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Keywords:

  • cerebellar stimulation;
  • swallowing;
  • transcranial magnetic stimulation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. Conflicts of interest
  10. Study sponsor
  11. Author contributions
  12. References

Background  Animal and human brain imaging studies suggest that the cerebellum plays an important role in the control of swallowing. In this study, we probed the interaction between cerebellar and pharyngeal motor cortical activity with transcranial magnetic stimulation (TMS) to determine if the cerebellum can modulate cortical swallowing motor circuitry.

Methods  Healthy volunteers (= 16, eight men, mean age = 32, range 19–57 years) underwent TMS measurements of pharyngeal electromyography (EMG) recorded from a swallowed intraluminal catheter to assess cortical and cerebellar excitability. Subjects then underwent a paired pulse paradigm, where active or sham TMS conditioning pulses over the cerebellum and control sites were followed by suprathreshold TMS over the cortical pharyngeal area. Paired pulses were delivered at varying inter-stimulus intervals (ISIs) with the cortical response amplitudes being assessed.

Key Results  Stimulation of the cerebellum over its midline or hemispheres evoked distinct pharyngeal EMG responses. There was no difference in EMG amplitudes following cerebellar hemispheric or midline stimulation (mean 55.5 ± 6.9 vs 42.8 ± 5.9 μV, = 0.08). In contrast, after cerebellar preconditioning, the cortically evoked responses underwent maximal facilitation at ISIs of 50–200 ms (< 0.05), an effect not seen with sham or trigeminal nerve preconditioning.

Conclusions & Inferences  Posterior fossa stimulation excites the cerebellum and evokes direct motor responses within the pharynx. When conditioned with TMS, the cerebellum strongly facilitates the cortical swallowing motor pathways. This finding suggests that the cerebellum exerts a modulatory effect on human swallowing and raises the possibility that excitatory neurostimulation of the cerebellum may be therapeutically useful in promoting recovery of dysphagia after neural damage.


Abbreviations:
EMG

electromyography

GLM

generalized linear model

ISIs

inter-stimulus intervals

MEPs

motor evoked potentials

MRI

magnetic resonance imaging

ms

milliseconds

SEM

standard error of mean

TMS

transcranial magnetic stimulation

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. Conflicts of interest
  10. Study sponsor
  11. Author contributions
  12. References

There is well-described evidence that multiple regions within the central nervous system such as the cortex, brainstem, and cranial nerves play important roles in carrying out normal swallowing function. However, the physiologic role of the cerebellum in the control of human swallowing is ill-defined. Indeed, little is known about the contribution of the cerebellum in the regulation of swallow motor function. The cerebellum is thought to have a modulatory role in the coordination of movement, posture, and motor balance.1 The motor symptomatology of cerebellectomized animals, described by Luigi Luciani as early as 1891, provided limited knowledge implicating cerebellar involvement in swallowing, and more recently case studies of cerebellar pathology2,3 and functional human brain mapping have added further circumstantial evidence.4

In a feline model, Mussen5,6 was one of the earliest to suggest a role for the cerebellum in swallowing when he observed movements relating to swallowing (for e.g., contraction of the throat and overt swallowing) following cerebellar stimulation. Furthermore, electrical stimulation of the cerebellar midline structure in cats7 and deep cerebellar nuclei in rats8 was shown to alter feeding behavior and feeding regulation, respectively. More recently, a study by Zhu et al.8 on rodents suggested a wider role of the cerebellum in modulating gastrointestinal functions via interactions with gut hormones such as cholecystokinin9 and via projections to the hypothalamus.10 However, despite these findings, further detailed neurophysiologic experiments in animals in the evaluation of the cerebellum are still required to further probe the role of the cerebellum in the swallowing process.

