Pharyngeal electrical stimulation can modulate swallowing in cortical processing and behavior — Magnetoencephalographic evidence

NeuroImage 104 (2015) 117–124

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Pharyngeal electrical stimulation can modulate swallowing in cortical processing and behavior — Magnetoencephalographic evidence Sonja Suntrup a,b,⁎, Inga Teismann a, Andreas Wollbrink b, Martin Winkels b, Tobias Warnecke a, Christo Pantev b, Rainer Dziewas a a b

Department of Neurology, University of Muenster, Albert-Schweitzer-Campus 1, Gebäude A1, 48149 Münster, Germany Institute for Biomagnetism and Biosignalanalysis, University of Muenster, Malmedyweg 15, 48149 Muenster, Germany

a r t i c l e

i n f o

Article history: Accepted 6 October 2014 Available online 16 October 2014

a b s t r a c t Background: The act of swallowing is a complex neuromuscular function that is processed in a distributed network involving cortical, subcortical and brainstem structures. Difficulty in swallowing arises from a variety of neurologic diseases for which therapeutic options are currently limited. Pharyngeal electrical stimulation (PES) is a novel intervention designed to promote plastic changes in the pharyngeal motor cortex to aid dysphagia rehabilitation. In the present study we evaluate the effect of PES on cortical swallowing network activity and associated changes in swallowing performance. Methods: In a randomized, crossover study design 10 min of real (0.2-ms pulses, 5 Hz, 280 V, stimulation intensity at 75% of maximum tolerated threshold) or sham PES were delivered to 14 healthy volunteers in two separate sessions. Stimulation was delivered via a pair of bipolar ring electrodes mounted on an intraluminal catheter positioned in the pharynx. Before and after each intervention swallowing capacity (ml/s) was tested using a 150 mlwater swallowing stress test. Event-related desynchronization (ERD) of cortical oscillatory activity during volitional swallowing was recorded applying whole-head magnetoencephalography before, immediately after and 45 min past the intervention. Results: A prominent reduction of ERD in sensorimotor brain areas occurred in the alpha and beta frequency ranges immediately after real PES but not after sham stimulation (p b 0.05) and had faded after 45 min. Volume per swallow and swallowing capacity significantly increased following real stimulation only. Conclusion: Attenuation of ERD following PES reflects stimulation-induced increased swallowing processing efficiency, which is associated with subtle changes in swallowing function in healthy subjects. Our data contribute evidence that swallowing network organization and behavior can effectively be modulated by PES. © 2014 Elsevier Inc. All rights reserved.

Introduction Swallowing is a complex motor task involving a widely distributed neuronal network (Jean, 2001; Michou and Hamdy, 2009). Dysphagia is a symptom of manifold neurologic disorders, which has huge impact on the quality of life and is associated with malnutrition, aspiration pneumonia, care dependency and significant mortality. Efficient treatment options for dysphagia rehabilitation are still limited. Spontaneous compensatory changes in swallowing-related cortical areas have, however, been reported for example after stroke (Hamdy et al., 1998a; Teismann et al., 2011a), in Parkinson's disease (Suntrup et al., 2013b) and motor neuron disorder (Dziewas et al., 2009). This natural ability of the brain to reorganize and functionally adapt to altered requirements is known to be driven by experience. Therefore, application of tactile, thermal (Hamdy et al., 2003; Kaatzke-McDonald et al., 1996; ⁎ Corresponding author at: Department of Neurology, University of Muenster, AlbertSchweitzer-Campus 1, Gebäude A1, 48149 Münster, Germany. Fax: +49 251 8344414. E-mail address: [email protected] (S. Suntrup).

http://dx.doi.org/10.1016/j.neuroimage.2014.10.016 1053-8119/© 2014 Elsevier Inc. All rights reserved.

Rosenbek et al., 1996; Sciortino et al., 2003; Teismann et al., 2009b) or gustatory stimuli (Babaei et al., 2010; Logemann et al., 1995; Mistry et al., 2006; Pelletier and Lawless, 2003) to oropharyngeal receptors has been tested in a variety of clinical and neurophysiological studies on swallowing function in health and disease. Hamdy and co-workers developed and evaluated pharyngeal electrical stimulation (PES) as a neuromodulation device to enhance cortical reorganization for the restoration of swallowing function after brain injury. They showed that following electrical stimulation of the base of the tongue and posterior pharyngeal wall via a pair of catheter-mounted electrodes, motor cortex excitability and pharyngeal cortical representation area as mapped with repetitive transcranial magnetic stimulation (TMS) increased for at least 30 min (Hamdy et al., 1998b). Prior to clinical application the same group demonstrated the reversal of a virtual lesion induced by TMS in healthy pharyngeal motor cortex by subsequent PES. Finally, in a sham-controlled clinical pilot study on 50 stroke patients daily PES for three days led to reduced aspiration severity rates, improved feeding status and resulted in a shorter time to discharge from the hospital compared to the control group (Jayasekeran et al., 2010),

