Neural coordination of bilateral hand movements: evidence for an involvement of brainstem motor centres

Bilateral hand movements are assumed to be coordinated by a neural coupling mechanism. Neural coupling is experimentally reflected in complex electromyographic (EMG) responses in the forearm muscles of both sides to unilateral electrical arm nerve stimulation (ES). The aim of this study was to examine a potential involvement of the reticulospinal system in neural coupling by the application of loud acoustic stimuli (LAS) known to activate neurons of this system. LAS, ES and combined LAS/ES were applied to healthy subjects during visually guided bilateral hand flexion–extension movements. Muscle responses to the different stimuli were evaluated by electrophysiological recordings. Unilateral electrical ulnar nerve stimulation resulted in neural coupling responses in the forearm extensors (FE) of both sides. Interestingly, LAS evoked bilateral EMG responses that were similar in their configuration to those induced by ES. The presence of startles was associated with a shift of the onset and enhanced amplitude of LAS‐induced coupling‐like responses. Upon combined LAS/ES application, ES facilitated ipsilateral startles and coupling‐like responses. Modulation of coupling‐like responses by startles, the similarity of the responses to ES and LAS, and their interaction following combined stimulation suggests that both responses are mediated by the reticulospinal system. Our findings provide novel indirect evidence that the reticulospinal system is involved in the neural coupling of hand movements. This becomes clinically relevant in subjects with a damaged corticospinal system where a dominant reticulospinal system leads to involuntary limb coupling, referred to as associated movements.


Introduction
Bimanual motor tasks are an integral part of many activities of daily living.It is suggested that automatic coordination of bilateral hand movements is achieved by a neural coupling (Dietz et al., 2015).This neural coupling is reflected by the appearance of a bilateral response pattern in the forearm muscles following unilateral electrical arm nerve stimulation.Automatic inter-limb coordination by neural coupling is not limited to bimanual tasks, but is also present in other tasks of daily living, such as locomotion (Balter & Zehr, 2007;Dietz, 2002).
It has been suggested that different neural mechanisms are involved in the coordination of hand movements by neural coupling, such as homologous motor cortices connected by the corpus callosum (Carson et al., 2004;Eliassen et al., 2000;Liuzzi et al., 2011).However, this assumption seems rather unlikely as in post-stroke subjects, bilateral coupling responses are preserved if the arm nerve of the unaffected side is stimulated, but absent if the nerve of the paretic side becomes stimulated (Schrafl-Altermatt & Dietz, 2016).This observation suggests that neural coupling is generated by one hemisphere using both ipsi-and contralateral corticospinal pathways.However, electrophysiological studies indicate that the non-crossing corticospinal tract is of minor relevance for the control of hand movements (Morecraft et al., 2013;Ziemann et al., 1999).
A structure that might mediate neural coupling is the reticulospinal system, a phylogenetically conserved motor system with an important functional role in the coordination of multijoint, bilateral limb movements throughout mammalian species (Baker, 2011).Electrical microstimulation of reticulospinal neurons evokes bilateral muscle responses in cats and primates, reflecting the divergent reticulospinal projections to inter-and motoneurons of both hemicords (Drew & Rossignol, 1990a, b;Davidson & Buford, 2004, 2006).Accordingly, preclinical experiments demonstrated that unilateral motor commands are distributed to the two body sites by the reticulospinal system (Brocard et al., 2010).In line with these observations, recent experiments showed that reticulospinal drive is greater during bilateral than unilateral limb movements in healthy subjects (Maslovat et al., 2020).These findings support the idea that the reticulospinal system is a structure that might be involved in the coordination of bilateral hand movements by neural limb coupling.
Application of loud acoustic stimuli (LAS) represents a non-invasive approach to activate brainstem networks in humans (Rothwell, 2006); particularly, neurons within different nuclei of the reticulospinal motor system (Fisher et al., 2012;Tapia et al., 2022).Electrophysiological recordings in cats and primates demonstrated that reticulospinal neurons are activated by loud sound (Irvine & Jackson, 1983;Fisher et al., 2012;Tapia et al., 2022).One effect of LAS-induced activation of reticulospinal neurons is the appearance of stereotypical startle responses in axial and proximal limb muscles (Brown et al., 1991;Valls-Sole, 2012).In addition, LAS lead to a shortening of motor reaction time (Valls-Sole et al., 1999) which, it is suggested, reflects a tone-induced activation of reticulospinal neurons (Honeycutt et al., 2013;Nonnekes et al., 2014;Germann & Baker, 2021;Tapia et al., 2022).
The aim of this study was to examine a potential involvement of the reticulospinal system in the neural coupling of bilateral hand movements.For this purpose, a new experimental setup was designed that allowed the application of: (1) LAS; (2) unilateral arm nerve electrical stimuli (ES); and (3) combined LAS/ES to healthy volunteers performing continuous visually guided hand flexion-extension movements (in contrast to LAS applied during a reaction time task in the StartReact paradigm (Valls-Sole et al., 1999)).This novel approach might clarify whether the reticulospinal system is involved in the generation of neural coupling responses that are suggested to underly the coordination of bilateral hand movements.It is hypothesized that, if neural coupling responses are mediated by the reticulospinal system, the activation of the reticular system by LAS should evoke similar bilateral reflex responses in the activated forearm muscles as following unilateral nerve stimulation.Furthermore, it is expected that the responses to ES and LAS interact during combined stimulation indicating their generation in related neural structures.