Neuroimaging of the healthy human brain with positron emission tomography has demonstrated increased regional cerebral blood flow in the cerebellum during swallowing.11,12 Several studies using functional magnetic resonance imaging have also reported cerebellar involvement in swallowing.4,13 More recently, both cerebellar hemispheres were seen to activate during tasks that induced elevation of the larynx, an action crucial for airway protection during swallowing.14

The third source of evidence to implicate an interaction between the cerebellum and swallowing comes from pathophysiologic observations. Several studies in stroke have shown that cerebellar infarcts can be associated with dysphagia.2,3 The rare neurodegenerative condition of spinocerebellar ataxia is sometimes associated with dysphagia, and patients often die following aspiration pneumonia.15 There is also evidence that surgical removal of posterior fossa tumors in children can increase the chances of postsurgical dysphagia, albeit with good long-term recovery.16

Although the above evidence suggests a role for the cerebellum in the swallowing process, the physiologic relationships within cerebellar-cortical pathways are yet to be reported in human literature. One method to probe this interaction is non-invasive transcranial brain stimulation. Indeed, Ugawa et al. demonstrated that it is possible to non-invasively stimulate hand areas of the cerebellum using electrical stimulation. This group then successfully applied transcranial magnetic stimulation (TMS) to the intact human cerebellum while recording from the upper limb.17,18 The latter study showed that pairing TMS pulses over hand motor cortex with prior magnetic stimulation over the cerebellum induced inhibitory changes to hand motor responses.18 This suppressive effect of the cerebellum on hand motor function has been reproduced by others using similar paired pulse paradigms.19 Although there have been no equivalent experimentation in the swallowing motor system, short latency responses to cortical stimulation have been used to study swallowing and explore facilitation to cranial nerve stimulation.20

Thus, the aim of this study was to firstly determine if stimulation of the cerebellum could induce activation of the pharyngeal muscles. Thereafter, the study assessed the effects of prior cerebellar stimulation on cortico-bulbar projections to the pharynx using a paired pulse paradigm. Our hypotheses were that (i) cerebellar stimulation would induce motor responses in the pharynx, and that (ii) preconditioning with cerebellar stimulation would facilitate the cortical swallowing projections.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. Conflicts of interest
  10. Study sponsor
  11. Author contributions
  12. References

Participants

Sixteen healthy right handed subjects participated in this study (age range 19–57 years, mean 32 years, eight men, eight women). All subjects were in good health, gave written consent and complied with exclusion criteria (history of epilepsy, cardiac pacemaker, swallowing problems, pregnancy, metal in the head or eyes or use of medication which decreases seizure threshold). The experimental procedures conformed to the World Medical Association Declaration of Helsinki, and approval was granted by the Stockport Research Ethics Committee. Studies were conducted in the clinical laboratories of the Gastrointestinal Sciences Department of Salford Royal NHS Foundation Trust, United Kingdom.

Power calculation

From previous work with TMS suggesting a 40% effect size, a sample size calculation revealed that a minimum of 12 subjects were needed to achieve a power of 80% and statistical significance of 5% to demonstrate difference in effects between interventions, Hence, 16 people were recruited to allow for subject dropout.

Procedures

Cortical and cerebellar single pulse TMS  Cortical single pulse TMS over the vertex was delivered through a Magstim 2002 stimulator (Magstim Company, Whitland, Wales, UK) connected to circular magnetic coil with an outer diameter of 90 mm, providing stimulation of the pharyngeal motor cortices up to a maximum of 2.0 T as previously described.20

Single pulse TMS of the cerebellum was achieved through a figure of eight magnetic coil with an outer diameter of 70 mm, which produced a maximum output of 2.2 T. The coil was positioned over the posterior fossa of the head, tangentially to the scalp with the handle pointing superiorly, which induced downward current in the cerebellar cortex as described elsewhere21 (Fig. 1). Sham stimulation was delivered to the cerebellum by tilting the coil 90º with only the edge of one wing of the figure of eight coil in contact with the head.22

image

Figure 1.  Schematic representation showing the position of the figure of eight magnetic coil over the posterior fossa for cerebellar stimulation. In (A) the central part of the coil is positioned 1 cm below the inion to stimulate the midline region of the cerebellum. In (B), the coil is positioned either to the right or to the left of the inion for the stimulation of cerebellar hemispheres. In both cases, the coil was positioned tangentially to the scalp, with the handle pointing superiorly.