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implying that neuroplastic changes lead to clinically meaningful functional improvement. Apart from these promising results, the underlying mechanism of action of PES, the neurophysiological correlate of behavioral gains and the temporal dynamics of the effects are not completely understood yet. In previous studies, PES-related excitability changes have predominantly been detected with evoked responses by using TMS. Here we contribute further knowledge on the cortical topography and frequency–specificity of activation pattern changes during the act of swallowing by taking advantage of a different functional neuroimaging modality. Magnetoencephalography (MEG) is capable of detecting and localizing power changes in oscillatory cortex activity (Taniguchi et al., 2000) evoked by a decrease or increase in synchrony of the underlying neuronal populations, otherwise known as event-related desynchronization (ERD) or synchronization (ERS) (Pfurtscheller and Lopes da Silva, 1999; Pfurtscheller, 2001). Voluntary movements are generally accompanied by a modulation in the alpha and beta frequency ranges, which is characterized by a power decrease (ERD) during the task, followed by a power increase (ERS) after movement execution. There is general agreement that ERD reflects cortical activation or arousal while ERS has been associated more with inhibitory activities (Pfurtscheller and Lopes da Silva, 1999). Being able to analyze the complete act of swallowing instead of a pharyngeal evoked potential the method allows for exploring stimulation-induced alterations in the cortical large-scale oscillatory swallowing network beyond the pharyngeal motor cortex. Methods Subjects Fourteen healthy volunteers (8 male, 6 female, mean age 30.3 ± 4.7 years) participated in this study. Subjects were free of any neurologic, psychiatric or ear–nose–throat disorder, did not take any medication affecting the central nervous system and were right-handed (mean handedness score 90.0 in the Edinburgh Handedness Inventory (Oldfield, 1971)). The local ethics committee approved the protocol of the study. Informed consent was obtained from each subject after the nature of the study was explained in accordance with the principles of the declaration of Helsinki. Experimental outline Each subject participated in two experimental sessions to determine the effects of real or sham PES on swallowing performance as well as on cortical swallowing-related activation as measured by MEG. The order of the interventions was randomized and sessions were at least one week apart to avoid any carryover effects. In each session (see Fig. 1), MEG data were recorded for 15 min before (block 1) and immediately after the intervention (block 2) and, in the majority of subjects, another 15 min past the end of the second MEG measurement (block 3) as described below. Moreover, a simple 150 ml-water swallowing stress test was performed by all participants according to the protocol of Hughes and Wiles (Hughes and Wiles, 1996) before and after stimulation. Each subject drank 150 ml of water from a plastic beaker ‘as quickly as is comfortably possible’. The number of swallows was counted by observing the movements of the thyroid cartilage. A stopwatch was started by a person

experienced in swallow screening when the water first touched the bottom lip, and stopped when the larynx came to rest for the last time. The volume per swallow (ml), time per swallow (s) and swallowing capacity (ml/s) were calculated in each subject. Medical technical assistants involved in data collection and the investigators performing nonautomated steps in data preprocessing and analysis were blinded towards the order and type of intervention. Pharyngeal electrical stimulation For PES an intraluminal catheter of 3.2 mm in diameter housing a pair of bipolar platinum ring electrodes with a distance of 10 mm in between (Gaeltec, Ltd, Dunvegan, Isle of Skye, UK) was inserted transorally or transnasally as to the subject's preference. The electrodes were positioned in the pharynx with the upper electrode about 15 to 16 cm aboral from the incisors or nostril. During catheter insertion, mild gagging occurred in about 40% of the subjects but was reported to cause only minor discomfort. The catheter was connected to a constant current stimulator (Model DS7A) controlled by a trigger generator (Model DG2A, both Digitimer Limited, Welwyn-Garden City, Herts, UK) to deliver stimuli of 0.2 ms pulse duration at a frequency of 5 Hz with 280 V, which had previously been found to be the most effective stimulation parameters (Fraser et al., 2002). The current intensity (mA) was individually adjusted. Therefore prior to the actual intervention the perceptual threshold (PT) and the maximum tolerated threshold (MTT) were in any intervention condition determined repeatedly by slowly increasing the current (max. 30 mA). The average values of three trials were taken into account for the calculation of the optimal stimulation intensity according to the formula PT + 0.75 × (MTT − PT) (Fraser et al., 2003). In the real PES condition stimulation was afterwards delivered for a total of 10 min whereas in the sham condition the catheter was left in place for a further 10 min without current flow between the electrodes. MEG data acquisition MEG data were collected using a whole head 275-channel SQUID sensor array (Omega 275, CTF Systems Inc.) installed within a magnetically shielded room. During single MEG measurements, each of 15 minute duration, subjects swallowed self-paced without external cueing. To facilitate volitional swallowing water was infused into the oral cavity via a flexible plastic tube 4.7 mm in diameter attached to a fluid reservoir. The reservoir bag was positioned about 1 m above the mouth of each subject when seated. The tip of the tube was randomly placed in the left or right corner of the mouth between the buccal part of the teeth and the cheek and gently fixed to the skin with tape. The infusion flow was 12 ml/min. Swallowing acts were identified by surface electromyographic (EMG) recording with bipolar skin electrodes (Ag-AgCl) placed on the submental muscle groups (Ding et al., 2002; Vaiman, 2007). The electrodes were connected to a bipolar amplifier (DSQ 2017E EOG/EMG system, CTF Systems Inc., Canada). Magnetic fields were recorded with a sample frequency of 600 Hz. During acquisition the data were filtered using a 150 Hz low-pass filter. As later coregistration between MEG and MRI data was based on three prominent head landmarks (nasion, left and right ear canal) a current was sent through three small coils placed at these fiducial positions (fixed in