Methods
Nineteen healthy participants were included in the study and performed all experiments.Only participants without musculoskeletal impairments and hearing deficits were included in the study.Data from three subjects were excluded due to difficulties in tolerating the loud tones combined with exaggerated startle responses (i.e.startle incidence >60% in the sternocleidomastoid (SCM) muscle over all trials of the four experimental blocks).Hence, data from 16 healthy subjects (nine female; age (mean ± standard deviation): 25.9 ± 2.6 years; all right-handed) were further analysed.The study was approved by the Ethics Committee of the Canton Zurich (Study-ID: 2021-00973) and is registered online (clinicaltrials.gov;NCT04967274).The study was conducted according to the guidelines of the Declaration of Helsinki and Good Clinical Practice.Written informed consent was obtained from all participants.

Experimental procedures
The participants were comfortably sitting in a chair with their forearms placed on adjustable arm rests.They were instructed to perform bilateral, visually guided hand flexion-extension movements while holding 1 kg dumbbells in a pronated forearm/hand position (Fig. 1A).The forearms were fixed to the arm rest by velcro tape in order to prevent movement of the elbow joint.Volunteers were instructed to flex and extend their wrist joints according to a trajectory displayed in front of them using the GRAIL immersive virtual reality system (Motek Medical B.V., CL Houten, Netherlands).Three different stimulus conditions were randomly applied to the volunteers at the end of the extension phase of the visuomotor task: (1) LAS were released to activate the reticulospinal system; (2) unilateral ES to the right ulnar nerve to evoke a bilateral neural coupling response (Dietz et al., 2015); and (3) combined application of LAS and ES (Fig. 1A).During combined stimulation, the LAS preceded the ES by 10 ms.The earlier release of the LAS should ensure that the afferent input induced by ES encounters an already activated reticulospinal system.
At the beginning of the experiment, five LAS were delivered in a resting position to assess LAS-induced muscle responses in relaxed forearm extensor muscles (FE) and to familiarize the volunteers with the LAS.Afterwards, the participants performed a practice trial consisting of 20 flexion-extension movements to get familiarized with the visuomotor task.In five out of V. Dietz and others J Physiol 602.2 20 movements, ES were released to familiarize subjects with the ES.Thereafter, the participants performed four experimental blocks each consisting of 35 bilateral hand flexion-extension movements over a period of 140 s.The different stimulus conditions were presented in a non-predictable, pseudo-randomized order that varied between the four assessment blocks.Each assessment block consisted of 17 'dummy' stimuli (no ES or LAS), six LAS, six ES and six combined stimuli (LAS and ES).In total, 68 dummy trials, 24 LAS, 24 ES and 24 combined stimulation trials were performed on each subject during the whole experiment.There were breaks of 5 min between the blocks to prevent muscle fatigue.In a subset of the cohort (n = 7), LAS and ES were applied during unilateral left-hand movements.In this part of the experiment, a total of 36 LAS and 36 ES were released over a total of four experimental blocks.ES were applied to the resting right arm.
A marker-based motion capture system (Vicon system, Oxford, UK) was coupled to the GRAIL system which allowed the real-time motion capture data (hand movements) to be integrated into the virtual scenery (Fig. 1B).Hand flexion-extension was performed according to a projected target moving with a flexion-extension amplitude of 90°(ranging from −45°(flexion) to 45°(extension)) at a velocity of 0.25 Hz (Fig. 1B,C).The stimuli (i.e.LAS and ES) were released at 80% of maximal hand extension (i.e. at 36°h and extension, Fig. 1D) via an analogue input voltage generated by the D-Flow software (Motek Medical B.V.).The standardized, event-based triggering of LAS and ES assured a high level of muscle activity (Fig. 1D) that was comparable between stimulation modes and subjects.5).