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Cranial nerve stimulation  Cranial nerve stimulation was achieved using a smaller, 50 mm diameter, figure-eight coil, which allowed focal stimulation of an area of tissue 2 cm2 beneath it, to a maximum intensity of 2.0 T. To stimulate the trigeminal nerve, we used the supraorbital nerve on each side, with the coil windings positioned to cross the supraorbital foramen tangentially with the handle of the coil pointing laterally. The choice of nerve branch relates to its anatomic accessibility that allowed the selective activation of a purely afferent branch of the trigeminal nerve that consistently evoked a stable and bilateral blink reflex.23 This observation confirmed that stimulation of the nerve had occurred. The medial border of the coil was placed 2 cm from the midline to minimize the chance of bilateral stimulation of trigeminal afferents, as described elsewhere.24

Paired pulse stimulation  Paired pulse stimulation was used to assess the effects of cerebellar stimulation on cortico-pharyngeal pathway excitability. In this paradigm, comprising both conditioning and test pulses, TMS of either the cerebellum or cranial nerve was followed by TMS of pharyngeal motor cortex and performed with two Magstim 2002 stimulators that were connected via a Magstim Bistim module (Magstim Company). The output of each of the two Magstim 2002 stimulators were connected to the 90 mm round coil and the figure of eight coils, respectively, and used to stimulate the trigeminal nerve or cerebellum followed by the cortex. In a similar way as described by Kujirai et al.,25 paired pulses of TMS consisting of a conditioning pulse and test pulse were delivered sequentially at ISIs of 3, 5, 9, 20, 50, 100, and 200 ms in a random order as assigned by Signal software version 4.0 (Cambridge Electronic Design Ltd, Cambridge, UK). Conditioning pulses to the cerebellar hemisphere with the greatest pharyngeal amplitude were delivered at suprathreshold intensity (110% of pharyngeal motor threshold). Test pulse stimulations to the pharyngeal motor cortex were also delivered at 110% of pharyngeal motor threshold. The Signal software was programmed to discharge three pairs of TMS pulses at each ISI in addition to three single test pulses, giving 24 pulses that were randomly assigned.

Pharyngeal EMG measurements  A 3.2 mm diameter intraluminal catheter (Gaeltec Ltd, Dunvegan, Isle of Skye, UK) was swallowed by subjects either transnasally or transorally depending on subject’s preference. The catheter housed a pair of bipolar platinum ring electrodes that were positioned in the pharynx (∼14–15 cm aboral from the incisors or nasal flare) to record electromyographic (EMG) traces. The catheter was connected via a preamplifier and interface to a personal computer that recorded the traces using ‘Signal’ Application Program. An earth lead was connected to a skin electrode sited over the upper portion of one of the sternocleidomastoid muscles of the neck. The catheter was connected via a preamplifier (CED, Cambridge Electronic Design Ltd), amplifier (CED 1902) and interface (CED 1401) to a personal computer that allowed real time visualization and recording of the traces using the Signal Application Program (CED). This had filters set at 200 Hz–2 kHz and allowed a sampling rate of 4–8 kHz. Response signals were processed through a 50/60 Hz noise eliminator (HumBug; Quest Scientific, North Vancouver, British Columbia, Canada) to remove any unwanted electrical interference. Analysis of the amplitude and latencies of the traces was conducted using Signal program.

Experimental protocol 1: effects of cerebellar and cortical stimulation on pharyngeal muscle excitation  All subjects were seated in a comfortable chair and had a pharyngeal catheter inserted as previously described. The cranial vertex and inion were identified and marked on the scalp. The round coil was positioned at the optimal site overlying the vertex and discharged to identify the baseline pharyngeal motor threshold with cortical stimulation. Cortical stimulation was commenced at an intensity of 30% of stimulator output and increased by steps of 5% until responses were obtained. The pharyngeal motor threshold was defined as the minimum stimulation intensity that evoked pharyngeal motor evoked potential (MEP) >20 μV in at least 5 of 10 trials. Ten pharyngeal MEPs (PMEP) were then acquired with TMS at 110% of motor threshold at the pharyngeal site.

To identify the pharyngeal motor threshold to cerebellar stimulation, the figure of eight coil was positioned at the back of the scalp in the orientation described previously. The point where the windings of the coil intersected was positioned 1 cm below the inion (for stimulation of cerebellar midline) and between 2 cm and 4 cm lateral to midline (for stimulation of cerebellar hemispheres) to elicit PMEPs. The position of TMS coil to stimulate the cerebellum was also confirmed in two subjects with the use of MRI guided frameless stereotaxy (Brainsight 2; Rogue Research, Montreal, Canada) by co-registering the TMS coil positions over the midline and cerebellar hemispheres with the subjects’ whole brain magnetic resonance image (Fig. 2). Pharyngeal motor threshold was defined as the minimum stimulation intensity that evoked PMEP >20 μV in at least 5 of 10 trials of single pulse TMS over each cerebellar site (midline, right, and left hemispheres). Pharyngeal motor evoked potential was assessed by giving TMS at 110% of motor threshold, with 10 stimuli given at the three cerebellar sites. The cerebellar site that had the lowest motor threshold and most easily identifiable EMG responses to stimulation was then used during paired pulse stimulation in experiment 3 (see below).