Fig. 1. Outline of a single experimental session.

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the left and right ear canal by ear plugs) which were hence detectable by the MEG measuring system. Moreover, the participants' head movements were continuously recorded. Data analysis Behavioral data Analysis of behavioral data was carried out using SPSS Statistics 22.0 (IBM Corp., USA). To evaluate the effect of stimulation (real PES, sham PES) and time point (pre, post) on performance in the 150 ml-water swallowing test repeated measures ANOVA was performed. Separate repeated measures ANOVAs were also applied to test the influences of intervention (real PES, sham PES) and MEG measurement block (1, 2, 3) on head movement and EMG characteristics of the swallowing acts conducted during MEG acquisition (see below). For every ANOVA Mauchly's test evaluated the sphericity assumption and Greenhouse– Geisser correction was applied in case of violation. A p-value of b 0.05 was used to indicate statistical significance. MEG data preprocessing MEG data analysis was carried out with custom-made Matlab (MathWorks Inc., USA) scripts based on FieldTrip (http://www.ru.nl/ fcdonders/fieldtrip), an open source Matlab toolbox for the analysis of neurophysiological data (Oostenveld et al., 2011). For event-related analysis of the MEG recordings each individual's coregistered EMG signal was used to identify the beginning of main muscle activation (M1) and the end of the task-specific muscle activity (M2) for every single swallow as previously published (Dziewas et al., 2009; Suntrup et al., 2013a; Teismann et al., 2011b) (see Fig. 2): The beginning of the main muscle activation was defined as an enduring N 100% increase in amplitude or frequency of the EMG signal. The end of task-specific muscle activity was defined as a decrease in amplitude or frequency of the EMG signal greater than 50%. For further event-related MEG data analyses time intervals were defined as follows: (1) Activation stage: −0.4 to 0.6 s in reference to M1 (2) Resting stage: 0 to 1 s in reference to M2 For comparison of swallowing behavior between single MEG data acquisition sessions the mean power (root-mean-square value) and peak-to-peak amplitude of the submental EMG recordings were calculated across the swallowing activation stage (1) in all subjects. Moreover, a third marker (M0) was manually set at the first visible oral swallowing movement in the EMG to determine mean total swallow duration (from M0 to M2) per subject. MEG data were filtered within five different frequency bands by applying a fourth order two-pass Butterworth filter prior to further computations: theta (4–8 Hz), alpha (8–13 Hz), beta (13–30 Hz), low gamma (30–60 Hz), and high gamma (60–80 Hz).

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MEG source reconstruction Source localization of each single subject's swallowing-associated cortical activity was done separately in all frequency bands for pre and post intervention measurements applying a linear constrained minimum variance (LCMV) beamformer. This technique is characterized by an adaptive spatial filter designed to let pass brain activity originating from a specified location while attenuating activity from other locations. A 3D map of neural power as a function of location is obtained by designing multiple spatial filters (Van Veen et al., 1997). This approach does not require any prior assumptions about the number or spatial distribution of active sources. As it does not rely on averaging across trials to increase the signal-to-noise ratio this method is capable of analyzing induced brain activity such as ERD of cortical rhythms which occur during motor tasks (Pfurtscheller and Aranibar, 1979; Pfurtscheller and Lopes da Silva, 1999; Taniguchi et al., 2000). It has proved to be a reliable method to examine the complex function of swallowing in humans (Dziewas et al., 2009; Furlong et al., 2004; Teismann et al., 2009a, 2010, 2011b). An individual anatomical MRI was not available for all subjects. Therefore a single shell volume conduction model (Nolte, 2003) was generated from the canonical single subject T1-weighted MRI (SPM8) as implemented in FieldTrip and used for source reconstruction. This template MRI was segmented and realigned to the same coordinate system that the functional MEG data were related to. To do so the coordinates of the three anatomical fiducial points that had also been specified during MEG data acquisition were determined manually from the template MRI. Afterwards the segmented brain volumes were discretized into a grid with a 1 cm resolution and leadfield matrices were calculated for each grid point. A spatial filter was then computed from the data covariance matrix and the lead field matrices for each grid point to estimate source power. To determine relative power changes the active stage was contrasted against baseline and normalized by the baseline power. For each condition volumetric source estimates of the individual subjects' functional data were spatially normalized to a template brain (T1, Montreal Neurological Institute, Canada) using SPM8 (http://www.fil.ion.ucl.ac.uk/spm). Grandaverages of normalized source activation maps were computed across all subjects for pre and post intervention data for all frequency bands. Grandaverages were subsequently interpolated onto the template MRI for localization and visualization of active regions using the WFU-PickAtlas tool (http://fmri. wfubmc.edu) implemented in FieldTrip.