Acoustic stimulation
LAS (120 dB, 50 ms, 1000 Hz) were generated by a custom-made Simulink application (Matlab R2021b) that was triggered by the D-Flow software.Acoustic stimuli were presented by a large speaker box (Electro-Voice, ELX200, USA) located 0.3 m behind the subject's head.Sound intensity was monitored regularly and adjusted to 120 dB using a high-precision sound level meter (Cirrus research, CR162B).

Electrical ulnar nerve stimulation
Non-noxious electrical stimuli (ES) were applied transcutaneously to the distal part of the right ulnar nerve through self-adhesive surface electrodes (Ambu A/S Neuroline 700, Denmark).Each ulnar nerve stimulation consisted of a train of five monophasic pulses (duration 1 ms), separated by 2 ms, resulting in a burst stimulation of 13 ms.The stimuli were released by an electrical current stimulator (Dantec KEYPOINT G4, Neurolite, Belp, Switzerland) that was triggered by the D-Flow software.The stimulation intensity was set to 150% of the motor threshold which was defined as the first visible twitch of the little finger.

Data analysis
The acoustic and electrical stimuli were incorporated as analogue input into   Furthermore, the similarity of coupling-like and coupling response was assessed by analysing the time series data from 60 to 140 ms after stimulus onset.For this purpose, one-dimensional statistical parametric mapping (SPM) was applied (Pataky et al., 2013).All SPM analyses were performed with the open source spm1d package (v.0.4,https://spm1d.org/)(Pataky, 2012) in Matlab.Normality of the data was assessed by the spm1d build in function (spm1d.stats.normality.ttest_paired).As all our data were non-normally distributed, statistical non-parametric mapping (SnPM) was applied (spm1d.stats.nonparam.ttest_paired).The null hypothesis was rejected if the SnPM {t} exceeded the critical threshold t * at alpha = 0.05.
Frequentist statistical approaches (e.g.t test, Wilcoxon's matched-pairs signed-rank test) only indicate whether a null hypothesis (H0) can be rejected and do not allow inferences about the likelihood of H0.Hence, Bayesian analysis (Bayesian paired sample t tests, Bayesian Wilcoxon's signed-rank tests) was performed to assess the strength of evidence for H0 (i.e. for the similarity of the coupling and coupling-like response).Bayesian analysis was performed using Jeffreys's Amazing Statistics Program (JASP, 0.17.2.1 version).Bayes factors reflect the ratio (BF 01 ) between the likelihood of H0 and the likelihood of the alternative hypothesis (H1).BF 01 values between 1 and 3 indicate anecdotal evidence for H0 and values between 3 and 10 are considered to reflect moderate evidence for H0.BF 01 values between 1 and 0.33 suggest anecdotal evidence for H1 and values between 0.33 and 0.1 indicate moderate evidence for H1 (Jeffreys, 1961).The priors on the effect were defined by a zero-centred Cauchy distribution as reported previously (Jeffreys, 1961).Standard medium-width priors (i.e.r = 0.707) were used for interpretation of Bayes factors.
During bilateral hand movements, startle responses were also present in both the SCM and FE muscles (Fig. 2B).In the SCM, the overall incidence of startle reflexes was 34.8 ± 22.5%.There was a significant habituation of the startle response in the SCM over the four experimental blocks (Friedman's test, Friedman chi square(3) = 29.894,P < 0.001, W = 0.623).In the FE muscles, startle incidence over the four experimental blocks was 42.6 ± 15.1%.The startle response exhibited a peak latency of 73.1 ± 2.2 ms (left FE) and 78.0 ± 6.4 ms (right FE; Fig. 2B).Startle responses in the FE were mainly present during the first experimental block reflecting the significant habituation of the startle response over time (Friedman's test, Friedman chi square(3) = 15.538,P = 0.001, W = 0.324).
In contrast to the resting condition, additional reflex responses occurred in the FE muscles during bilateral hand movements.First, a bilateral early positive-to-negative response appeared, most likely corresponding to a vestibular-evoked response (VEMP; Fig. 2B).The early positive vestibular peak (P V ) in the left FE occurred at 36.7 ± 7.8 ms and the negative peak (N V ) at 52.8 ± 4.7 ms.The incidence of VEMP across LAS trials was 55.7 ± 32.7%.Second, a prominent complex response occurred after the vestibular response in the FE of both sides.This response closely resembles the bilateral responses evoked by unilateral ulnar nerve stimulation and, therefore, is called a coupling-like response (Fig. 2B; N CL , P CL; ).The coupling-like response was present in trials with and without VEMP.