image

Figure 2.  Cerebellar stimulation sites co-registered to a subject’s magnetic resonance imaging (MRI), using frameless fMRI stereotaxy (Brainsight 2; Rogue Research, Montreal, Canada). The external colored cone marker on the head reconstruction indicated the central point of the coil, and the grey arrows indicate the shortest perpendicular distance between the coil and the cerebellar region targeted. In the MRI brain slices, the cerebellum is outlined in grey line. In (A), the figure of eight magnetic coil was positioned as such that the central windings were 1 cm below the inion to stimulate the cerebellar midline. In (B), the coil was positioned 1 cm below the inion and 4 cm to the right of the midline for the stimulation of the right cerebellar hemisphere.

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Experimental protocol 2: effects of cranial nerve stimulation on pharyngeal muscle excitation  Following experiment 1, each subject proceeded to experiment 2. The 50-mm diameter, figure-eight coil was discharged over the supraorbital nerve on both sides as previously described. Stimulation was commenced at 30% of stimulator output and increased by steps of 5% of stimulator output until stable and quantifiable EMG responses were evoked in at least 5 of 10 trials. This intensity was then defined as the motor threshold stimulus intensity for the nerve. Each nerve was then studied by the application of 10 single pulse TMS at 110% of motor threshold, ensuring that consistent EMG responses were obtained while confirming stable bilateral blink reflex. Data from the supraorbital nerve that had the lowest motor threshold and most easily identifiable EMG morphology of the two nerves was then used during paired pulse stimulation in experiment 3 (see below).

Experimental protocol 3: effects of cerebellar and cranial nerve stimulation on TMS evoked pharyngeal cortical excitability  Following experiments 1 and 2, each subject then proceeded to experiment 3. The paired pulse stimulation experiments consisted of a conditioning pulse followed by a test pulse. In all three conditions studied (cerebellar, trigeminal, sham), the TMS test pulse was applied to pharyngeal motor cortex at 110% of motor threshold. In condition 1, the conditioning pulse was discharged at the cerebellar site identified earlier. In condition 2, the sham conditioning pulse with coil tilt was discharged over the cerebellar site. In condition 3, the conditioning pulse was discharged over the supraorbital nerve. As describe above, paired pulses of TMS consisting of a conditioning pulse and test (cortical) pulse were delivered sequentially at ISIs of 3, 5, 9, 20, 50, 100, and 200 ms in a random order as assigned by Signal software version 4.0. Three pairs of TMS pulses at each ISI in addition to three unconditioned single test pulses were randomly delivered giving a total of 24 pairs of pulses.

Data analysis

Response amplitude: the maximum peak to peak voltage of the EMG response is expressed in microvolts (μV).

Response latency: the interval between the onset of the stimulus and the onset of the EMG response is expressed in milliseconds (ms).

The latencies and amplitudes of individual PMEPs in each group of 10 EMG traces for single pulse TMS were determined and then averaged. Graphs show data normalized to baseline and expressed in the results as a percentage change from baseline, whereas error bars refer to standard error of mean (±SEM).

In experiment 3, each TMS pulse over the pharyngeal cortex was preconditioned with pulses over either the cerebellum or cranial nerve. As described previously, preconditioning was also tested with a sham stimulus over the cerebellum. Percentage change in amplitude and latency of pharyngeal MEPs for the three conditions (cerebellum, sham, and trigeminal nerve) were recorded across the seven different ISIs (see Procedures above) and compared with the pharyngeal MEPs elicited with test pulse over cortex alone (baseline).