MEG statistical analysis To identify source locations that were significantly modulated by the intervention a cluster-based nonparametric randomization approach built into FieldTrip was applied (Maris and Oostenveld, 2007). In this procedure clusters are defined on the basis of the actual data distribution and the statistical significance of these clusters is tested using a Monte-Carlo randomization method to control for the type I error

Fig. 2. Definition of activation and resting stage according to swallowing-related submental muscle activity. The surface EMG trace of a single swallowing act is shown. M0 depicts swallow initiation, M1 marks the beginning of main muscle activation and M2 defines return to baseline.

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with respect to multiple comparisons. At first a two-sided dependent samples t-statistic was calculated on a cluster-level by taking the sum of the t-values within the respective cluster. By randomly permuting the data across the pre and post conditions 1000 times and recalculating the test statistic each time, we obtained a data driven reference distribution of maximum cluster t-values to evaluate the statistic of the observed data. A p-value was obtained by calculating the proportion of random partitions that resulted in a larger test statistic than the observed one. Pre and post intervention data were concluded to be significantly different if the p-value was smaller than the critical alpha-level (0.025 for the two-sided test). As known from former MEG-studies on swallowing (Furlong et al., 2004; Loose et al., 2001; Teismann et al., 2011a) tongue movement during swallowing produced an ERS-like source reconstruction artifact located at the base of the brain and reaching into the inferior temporal lobe bilaterally, making it difficult to study activation in subcortical and bulbar structures. It had to be excluded from statistical comparison because its power exceeded that of task-related cortical ERD. Therefore the statistical test was calculated within a predefined region of interest (ROI) consisting of frontal and parietal cortical areas as previously published (Suntrup et al., 2013a). Thereby we included those brain regions that had consistently been found to be associated with swallowing in prior neuroimaging studies (Dziewas et al., 2003; Furlong et al., 2004; Hamdy et al., 1999a; Kern et al., 2001b; Martin et al., 2001; Teismann et al., 2009a) and can reliably be localized with MEG. The ROI was defined using the WFU-PickAtlas tool as follows: Medial Frontal Gyrus, Middle Frontal Gyrus, Superior Frontal Gyrus, Anterior Cingulate, Insula, Precentral Gyrus, Postcentral Gyrus, Paracentral Lobule, Superior Parietal Lobule and Inferior Parietal Lobule. Results Pharyngeal electrical stimulation Table 1 shows group mean values for perceptual threshold, maximum tolerated threshold and calculated optimal stimulation intensity. The latter is equal to the actually delivered stimulation intensity in the real intervention group. A paired samples t-test did not show any significant differences between both study groups (p N 0.05). Water swallowing test Group mean values of the 150 ml-water swallowing stress test pre and post intervention are shown in Table 2. Univariate 2 × 2 ANOVA revealed a significant main effect of intervention on swallowing capacity (F(1,13) = 5.193 p = 0.040) and a significant interaction (intervention × measurement block) on volume per swallow (F(1,13) = 6.216; p = 0.027) and swallowing capacity (F(1,13) = 14.592; p = 0.002) with greater volume per swallow and swallowing capacity after real PES compared to the sham intervention. There was also a significant main effect of the measurement block (pre vs. post intervention) with longer swallow duration in the second measurement block in both stimulation conditions (F(1,13) = 4.911; p = 0.045). Baseline differences between the two sessions were excluded by dependent samples t-tests on pre intervention swallowing test parameters, which did not yield significance.

Table 1 Results of the PES thresholding procedure to obtain optimal stimulation intensity (n = 14 subjects; mean ± SD).