Coupling-like vs. coupling response
LAS-induced coupling-like responses were compared with ES-induced coupling responses (Fig. 3B).Comparison between the coupling-like and coupling response were confined to the left FE (i.e.contralateral to ES).There were no differences between the LAS-induced coupling-like and the ES-induced coupling response: The response latencies of the negative (Wilcoxon's signed-rank test, Z = 0.879, P = 0.4, rs = 0.220) and positive (paired t test, T(15) = 0.076, P = 0.941, r = −0.514;see Table 1) components of the coupling-like response did not differ from those of the ES-induced coupling response (see Table 1).Furthermore, the size of the coupling-like   3C).

Response interactions to combined stimuli
Responses to combined application of LAS and ES differed between the left and right sides (Fig. 4).

Coupling responses during unilateral hand movements
In a subset of subjects (n = 7), the neural coupling mechanism was investigated during unilateral left-hand movements.Coupling (ES) and coupling-like (LAS) responses were present in the FE of the moving hand.
In contrast, no responses appeared in the FE of the relaxed hand (Fig. 5).The coupling, as well as the coupling-like responses, did not differ in latency and configuration between unilateral and bilateral hand movements (coupling-like response: latency N CL unilat.Wilcoxon's signed-rank test, Z = 1.352,P = 0.219, rs = 0.571).These findings were supported by the Bayes factors analysis providing anecdotal evidence for the similarity of the coupling-like and the coupling response latency (N C : BF 01 = 2.561; latency P C : BF 01 = 2.600; RMS 60-140 : BF 01 = 1.076).In the SnPM analysis of coupling-like and coupling response from 60 to 140 ms after stimulus release, only two points of the data series (119.0 ms; 119.5 ms) were different (t * > 6.191).

Discussion
The aim of this study was to investigate an involvement of the reticulospinal system in the neural coupling of hand movements.Experimentally, neural coupling is reflected in the appearance of a complex EMG response in the FE muscles of both sides to unilateral, electrical ulnar nerve stimulation during bilateral hand movements (Dietz et al., 2015).This bilateral response pattern allows a rapid compensation of a unilateral displacement by both hands (Thomas et al., 2018).In the present study, an involvement of the reticulospinal system in neural coupling was investigated by the application of LAS, known to activate neurons of the reticulospinal system.The main findings are that (1) LAS evoked a response pattern that closely resembled in its configuration the ES-induced coupling response.This LAS-induced coupling-like response could be distinguished from startle responses.
(2) The presence of a startle response was associated with a delayed onset and enhanced amplitude of the coupling-like response.
(3) Application of combined acoustic and electrical stimuli resulted in a unilateral facilitation of startle and coupling-like responses indicating an interaction of ES and LAS.These findings support the idea that the reticulospinal system is involved in the generation of coupling responses that are assumed to contribute to the coordination of bilateral hand movements.

Neural structures underlying inter-limb coordination
Different neural mechanisms might underlie neural coupling.For example, electrophysiological recordings indicate that hand coordination is achieved by an interaction of cortical motor areas of both hemispheres (Meyer et al., 1995;Carson et al., 2004).This assumption is based on the observation that the excitability of the motor cortex by transcranial magnetic stimulation (TMS) is enhanced ipsilateral to a unilaterally moving hand (Carson et al., 2004).This observation does, however, not exclude an involvement of the brainstem in the facilitation of the motor response.The fact that this facilitation is also present in patients with agenesis of the corpus callosum (Meyer et al., 1995) and that temporal coupling is preserved in these patients (Franz, 1996) indicates an involvement of subcortical neural structures in the neural coupling.