Statistical methods

Statistical analysis was performed using spss 15.0 (SPSS Inc., Chicago, IL, USA). Non-sphericity was corrected using Greenhouse-Geisser when necessary. In study protocols 1 and 2, statistical analysis of untransformed data was carried out using Friedman’s anova to compare means of amplitude and latency elicited from the cortex, cerebellar, and cranial nerve sites. Post hoc head to head comparisons of amplitude and latency between cerebellar stimulation and cortical or cranial nerve stimulation were conducted using Wilcoxon’s signed-rank sum test. In study protocol 3, percentage change of excitability (in amplitude and latency) with paired pulse stimulation was analyzed using a generalized linear model (GLM) repeated measures anova with inter-stimulus interval (ISI) (3–200 ms) and SITE (cerebellum, sham cerebellum, and cranial nerve) as within patient variables. Post hoc comparisons of individual ISIs with test pulse were analyzed with paired t-test. Statistical significance was set at < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. Conflicts of interest
  10. Study sponsor
  11. Author contributions
  12. References

There were no adverse incidents associated with TMS to any of the brain and nerve sites studied. In some subjects, cerebellar stimulation produced a twitch of posterior neck muscles, which was well tolerated. In all subjects, TMS over the cerebellum sites evoked biphasic or triphasic PMEPs that were reproducible, Fig. 3. Stimulation over the branch of the trigeminal nerve produced both an early and late PMEP responses that had distinctive and different morphology and latency from cerebral and cerebellar stimulation, Fig. 4. The late PMEP responses from trigeminal nerve branch stimulation were more consistently evoked compared with the early responses.

image

Figure 3.  Sample pharyngeal motor evoked potential (MEP) traces from a subject obtained with single pulse TMS over the cortex (representing the pharyngeal area), cerebellar midline and cerebellar hemispheres. The gray traces indicate overdrawing of traces from 10 trials, whereas the red trace is the averaged MEP.

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image

Figure 4.  Sample pharyngeal motor evoked potential (MEP) traces from a subject obtained with single pulse transcranial magnetic stimulation over the supraorbital nerves (branch of trigeminal nerve). The gray colored traces indicate overdrawing of traces from 10 trials, whereas the red trace is the averaged MEP.

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The mean (±SEM) horizontal distance between the midline of the back of the head (inion) to the site for right and left cerebellar stimulation was 4.2 cm (±0.4) and 4.5 cm (±0.3), respectively. The average resting motor thresholds were ∼70% of stimulator output (range 54–85%) for pharyngeal motor cortex, ∼65% (range 52–85%) for cerebellar midline, ∼60% (range 46–78%) for right cerebellar hemisphere and ∼61% (range 44–88%) for left cerebellar hemisphere, respectively. The average resting motor threshold for the right and left cranial nerve branches were ∼46% (range 34–73%) and ∼47% (range 28–63%) of stimulator output, respectively.

Experiments 1 and 2: effects of single pulse TMS

Effects of cerebellar and cortical stimulation  The mean amplitudes (±SEM) for pharyngeal MEP following stimulation of the cerebellar midline (vermis), right cerebellum and left cerebellum were 42.8 (±6.3), 59.3 (±10.0), and 51.7 (±7.0) μV, respectively. In comparison, the mean amplitude (±SEM) for pharyngeal MEP following cortical stimulation was 72.4 (±6.3) μV. There was no significant difference in amplitude across the three cerebellar stimulation sites as suggested by Friedman’s anova, χ2(2) = 5.4, = 0.072. However, pharyngeal MEP amplitudes from the cerebellum were smaller than from the cortex, = 5, = 0.003, r = −0.85 (Wilcoxon’s signed-rank sum test).

The mean latencies (±SEM) for pharyngeal MEP following stimulation of the cerebellar midline (vermis), right cerebellum and left cerebellum were 10.0 (±1.2), 8.3 (±0.7) and 8.5 (±1.1) ms, respectively. In comparison, the mean latency (±SEM) for pharyngeal MEP following cortical stimulation was 9.5 (±0.4) ms. There was no significant difference in latency between all three cerebellar stimulation sites as shown by a Friedman’s anova, χ2(2) = 3.0, = 0.23. Moreover, pharyngeal MEP latencies from the cerebellum and cortex were similar, = 7, = 0.096, r = −0.43 (Wilcoxon’s signed-rank sum test).

