannot exclude the possibility that muscular changes may have taken place, the findings suggest that the decrease in maximal voluntary torque was not explained by muscular changes. The decrease in MVC is then more likely attributed to a "voluntary deficit" due to changes in the functional properties of the central nervous system. This is in line with previous studies suggesting that a pronounced part of this decrease of strength is causally related to changes within the nervous system (8, 21, 23, 33, 48) , but deeper insight into the specific mechanisms underlying this central nervous plasticity is limited. On the basis of the obtained measurements, it is not possible to specify the underlying cause of the observed reduction in maximal voluntary strength. Considering the complexity of the central nervous system, there are many ways and mechanisms that could potentially be involved in this change. DISCUSSION The second behavioral finding of the study was that the variability of the submaximal static contractions increased following immobilization. Subjects were instructed to maintain the target level of contraction with maximal accuracy, and during the contraction, EEG and EMG measurements were obtained. This increased variability does not likely relate to the decrease in MVC, since the target was specified as a relative Fig. 5 . TMS stimulus-response curves were generated based on motor-evoked potential (MEP) amplitudes obtained in FCR (n ϭ 10) and APB (n ϭ 10) at rest and during tonic contraction of 10% MVC before [pretests 1 and 2 (pre)] and after immobilization (cast removal) and again 1 wk after cast removal (retest). A and B show MEP amplitudes obtained in FCR at rest (A) and during tonic contraction (B). C and D show MEP amplitudes obtained in APB at rest (C) and during tonic contraction (D). All MEP amplitudes are normalized to the corresponding individual Mmax. In all panels, the abscissa represents the stimulation intensity normalized to the MEP threshold in pretest 1. Data are plotted as group averages Ϯ SE. level of contraction; i.e., 10% of maximum iEMG obtained during MVC on the day of the test. The finding of increased variability of contraction levels following immobilization is consistent with previous studies documenting increased torque fluctuations during low-intensity isometric contractions following bed rest (85, 94) . Fluctuations in motor output are influenced by a number of factors, and the magnitude of the fluctuations has been shown to vary with the contraction level, specific muscle, type of contraction, and age of the subject (25) . Several potential neural mechanisms may underlie increased contraction variability. Torque fluctuations may relate to alterations in muscle activation strategy among agonist, synergist, and antagonist muscles, but in the present study, variability of FCR or APB iEMG rather than force fluctuations was used as a measure of motor performance. Based on this, the increased contraction variability more likely relates to changes in the discharge behavior (or characteristics) of FCR and APB motor units. It may be possible that the observed increased variability of the static contractions relate to the neurophysiological findings of the study. DISCUSSION The most consistent neurophysiological finding in this study is that the maximal amplitude of the FCR (and APB) H-reflex as well as the H slope /M slope ratios increased significantly following immobilization both at rest and during tonic contraction. This finding is in agreement with the previous studies by Anderson et al. (2) , who observed an increased response following 3 wk of hindlimb unloading in rats, and Clark et al. (18) , who observed increased soleus H-reflexes at rest following 4 wk of unilateral lower limb suspension. Since the increase in the H-reflex amplitudes in both the present study and the studies by Andersson et al. (2) and Clark et al. (18) was in all likelihood caused by the reduction in sensorimotor activity of the immobilized or unloaded limbs, it is not surprising that similar changes were not seen in the study by Yamanaka et al. (93) , since in this study the subjects trained with a dynamic leg press machine every day during the 20 days of bed rest. DISCUSSION The H-reflex is mediated by a largely monosynaptic pathway that includes the Ia-afferent neuron, its synapse on the a-motoneuron, and the ␣-motoneuron (73) and as such the H-reflex circuitry comprises the central part of the monosynaptic stretch reflex. Previous studies have shown that the H-reflex is modulated during different motor tasks (14, 98) and that the amplitude of H-reflex is affected by motor training (34, 61, 71, 89, 92) . Perez et al. (71) have previously provided evidence suggesting that the decrease of H-reflex amplitude accompanying motor skill learning is due to increased presynaptic inhibition of the Ia afferents. It would then not be surprising if presynaptic inhibition were reduced following immobilization, and this would indeed be consistent with our observation of increased H-reflex amplitude. It has been demonstrated that there is a significant presynaptic inhibition of Ia afferents in resting subjects, which maintains the H-reflex at a relatively suppressed level (44) , and it would not be unlikely that the changes in sensory and motor activity during the immobilization lead to a reduction in this inhibition. This would indeed be consistent with the findings of Manabe et al. (56) , who observed increased Ia-afferent monosynaptic excitatory postsynaptic potentials (EPSPs) in the ␣-motoneurons following a period of nerve block with tetrodotoxin. DISCUSSION The lack of change in the MEP amplitude also supports this possibility, since we would have expected parallel changes in H-reflex and MEP amplitude if the increase of the H-reflex had been caused by increased excitability of the spinal motoneurons following immobilization. However, as pointed out by Nielsen et al. (62) , it is not necessarily the same motoneurons that are recruited in the H-reflexes and MEPs, and the MEPs also may be influenced by changes in cortical excitability as well as the excitability of other neurons downstream from the motor cortex, which might counteract the changes in spinal motoneuronal excitability. It also should be noted that a small, but nonsignificant, increase of the MEPs was observed. It may be that the lack of statistical significance was caused by the higher variability of the MEPs than the H-reflexes. In this case, increased spinal motoneuronal excitability could be involved. In addition to changes in GABAergic presynaptic inhibition, the observed spinal excitability