Responses
In fact, preclinical neuroanatomical and electrophysiological research favours the idea that the reticulospinal system is involved in the neural coupling mechanism.This neural structure represents a major descending motor system in vertebrate species.Single reticulospinal neurons originating from the ponto-medullary reticular formation convey motor commands to spinal interneurons and motoneurons of both ipsi-and contralateral sides, thus driving bilateral muscle activation (Drew & Rossignol, 1990a;Matsuyama et al., 1997;Davidson & Buford, 2006;Riddle et al., 2009).The strong bilateral motor drive of the reticulospinal system to the spinal cord represents an ideal substrate for the coordination of inter-limb movements.
Involvement of the reticulospinal system in the coupling of hand movements LAS are known to activate motor-related reticulospinal neurons (Irvine & Jackson, 1983;Fisher et al., 2012;Germann & Baker, 2021;Tapia et al., 2022).In the present study, LAS were applied in order to investigate a potential involvement of reticulospinal system in the generation of coupling responses elicited by unilateral nerve stimulation.The main observation was that LAS elicit a complex EMG response pattern in the activated FE of both sides, i.e. a coupling-like response.The response pattern did not differ in size, latency and configuration from the coupling response induced by ES, and therefore points to a generation in related neural structures.Response similarity was confirmed by Bayesian statistics that provided evidence for the similarity of the coupling and LAS-evoked coupling-like response.
LAS are known to activate or suppress neural structures besides reticulospinal neurons.In our study, startles were frequently preceded by small earlier responses that most likely reflect auditory-evoked vestibular response (i.e.VEMP) (Naranjo et al., 2015;Ashford et al., 2016).Although the late VEMP components (Colebatch et al., 2016) temporally coincide, it appears rather unlikely that they represent coupling-like responses: first, coupling-like response were also present in trials where vestibular P V -N V responses were lacking and second, the amplitude of late VEMP components appear too small to reflect coupling-like responses.LAS are also known to result in a short-term suppression of motor cortex activity (Tapia et al., 2022).Several facts speak against a cortical influence on the coupling-like response: first, LAS-related effects seem to be weak during muscle contraction (Chen et al., 2016).Second, a relevant cortical suppression at 50 ms after LAS (Furubayashi et al., 2000;Tazoe & Perez, 2017;Germann et al., 2023) should modulate the coupling-like response without startle, but not the shifted one with startles.Third, the similarity of the responses to LAS and ES makes a LAS-induced cortical extra-effect implausible.
The coupling-like response could be clearly differentiated from the startle response: first, startle responses appeared during both resting and movement conditions, while coupling-like responses were only present during hand movements.Second, startle responses showed a different configuration and latency from the coupling-like responses.Third, startle responses showed a characteristic habituation over the course of the experiment (Ornitz & Guthrie, 1989;Valls-Sole et al., 2008), while the coupling-like responses did not habituate.These differences might point towards a generation of the two responses in different neural structures.However, several aspects support the assumption that the responses are generated in related brainstem structures.First, startle responses and StartReact effects, both assumed to be mediated by reticulospinal neurons (Nonnekes et al., 2015), also habituate differentially.Second, there was an interaction between the startle and coupling-like response: The onset of the coupling-like response was shifted by the duration of the startle.This shift suggests that the two reflex responses are released serially, and not in parallel.Moreover, the presence of startles facilitated the subsequent coupling-like responses.Third, the application of combined LAS/ES resulted in a facilitation of both startle and coupling(-like) responses ipsilateral to ES.All these interactions indicate that both startle and coupling-like responses are mediated by related neural structures.
There is a general consensus that the startle reflex is mediated by reticulospinal neurons in the caudal pons (Davis et al., 1982;Brown et al., 1991;Lingenhohl & Friauf, 1994;Yeomans & Frankland, 1995).Hence, the strong interactions of coupling-like with the startle response makes it likely that the coupling-like response is also mediated by neurons within this system.In fact, the reticular formation is a highly heterogenous neural structure consisting of nuclei distributed across the mesencephalon, pons and medulla.Therefore, it is suggested that not only motor system functions such as startles, coupling-like responses or StartReact effect, but also the coupling responses are mediated by specific neural circuits within the reticular formation.
Neural coupling is thought to be involved in the coordination of bilateral hand movements.However, even during unilateral hand movements a 'coupling' response appeared in the extensor muscles of the moving hand (i.e.contralateral to ES).Again, the configuration of the ES-evoked response was similar to the one evoked by LAS in the extensors of the moving hand.However, the data basis for statistical analysis might be underpowered and the results should therefore be interpreted with caution (Keysers et al., 2020).In the relaxed extensor muscles, neither ES nor LAS induced a response, as reflex strength did not exceed motor threshold.This assumption is supported by the finding of a facilitated motor-evoked potential to TMS in the relaxed forearm muscles contralateral to the moving hand (Carson et al., 2004).These observations indicate that neural coupling is a robust mechanism that is present even without a functional context.In contrast, non-coordinated/asynchronous movements are difficult to perform, as they require separate voluntary control of the hands (Kochli et al., 2020).
Reticulospinal neurons are assumed to be involved in the generation of neural coupling responses.This structure might, however, not be solely responsible for the coordination of hand movements.The observation that callosotomy patients can exhibit a temporal uncoupling during continuous bimanual movements (Kennerley et al., 2002) implies that interhemispheric interaction can contribute to hand coordination.Thus, different neural structures (subcortical, cortical) might be involved in inter-limb coordination, depending on the specific motor task.
The role of the reticulospinal system in normal and impaired motor control Preclinical and clinical evidence indicate that the reticulospinal system plays a key role in the automatic coordination of limb movements, not only in animals but also in humans (Maslovat et al., 2017;Maslovat et al., 2020).Therefore, the role of this system in human motor control might not be restricted to the coordination of hand movements, but might also contribute to the coordination of arm and leg movements during stepping (Dietz, 2002), for instance.In the latter task, neural coupling was interpreted as a residual function of quadrupedal coordination of stepping movements.Therefore, neural coupling of limbs seems to be essential for the automatic execution of many activities of daily living.
The coupling of upper limbs might be inherited, as skilled single limb movements develop only with the maturation of the corticospinal tract during early infancy (Connolly & Stratton, 1968;Koerte et al., 2010).Infants up to about 6 years of age show involuntary movements of the contralateral hand that mirror the intended movements of the primarily activated side, e.g. during reaching and grasping hand movements (Connolly & Stratton, 1968;Licari & Larkin, 2008;Soska et al., 2012).These coupled movements decrease in frequency and intensity during adolescence, likely reflecting the maturation of the corticospinal system that enables the performance of precision movements (Connolly & Stratton, 1968;Koerte et al., 2010).Patients suffering damage of the corticospinal tract (e.g.stroke) often show 'associated movements' characterized by involuntary activation of contralateral limb muscles (Ejaz et al., 2018;McPherson et al., 2018).These coupled movements have been attributed to an upregulated reticulospinal motor drive in response to corticospinal tract damage (McPherson et al., 2018).When fine motor control is lost after damage of the corticospinal tract, the reticulospinal system can still provide a 'lower-level' motor control.
Our conclusion that the reticulospinal system is involved in the generation of neural coupling responses and, therefore, might contribute to inter-limb coordination is supported by the following findings: first, acoustic stimulation evoked responses that were similar in their configuration to the neural coupling responses elicited by electrical stimulation.Second, coupling-like responses were modified by startles known to be generated by the reticulospinal system.Third, combined stimulation led to a response pattern suggesting an interaction of the stimuli on a brainstem level.These findings are in line with the increasing evidence that the reticulospinal system represents an essential neural structure in motor control not only in animals but also in humans.