Effects of cranial nerve stimulation  Stimulation over the right and left supraorbital branches of the trigeminal nerves produced typical early pharyngeal MEP responses with a mean amplitude of 33.8 (±5.9) and 23.7 (±2.4) μV, respectively. The late pharyngeal MEP responses from the right and left supraorbital nerves were generally larger with mean amplitude of 38.6 μV (±5.2) and 33.0 μV (±4.6), respectively. The mean latencies for the early pharyngeal MEP responses were 24.6 (±1.3) and 24.5 (±1.8) ms, respectively, for right and left nerves. By comparison, mean latencies for the late PMEP responses were 60.2 (±2.1) and 60.1 (±2.5) ms for the right and left sides, respectively.

Comparison between cerebellar and cranial nerve stimulation  Comparisons of PMEP amplitudes following stimulation of the cerebellar midline (vermis), left cerebellar hemisphere, right cerebellar hemisphere and early responses of cranial nerves was undertaken with Friedman’s anova. This showed a significant difference in amplitude across the stimulation sites, χ2(2) = 9.3, = 0.022. A post hoc analysis with Wilcoxon signed-rank test comparing the mean amplitudes of the grouped cerebellar sites with mean amplitudes of early cranial nerve responses showed a significant size difference between cerebellar and cranial nerve MEPs, = 8, = 0.010, r = −0.73.

Comparison of pharyngeal MEP latencies from stimulation of the cerebellar midline (vermis), left cerebellar hemisphere, right cerebellar hemisphere and early responses of cranial nerves was also undertaken with Friedman’s anova. This demonstrated a significant difference in latency across all the stimulation sites, χ2(2) = 24.7, < 0.0001. Furthermore, post hoc analysis with Wilcoxon signed-rank test comparing mean latencies of the grouped cerebellar sites with mean amplitudes of early cranial nerve responses showed a significant difference between cerebellar and cranial nerve MEPs, = 0, < 0.0001, r = −3.18.

Experiment 3: effects of paired pulse TMS stimulation

Cortical test pulse conditioned with cerebellar stimulation and trigeminal nerve stimulation  A representative example of PMEP traces obtained with TMS test pulse alone (cortex baseline) and with paired TMS pulses (cerebellum and cortex) from one subject is shown in Fig. 5. Paired pulse stimulation substantially increased the amplitude of MEPs following cerebellar conditioning pulses at ISIs of 50, 100, and 200 ms, as suggested by repeated measures anova that showed a significant ISI × SITE (cerebellum, sham cerebellum, cranial nerve) interaction; F(4, 66) = 4.45, = 0.003. Comparisons of individual ISIs with the cortically induced test pulse using paired t-test demonstrated that maximal facilitation in amplitude following conditioning with cerebellar stimulation occurred at ISIs of 50 ms (= 0.004), 100 ms (= 0.003) and 200 ms (= 0.001), Fig. 6. Significant facilitation in pharyngeal MEP amplitude was also seen with cranial nerve preconditioning at ISIs of 50 ms (= 0.04) and 100 ms (= 0.02) with a trend toward facilitation at 200 ms (= 0.07) when compared with test pulse stimulation using paired t-test (Fig. 6). Conditioning with sham cerebellar stimulation had no significant effect on amplitude at any ISI.

image

Figure 5.  Sample averaged pharyngeal motor evoked potential (MEP) traces from a subject following paired pulse stimulation; preconditioning pulse over cerebellum and test pulse over cortex. The baseline MEP trace was produced with only cortical stimulation (test pulse). The other traces resulted from preconditioning with cerebellar stimulation at inter-stimulus intervals (ISIs) of 3–200 ms. The cortical MEP trace of interest is highlighted in circles. Note the dramatic increase in MEP size compared with baseline at ISI of 50–100 ms.

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image

Figure 6.  Comparison of percentage change in cortical MEP amplitude (mean ± SEM) of pharyngeal motor evoked potential (MEP) from prior cerebellar (diamond inline image), sham cerebellar (square inline image) and trigeminal nerve (triangle inline image) stimulation, compared with test pulses at increasing inter-stimulus intervals (ISIs). Note the substantial increase in MEP size change at the longer ISIs to cerebellar stimulation compared with sham and trigeminal stimulation.