changes may also (at least at rest) have been caused by decreased postactivation (homosynaptic) depression (43) . This frequency-related depression of the H-reflex relates to presynaptic modulation of synaptic efficacy through changes in transmitter release probability, and postactivation depression recently was shown to increase following a single session of skill training (58) . Whether the increased H-reflex is caused by reduced presynaptic inhibition, postactivation depression or not, thus requires further experiments. Whatever the cause of the increase of the H-reflexes, it is possible that this may have influenced performance in the visuomotor task of maintaining a static contraction accurately. DISCUSSION As considered above, MEP measures and stimulus-response curve characteristics are influenced by changes in motor cortical representational patterns and corticospinal excitability. During recent years, several studies have demonstrated that the process of motor learning is associated with increased corticospinal excitability and expansion of motor cortical representational map areas (see e.g., Refs. 67-69; for review, see Refs. 46, and 72). Observations relating to immobilization or disuse have been less clear. Zanette et al. (96, 97) found that 30 -45 days of wrist and hand immobilization due to traumatic fracture were accompanied by increased MEP amplitudes without changes in MEP threshold and map areas. Immobilization was also accompanied by decreased intracortical inhibition and increased intracortical facilitation (96) . After immobilization, MEP recruitment was significantly greater on the immobilized side at rest, but this asymmetry disappeared during voluntary muscle contraction. In contrast, Facchini et al. (26) reported increased M threshold and decreased MEP amplitudes at rest after 3 days of finger immobilization with no changes in M-and F-wave amplitudes. For immobilization of the leg, Liepert et al. (55) found that the motor cortical map area of the inactivated tibialis anterior muscle diminished compared with the unaffected leg without changes in spinal excitability or M threshold . In contrast, Roberts et al. (76) recently reported that corticospinal excitability was increased after cast removal following 10 days of immobilization. It is likely that the inconsistent findings of the previous immobilization studies relate to differences in the immobilization paradigm, duration of immobilization, limb, and the conditions under which the measurements were obtained (rest vs. contraction). In the present study, TMS measurements were obtained both at rest and during voluntary contraction for a wrist (FCR) and a hand (APB) muscle to assess changes in corticospinal excitability. After a period of 1 wk of wrist and hand immobilization, no significant differences in TMS parameters were observed in either condition. Although this negative finding should be interpreted with caution, it points in the direction that 1 wk of immobilization does not lead to significant changes in corticospinal excitability. Change in Corticomuscular Coherence In humans, it was first suggested that coherence between magnetoencephalography (MEG) and electromyography (EMG) is in all likelihood mediated by fast corticospinal axons and their monosynaptic connections to spinal motoneurons (19) . This suggestion indicates that coupling between cortical activity recorded by EEG and muscle activity recorded by EMG in the 15-to 35-Hz frequency may reflect discharge of corticospinal cells in this frequency range (19, 27) . It is suggested that corticomuscular coupling plays a role in sensorimotor integration processes within the motor system, but although several hypotheses have been proposed, the functional significance of the coupling is still not fully understood (3, 4, 51, 75, 80) . Several studies have indicated that motor unit coherence results from oscillatory activity in the primary motor cortex and have demonstrated a phase delay between the coupled EEG and EMG oscillations (5, 11, 19, 35, 60) . This strongly suggests that EMG oscillations largely result from motor cortex neural activity transmitted to the spinal motoneurons via corticospinal pathways. On the basis of this evidence, it was recently argued that significant corticomuscular coherence is functionally relevant when associated with a negative cumulant at an appropriate lag (28) . Change in Corticomuscular Coherence Using coherence analysis, we recently demonstrated that visuomotor skill learning is accompanied by changes in corticospinal drive to spinal motoneurons as evidenced by a significant increase in EEG-EMG coherence around . In the present study we found that immobilization, i.e., reduced sensorimotor activity, is accompanied by the opposite changes, i.e., reduced corticomuscular coherence associated with maximum negative cumulant values at lags corresponding to the measured MEP latencies for FCR and APB, respectively. In some subjects the maximum negative cumulant also decreased significantly. The reduced coupling between the motor cortex (descending command) and the ␣-motoneuronal activity indicates that the relative contribution of the descending command to the excitation of spinal motoneurons was reduced during the submaximal static contractions. This likely relates to the increased difficulty in accurately maintaining the submaximal static contractions. Change in Corticomuscular Coherence The most straightforward (or simplistic) interpretation of this reduction in corticomusclar coupling is that the corticospinal drive to the spinal motoneurons is reduced following immobilization. However, the reduction of corticomuscular coherence may also reflect that the discharge frequency in the population of corticospinal cells has become more variable. This is also the likely explanation why corticomuscular coherence is absent during the dynamic phase of movement (49) . Only during static contractions do a sufficiently large number of neurons discharge steadily within the same 15-to 35-Hz frequency range in order for significant coherence to arise (5). Kilner et al. (49) previously suggested the possibility that cortical oscillations act as an efficient means of recruiting motoneurons while maintaining as low a corticospinal firing rate as possible. Theoretical studies show that a synchronous input can produce more output firing than an asynchronous one (4, 5). The hypothesis that motor cortical oscillations are used as a
{'paper_id': '15385799', 'title': 'Central nervous adaptations following 1 wk of wrist and hand immobilization', 's2_url': 'https://api.semanticscholar.org/CorpusID:15385799', 'source': 'S2ORC'}