Figure 1 .
Figure 1.Schematic drawing of the experimental setupA, healthy volunteers performed bilateral visually guided wrist flexion-extension movements in an immersive virtual reality system.Subjects were instructed to move their hands (grey circles) according to the displayed movement path (white circle; vertical arrows indicate movement direction).Loud acoustic stimuli (LAS; loudspeaker) and electrical stimuli (ES; flash) to the right ulnar nerve were applied during bilateral hand movements.B, hand movements were assessed in real time by a motion capture system tracking reflective markers (green circles) fixed at the joints of both forearms.LAS, ES or combined LAS/ES were randomly released at 80% of right-sided hand extension movements (red dotted line).Muscle EMG activity of the forearm extensors (FE) was recorded by a wireless multichannel EMG system (blue transmitters).C, sinusoidal movement trajectory (ROM: 90°, 0.25 Hz) that had to be followed by bilateral hand flexion-extension movements.The horizontal dotted line (red) indicates the hand position (i.e.80% of maximal wrist extension) at which stimuli (LAS, ES or combined LAS/ES) were randomly released, i.e. at the end of the hand extension phase (dotted vertical line).D, grand average of the right forearm extensor EMG activity during hand movements (16 subjects).Start (−45°hand position) and end (+45°hand position) of the extension movement are indicated by grey vertical lines.EMG data shown in this graph were not offset corrected.The release of LAS and ES (80% of hand extension) is indicated by the bold vertical dotted line.Abbreviations: EMG: electromyography; ROM: range of motion.[Colour figure can be viewed at wileyonlinelibrary.com]