Perceptual threshold (mA) Maximum tolerated threshold (mA) Calculated optimal stimulation intensity (mA)

Real PES

Sham PES

p-Value

5.9 ± 2.6 11.1 ± 4.4 8.8 ± 3.9

6.4 ± 2.9 10.5 ± 3.2 8.5 ± 3.1

0.396 0.638 0.761

Table 2 Results of the 150 ml-water swallowing stress test pre and post intervention (n = 14 subjects; mean ± SD). Pre (block 1)

Post (block 2)

Volume per swallow (ml) Real PES Sham PES

31.8 ± 10.4 34.5 ± 12.0

41.2 ± 17.2 33.3 ± 11.4

Time per swallow (ms) Real PES Sham PES

1.14 ± 0.21 1.25 ± 0.22

1.30 ± 0.34 1.27 ± 0.31

Swallowing capacity (ml/s) Real PES Sham PES

28.0 ± 7.3 27.1 ± 6.2

30.3 ± 7.4 26.0 ± 6.1

Behavioral data during MEG recordings Time from the end of PES to the beginning of the second 15-minute MEG measurement block was 6.7 ± 2.4 min. Therefore, the last MEG recording in block 3, starting another 15 min after the end of block 2, captured cortical activity from around 40 to 55 min after the end of PES. Due to technical reasons MEG measurement block 3 could successfully be recorded in both sessions with 9 out of 14 subjects only. Head movement during MEG recordings was 4.8 ± 3.0 mm in mean. Univariate ANOVA revealed no significant differences (main effects or interactions) in head movement between sessions or conditions in the 14 (F(1,13) = 0.219; p = 0.648) or 9 subject group (F(2,16) = 0.181; p = 0.212 (Greenhouse–Geisser corrected)). The participants' EMG swallowing parameters are presented in Table 3. There were no significant main effects or interactions (intervention × block) regarding swallow duration (14 subjects: F(1,13) = 2.050; p = 0.176; 9 subjects: F(2,16) = 0.842; p = 0.449), EMG power (14 subjects: F(1,13) = 0.546; p = 0.473; 9 subjects: F(2,16) = 0.834; p = 0.452) or EMG peak-to-peak amplitude (14 subjects: F(1,13) = 0.233; p = 0.637; 9 subjects: F(2,16) = 0.586; p = 0.568) between pre and post intervention measurements.

Cortical swallowing activation Baseline data Source distributions of averaged event-related desynchronization in cortical oscillatory activity are presented in Figs. 3a and b for the 14 (pre and post measurement) and 9 subjects (pre, post 1 and post 2 measurement) groups separately. Swallowing-related ERD ranged from theta up to the low gamma frequency band and was most prominent in the alpha and beta frequency range. No task related activation changes were Table 3 Electromyographic swallowing parameters (mean ± SD). Pre (block 1)

Post (block 2)

Post2 (block 3)

Swallow duration (s) Real PES (n = 14) Real PES (n = 9) Sham PES (n = 14) Sham PES (n = 9)

2.53 2.76 2.33 2.54

± ± ± ±

0.57 0.50 0.63 0.65

2.40 2.55 2.41 2.61

± ± ± ±

0.61 0.58 0.71 0.75

– 2.68 ± 0.73 – 2.68 ± 0.61

EMG power (μV) Real PES (n = 14) Real PES (n = 9) Sham PES (n = 14) Sham PES (n = 9)

77.1 88.9 70.0 80.8

± ± ± ±

48.0 54.5 31.5 32.6

77.6 92.2 56.0 61.5

± ± ± ±

57.7 63.0 20.5 20.8

– 107.3 ± 70.6 – 65.3 ± 32.1

147.4 138.6 150.7 116.0

466.7 483.6 417.9 403.9

± ± ± ±

206.7 210.6 140.7 118.7

– 541.6 ± 208.7 – 416.9 ± 111.0

EMG peak-to-peak-amplitude (μV) Real PES (n = 14) 463.2 ± Real PES (n = 9) 469.2 ± Sham PES (n = 14) 443.3 ± Sham PES (n = 9) 435.2 ±

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Fig. 3. a–b: Source distribution of group mean swallowing-associated activation per frequency band of all 14 subjects pre and immediately post intervention (a) and of the 9 subjects, who performed all three MEG measurements pre, immediately post and 45 min post intervention (b). The color bar indicates power changes relative to the resting stage. Negative values denote event-related desynchronization of oscillatory activity. The asterisks indicate statistically significant differences.

found in the high gamma range at all. Swallowing-associated activation was predominantly localized in the pericentral cortex bilaterally, congruent with primary and secondary sensorimotor areas.