B
Responses to acoustic stimulation during hand movements

AFigure 3 .
Figure 3. ES-evoked bilateral coupling responses in the FE muscles A, ES to the right ulnar nerve induced a bilateral coupling response consisting of negative (N C ) and positive (P C ) reflex components.An early reflex response (ER) was present on the stimulated right side.B, overlay of ES-induced coupling responses (blue line) and LAS-induced coupling-like responses without startles (green line).The coupling-like and coupling responses were almost congruent and did not differ with respect to response latency and configuration.Graphs represent grand averages (coloured lines) + SD (shaded area) of FE responses from 16 healthy volunteers.Stimulus onset is indicated by the vertical dotted line.C, left panel: average EMG activity of

Figure 4 .
Figure 4. Combined application of acoustic and electrical stimuli resulted in differential response patterns in the left and right FE On the right side, startles and coupling(-like) responses were enhanced compared with pure LAS responses (cf.Fig. 2B) with startles persisting until the end of the experiment.On the left side, coupling(-like) responses (without startles) were identical to pure LAS responses.Graphs represent grand averages (coloured lines) + SD (shaded area).Release of LAS (−10 ms) and ES (0 ms) are indicated by thin dotted (LAS) and bold dotted (ES) lines.Abbreviations: ES: electrical stimuli; FE: forearm extensor; LAS: loud acoustic stimuli; SD: standard deviation.

Figure 5 .
Figure 5. Overlay of LAS-and ES-evoked responses during unilateral right-handed movements Coupling-like (LAS; green line) and coupling (ES; blue line) responses appeared on the moving side, but not in the relaxed FE (side of electrical stimulation).Coupling-like and coupling responses were almost congruent in the FE of the moving hand.Graphs represent grand averages (coloured lines) + SD (shaded area).Stimulus onset is indicated by the vertical dotted line.EMG signals in this figure were not offset corrected.Abbreviations: ES: electrical stimuli; FE: forearm extensor; LAS: loud acoustic stimuli; SD: standard deviation.[Colour figure can be viewed at wileyonlinelibrary.com] The different components of muscle responses induced by LAS and ES were identified by a custom-made Matlab script calculating the maxima and minima of the rectified EMG responses within defined time windows after release of the stimuli.Acoustic startles were defined as bilateral EMG responses in both SCM and FE muscles within a time window of 50-80 ms (for SCM) and 60-90 ms (for FE) after LAS release.LAS trials were classified according to the presence or absence of a startle response in the FE muscle.LAS-evoked vestibular responses (vestibular-evoked myogenic potentials; VEMP) were assigned to a positive peak (P V ) between 20 and 55 ms and a negative peak (N V ) 40-60 ms after LAS in the FE muscles.Coupling-like and coupling responses were defined as negative peak (N CL , N C ) between 70 and 120 ms and positive peak (P CL , P Vicon Nexus (Vicon, Oxford, UK) allowing for a posteriori data synchronization.C ) between 80 and 150 ms after LAS and ES, respectively.Response size of the ES-induced coupling response was calculated by the root mean square (RMS) in the time window between 60 and 140 ms after stimulus onset.Response size of coupling-like responses to LAS and combined LAS/ES was calculated by RMS in the time window between 60 and 140 ms for trials without startles, and between 90 and 170 ms for trials with a preceding startle.Statistical analysisStatistical analysis was conducted using R version 4.2.1/RStudio2023.06.0 for windows.Statistics were based on the averaged outcomes from healthy subjects (n = 16).The level of significance was set at 0.05 for all statistical tests.All tests were adjusted for multiple comparisons via post hoc Bonferroni or Dunn's correction.Normality of data was assessed using the Shapiro-Wilk test.Data sets with normal distribution were assessed by parametric tests (paired t test, repeated measures one-way ANOVA), whereas non-normally distributed data were assessed by non-parametric statistical tests (Wilcoxon's matched-pairs signed-rank test; Friedman's test).Habituation of the acoustic startle response in both SCM and FE muscles over the four experimental blocks was assessed by repeated measures one-way ANOVA and Friedman's test.Differences in the reflex responses to LAS, ES and combined LAS/ES in the FE muscles were assessed by comparison of response latency and size (i.e.RMS over the respective time windows).These response differences were assessed by two-tailed, paired t test, repeated measures one-way ANOVA (normally distributed data) or the Wilcoxon matched-pairs signed-rank, Friedman's test (non-normally distributed data).T-scores (for paired t test), Z-scores (for Wilcoxon's signed-rank test), Friedman's chi square (for Friedman's test), and Mann-Whitney's U statistic (for the Mann-Whitney J Physiol 602.2