Intervention effect A significant power decrease of ERD was found in the alpha and beta frequency ranges in the MEG data recorded immediately after real PES within the 9 subject group (p b 0.05 and p b 0.001, respectively) and the 14 subject group (p b 0.05 for both frequency ranges). In contrast to that comparison of baseline MEG data with the last measurement block 45 min post intervention in the subgroup of the nine subjects, who performed all three MEG measurements, revealed no significant differences in cortical activation pattern after any stimulation condition. No significant effects occurred in any other frequency band or after

sham stimulation. Moreover, no areas with significant power increase of ERD were found. The attenuation of ERD in the alpha frequency range (Fig. 4, left) was restricted to the right hemisphere with a negative peak (t-value: −8.342) in the caudolateral precentral gyrus (Brodmann area (BA) 4). Prominent decrease of ERD power after stimulation was also found in the insula (BA 13), the primary sensorimotor cortex (BA 1–4), preand supplementary motor areas (PMC, SMA, BA 6), the inferior parietal lobe (IPL) including the supramarginal gyrus (BA 40), the frontoparietal operculum (BA 43, 44), and the dorsolateral prefrontal cortex (DLPFC, BA 9). In the beta frequency range (Fig. 4, right) areas showing a decrease of ERD were also predominantly found in the right hemisphere with a negative peak (t-value: − 3.999) in the superior frontal gyrus (PMC, BA 6). Reduction of oscillatory beta power was most prominent in the

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Fig. 4. Brain areas with significant decrease of task related ERD in the alpha (8–13 Hz, p b 0.05) and beta (13–30 Hz, p b 0.05) frequency bands immediately after real PES in all 14 subjects. Superimposed onto the MRI slices are the t-values for the difference in source power pre and post intervention, masked with the p-values obtained from the cluster-based randomization procedure.

adjacent SMA (BA 6), the right more than the left DLPFC (BA 9) and the anterior cingulate (BA 32) of both hemispheres as well as in the superior and medial frontal gyri (BA 8 and 10, right more than left). Attenuation was less pronounced, but still statistically significant in right primary sensorimotor cortices (BA 1–4) and parts of the superior (BA 5, 7) and inferior parietal lobe including the supramarginal gyrus (BA 40). Discussion This is the first study to investigate the effects of PES on the largescale oscillatory swallowing network. According to our findings PES induced a transient right-dominant attenuation of cortical swallowingrelated desynchronization (ERD) in oscillatory alpha and beta frequency ranges that were associated with subtle improvements of performance in a water swallowing stress test in healthy subjects. Baseline swallowing activation pattern Task-related activation changes were found in bilateral sensorimotor areas, which is a highly consistent finding among functional neuroimaging studies on swallowing (Michou and Hamdy, 2009) and therefore corroborates the validity of the observed activation pattern. In line with former MEG studies by our group (Dziewas et al., 2009; Suntrup et al., 2013a, b; Teismann et al., 2009a, 2010) and others using fMRI, PET and MEG (Furlong et al., 2004; Hamdy et al., 1999b; Malandraki et al., 2009) activation spread into secondary sensorimotor areas and regions involved in multisensory and sensorimotor integration as well. Similar to a previous study applying comparable methods in MEG data acquisition and analysis, swallowing-related ERD centered in the alpha and beta frequency bands but was also found in theta and low gamma frequency ranges (Suntrup et al., 2013a). PES-induced changes of swallowing network oscillations The extent of ERD during a motor task is related to task complexity and skill, reflecting energetic processes such as allocation of planning resources, arousal and effort (Manganotti et al., 1998; Neuper and Pfurtscheller, 2001; Studer et al., 2010). In a previous study by our group ERD increased with the complexity of different swallowing tasks (Suntrup et al., 2013a). Attenuation of movement-related oscillatory alpha and beta power was proven to be a neuronal correlate of motor learning (Kranczioch et al., 2008; Studer et al., 2010; Zhuang et al., 1997), indicating that task execution becomes less attention demanding and effortful as a result of increasing task automaticity (Studer et al., 2010). Although a variety of studies on motor training have been