Coupling-like responses with startles Startle responses FE Coupling-like responses without startles Figure 2. LAS-evoked reflex responses at rest and during bilateral hand movements
A, at rest, LAS evoked startle responses (SR) in the SCM (upper part) and FE muscles (lower part) on both sides.B, during bilateral wrist movements, LAS (in addition to SR) evoked vestibulospinal (P V : positive peak, N V : negative peak) and coupling-like responses (N CL : negative peak, P CL : positive peak) in the FE on both sides.Following LAS, the coupling-like response was shifted and its amplitude enhanced when a SR was present (upper part) compared with LAS responses without SR (lower part).Graphs represent grand averages (green line) + SD (shaded area) from all subjects (n = 16).EMG signals were not offset corrected.Abbreviations: FE: forearm extensor; LAS: loud acoustic stimuli; SCM: sternocleidomastoid; SD: standard deviation; SR: startle response.[Colour figure can be viewed at wileyonlinelibrary.com]

Table 1 . Latencies and size of coupling-like and coupling responses. Response latency and size are provided for LAS-evoked coupling-like responses (left column), for ES-evoked coupling responses (middle column) and for the responses evoked by combined LAS/ES (right column). For LAS trials, only coupling-like responses without startles were included in the analysis. For the combined LAS/ES condition, only responses without startles in the left FE were analysed (startles on the ride side persisted). Data represent mean values ± SD from healthy volunteers (n = 16). Abbreviations: ES: electrical stimuli; FE: forearm extensor; LAS: loud acoustic stimuli; RMS: root mean square; SD: standard deviation.
Z = 0.129, P = 0.911, rs = −0.131).Further analysis of the coupling-like response was confined to LAS trials without startle responses.
Physiol 602.2 coupling-like and coupling response from 60 to 140 ms after stimulus onset for bilateral hand movements.Right panel: results of SnPM {t} analysis comparing coupling-like and coupling responses from 60 to 140 ms after stimulus onset during bilateral hand movements.The red dotted lines represent the critical threshold t * at alpha = 0.05.Any clusters that exceed this threshold are considered significantly different.Abbreviation: ES: electrical stimuli; FE: forearm extensor; LAS: loud acoustic stimuli; SD: standard deviation; SnPM: statistical non-parametric mapping.[Colourfigure can be viewed at wileyonlinelibrary.com] ).The coupling-like and coupling responses were almost congruent and did not differ with respect to response latency and configuration.Graphs represent grand averages (coloured lines) + SD (shaded area) of FE responses from 16 healthy volunteers.Stimulus onset is indicated by the vertical dotted line.C, left panel: average EMG activity of J ± 32.2 µV vs. 108.5 ± 70.6 µV; Wilcoxon's signed-rank test, Z = 2.896, P = 0.002, rs = 0.815) were enhanced compared with the left side (Fig.4).In contrast to the left side (and to trials with LAS alone), startle responses in the right FE were larger and persisted until the end of the experiment.Similar to LAS trials, startles shifted the coupling(-like) response leading to a delayed onset of the response (N CL left vs. right: 89.6 ± 13.8 ms vs. 114.4± 22.4 ms; paired t test, T(15) = 4.694, P < 0.001, r = 0.396).