conducted, data on oscillatory power changes following sensory stimulation, as has been done here, is scarce. However it is known that sensory afferents strongly modulate motor output in swallowing (Lowell et al., 2008). Gow et al. (2004) observed that after a single PES pulse motor activity peaked 5 ms later than the equivalent sensory cortex activity, indicating an intra-cortical serial network, with input from the pharynx arriving in the sensory cortex before transmission to the motor cortex. Thus, in analogy to studies on motor skill acquisition by training one may conclude that attenuation of ERD following PES also reflects reduction of cortical swallowing processing demands and/or increased processing efficiency in our subjects. Because EMG swallowing characteristics did not differ between single MEG sessions, it is unlikely that the observed cortical changes can be attributed to varieties in task performance. In our study the PES-induced changes occurred in the alpha and beta bands. There is strong evidence that in the generation of alpha rhythms both thalamo-cortical as well as cortico-cortical loops play an important role, whereas beta activity appears only in the cortex (Lopes da Silva, 1991; Neuper and Pfurtscheller, 2001). Taken together, intracortical swallowing-relevant connections and pathways via the thalamus to interconnected subcortical areas may have been facilitated by PES resulting in an ‘optimal oscillatory state’ of the network (Zhuang et al., 1997). Similar changes in cortico-cortical coupling with regard to motor learning have previously been shown (Kranczioch et al., 2008). Our results are also compatible with the findings from earlier TMS studies showing that PES leads to changes in corticobulbar excitability (Fraser et al., 2002; Hamdy et al., 1998b) without changing excitability in the craniobulbar circuitry (Fraser et al., 2003). However, due to the insensitivity of MEG towards brain activation from deep sources we cannot contribute to this hypothesis any further. The finding of reduced ERD during swallowing after PES does not contradict former TMS studies, which showed increased excitability of the pharyngeal motor cortex (Fraser et al., 2002; Hamdy et al., 1998b). Several studies have demonstrated that ERD/ERS changes do not translate 1:1 into changes of cortical excitability (Rau et al., 2003). They may not correspond in time and location (Leocani et al., 2001) and ERD changes can be associated with both corticospinal facilitation and inhibition (Leocani et al., 2001). Discrepancies result from the specific sensitivity of each method to different electrophysiological phenomena: While MEG assesses the oscillatory aspects of cortical activity representing summed activation of apical dendrites from pyramidal cells, TMS is sensitive to parameters such as changes of the resting membrane potential and differential modulation of GABAergic and glutamatergic intracortical inhibition or facilitation (Rau et al., 2003). This may also explain, why activation changes in our study had almost returned to baseline at 40–55 min

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after PES, whereas Fraser et al. (2003) observed maximal facilitation of the pharynx at 60 min in their TMS study. As suggested by Romero et al. (2008) our MEG results may represent different aspects of PESinduced neuronal plasticity from those previously detected with TMS. Localization and lateralization of PES-induced changes Attenuation of ERD was clearly lateralized to the right hemisphere. There is considerable evidence from functional imaging (Li et al., 2009; Suntrup et al., 2013a) and lesion studies (Daniels et al., 1996; Robbins et al., 1993) that specific components of swallowing are differentially lateralized. Damage to the left hemisphere was found to be associated with oral-stage dysfunction whereas right-hemispheric stroke caused pharyngeal-stage dysfunction (Daniels et al., 1996; Robbins et al., 1993; Smithard et al., 1997). A time-dependent hemispheric shift of cortical swallowing-related activation from the left in the oral stage to the right in the pharyngeal stage has been demonstrated with MEG (Teismann et al., 2009a). Because electrical stimulation was applied to the pharynx in our study, it is conceivable that the intervention predominantly affected the pharyngeal phase of swallowing, which seems to be dominated by the right hemisphere. Changes were observed in the alpha and beta frequency ranges with alpha changes displaying stronger lateralization than beta alterations. While oscillations in the alpha frequency range have been linked to somatosensory and less to sensorimotor processing, the latter seems to be particularly sensitive to the motor components of a task (Studer et al., 2010). Because swallowing is a motor response that is evoked by peripheral sensory inputs to an integrative neural network (Theurer et al., 2009), one may hypothesize that pharyngeal afferent information primarily modulated sensory alpha activity in the right-dominant pharyngeal area which in turn affected motor beta oscillations, that – although still right-dominant – involved swallowing-relevant areas of both hemispheres, possibly via transcallosal connections. Most prominent activation changes were found in the caudolateral motor cortex, similar to Gow et al. (2004), who localized peak activation in the same area after applying single PES pulses. Besides primary sensorimotor regions, further swallowing-relevant areas that are known to be strongly interconnected showed an attenuation of ERD. Their functions shall shortly be discussed: The anterior cingulate has connections with brainstem autonomic nuclei and plays a role in visceromotor control (Aziz et al., 2000). The insula is a multifunctional region that is involved in the sensory processing of oropharyngeal stimuli (Rolls, 2005). Due to its connections with sensory and motor areas it is well suited for multisensory and sensorimotor integration (Augustine, 1996) and likely coordinates the temporal sequence of oropharyngeal movements during a swallow (Martin et al., 2001; Mosier et al., 1999). The (fronto-)parietal operculum cortex (BA 43, 44) is strongly interconnected with the insula and is said to constitute a secondary somatosensory cortex. Its damage is known to cause severe pharyngeal phase dysphagia (Alberts et al., 1992; Weller, 1993). The IPL and the adjacent supramarginal gyrus have been suggested to process proprioceptive feedback from the oral cavity and to integrate it with ongoing motor output (Malandraki et al., 2009). Together with the DLPFC and PMC, the IPL constitutes an interface between perception of body signals, attentional control and higher order sensorimotor processing (de Lange et al., 2010; Frith et al., 1991). Finally, movement initiation and execution are major functions of the SMA (Nachev et al., 2008). Taken together, PES effects were not restricted to the pharyngeal sensory and motor cortex but involved the whole swallowing network. Prominent attenuation of ERD was found in network hubs for multimodal integration and higher order sensorimotor processing. Methodological considerations There are more sophisticated methods such as endoscopy or videofluoroscopy than a simple water swallowing screening test available

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to evaluate the effect of PES on swallowing parameters. However, the focus of this study lay on the neuronal changes induced by PES. Changes in swallowing function were subtle but statistically significant. We attribute this to the fact that young healthy volunteers were tested, in whom intact swallowing is hard to improve with a single experimental intervention. Based upon our data the specificity of the PES-induced effect on swallowing processing cannot be confirmed because we did not use any control stimuli such as tactile or gustatory stimulation in this pilot MEG study. Moreover, it is known that swallowing-related motor tasks, e.g. tongue movements, contribute to the sum of activated cortical regions during swallowing (Kern et al., 2001a). In the present experiment we cannot definitely relate details of the observed activation pattern to separate components of the swallowing movement. In addition, it is conceivable that PES-induced changes in subcortical structures or brainstem have been missed because of the technical limitations of MEG. Detailed effects of PES on clinical swallowing parameters and neuromodulatory changes in deeper brain structures involved in swallowing will be subject to future investigations applying different methodologies. Conclusion In summary, we were able to show that a single session of pharyngeal electrical stimulation leads to beneficial temporary changes in cortical swallowing processing, which are associated with subtle effects on the swallowing function in healthy subjects. Our data contribute evidence that the swallowing network organization and behavior can effectively be modulated by PES and support further research on this promising tool for future dysphagia therapy. Funding This work was supported by the Deutsche Forschungsgemeinschaft (DFG) [grant number TE 840/1-1]. Disclosures R. Dziewas is a member of the clinical advisory board of Phagenesis Ltd. References Alberts, M.J., Horner, J., Gray, L., Brazer, S.R., 1992. Aspiration after stroke: lesion analysis by brain MRI. Dysphagia 7 (3), 170–173. Augustine, J.R., 1996. Circuitry and functional aspects of the insular lobe in primates including humans. Brain Research: Brain Res. Rev. 22 (3), 229–244. Aziz, Q., Thompson, D.G., Ng, V.W., Hamdy, S., Sarkar, S., Brammer, M.J., Bullmore, E.T., Hobson, A., Tracey, I., Gregory, L., et al., 2000. Cortical processing of human somatic and visceral sensation. J. Neurosci. Off. J. Soc. Neurosci. 20 (7), 2657–2663. Babaei, A., Kern, M., Antonik, S., Mepani, R., Ward, B.D., Li, S.J., Hyde, J., Shaker, R., 2010. Enhancing effects of flavored nutritive stimuli on cortical swallowing network activity. Am. J. Physiol. Gastrointest. Liver Physiol. 299 (2), G422–G429. Daniels, S.K., Foundas, A.L., Iglesia, G.C., Sullivan, M.A., 1996. Lesion site in unilateral stroke patients with dysphagia. J. Stroke Cerebrovasc. Dis. 6 (1), 30–34. de Lange, F.P., Toni, I., Roelofs, K., 2010. Altered connectivity between prefrontal and sensorimotor cortex in conversion paralysis. Neuropsychologia 48 (6), 1782–1788. Ding, R., Larson, C.R., Logemann, J.A., Rademaker, A.W., 2002. Surface electromyographic and electroglottographic studies in normal subjects under two swallow conditions: normal and during the Mendelsohn manuever. Dysphagia 17 (1), 1–12. Dziewas, R., Soros, P., Ishii, R., Chau, W., Henningsen, H., Ringelstein, E.B., Knecht, S., Pantev, C., 2003. Neuroimaging evidence for cortical involvement in the preparation and in the act of swallowing. Neuroimage 20 (1), 135–144. Dziewas, R., Teismann, I.K., Suntrup, S., Schiffbauer, H., Steinstraeter, O., Warnecke, T., Ringelstein, E.B., Pantev, C., 2009. Cortical compensation associated with dysphagia caused by selective degeneration of bulbar motor neurons. Hum. Brain Mapp. 30 (4), 1352–1360. Fraser, C., Power, M., Hamdy, S., Rothwell, J., Hobday, D., Hollander, I., Tyrell, P., Hobson, A., Williams, S., Thompson, D., 2002. Driving plasticity in human adult motor cortex is associated with improved motor function after brain injury. Neuron 34 (5), 831–840. Fraser, C., Rothwell, J., Power, M., Hobson, A., Thompson, D., Hamdy, S., 2003. Differential changes in human pharyngoesophageal motor excitability induced by swallowing,

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