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There was also concomitant shortening in cortical latency following cerebellar preconditioning at ISI of 20–200 ms (Fig. 7). Evaluation of latency changes with repeated measures anova found a significant interaction of ISI × SITE (cerebellum, sham cerebellum, cranial nerve); F(6, 114) = 2.26, = 0.042. Comparisons of individual ISIs with cortically induced test pulse demonstrated that maximal shortening in latency following conditioning with cerebellar stimulation occurred at ISI of 20 ms (= 0.0002), 50 ms (< 0.0001), 100 ms (= 0.002) and 200 ms (= 0.005) using paired t-test, Fig. 7. Conversely, there was small, but consistent lengthening of latency of pharyngeal MEP when preconditioned with cranial nerve stimulation at ISIs of 5 ms (= 0.03) and at 9 ms (= 0.03), on analysis with paired t-test (Fig. 7). Preconditioning with sham cerebellar stimulation had no significant effect on latency at any ISI.

image

Figure 7.  Comparison of percentage change in cortical MEP latency (mean ± SEM) of pharyngeal motor evoked potential (MEP) from prior cerebellar (diamond inline image), sham cerebellar (square inline image) and trigeminal nerve (triangle inline image) stimulation, compared with test pulses at increasing inter-stimulus intervals (ISIs). Note the reduced latency following cerebellar stimulation at the longer ISIs vs the subtle lengthening of latency to trigeminal stimulation at shorter ISIs.

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Comparison between cerebellar and cranial nerve stimulation  To further evaluate the facilitation response to cerebellar stimulation, cortico-pharyngeal response amplitudes conditioned following trigeminal stimulation at the various ISIs were directly compared with those following cerebellar stimulation at the respective ISIs using a further anova. Repeated measures anova comparing the change in amplitude for ISI × SITE (cerebellum vs. trigeminal) showed a significant interaction, F(6, 126) = 2.67, = 0.018. This interaction suggests that the facilitation in amplitude of pharyngeal MEP was contributed independently by preconditioning with cerebellar stimulation. Moreover, post hocanova analysis revealed that the difference between cerebellum and cranial nerve amplitude occurred at ISIs of 50 (= 0.024) and 200 (= 0.032).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. Conflicts of interest
  10. Study sponsor
  11. Author contributions
  12. References

Our study systematically evaluated the role of posterior fossa activation with TMS on pharyngeal motor pathway excitability. Although there have been several studies that have demonstrated activation of limb muscles with cerebellar TMS, this study was the first to explore the physiologic role of cerebellar activity with muscles involved in swallowing. Studies of hand muscle responses have indicated that when the cerebellum is conditioned by magnetic stimuli, cortico-thenar MEPs are inhibited, suggesting a suppression of excitability.19,26,27 In contrast, our study has demonstrated that stimulation of the cerebellum seems to facilitate the excitability of the motor output from the pharyngeal area of the motor cortex. These findings merit further consideration.

Direct effects of cerebellar stimulation

We found that the optimal distance to stimulate either cerebellar hemisphere was ∼4 cm lateral to the inion. Moreover, co-registration of cerebellar stimulation sites with magnetic resonance imaging in two subjects confirmed close proximity of the magnetic coils to the cerebellum. As there is no comparable cerebellar TMS study of the swallowing muscles, and indeed, there remains controversy concerning the precise role of the cerebellum in controlling swallowing, this information will be helpful in guiding future studies of cerebellar stimulation in health and disease. Of relevance, although cerebellar stimulation of vermis or hemisphere evoked similar pharyngeal MEPs responses, they were consistently smaller when compared with cortical stimulation, despite no inter-brain site difference in latency. One explanation for the relatively long latency seen to cerebellar stimulation might lie in the pauci-synaptic pathway that a cerebellar action potential may have to take to reach swallowing muscular, possibly via brainstem structures and hind-brain nuclei.28 Our data do not support the contention that a very fast and direct pathway exists between the cerebellum and the pharynx, which may be important in considering how the cerebellum and swallowing control mechanisms interact.

Comparison of cerebellar and cranial nerve responses

Stimulation of the scalp overlying the posterior fossa has become an established method of activating the cerebellum. However, several authors have raised the probability of extra-cerebellar activation that may be responsible for the resulting MEP. Of relevance, there is no evidence from hand muscle studies that it is possible to stimulate the cortico-spinal fibers directly, so activating cortico-bulbar fibers in the brainstem seems unlikely. Moreover, direct brainstem stimulation to TMS seems unlikely for the following reasons: Firstly, the TMS magnetic field is unlikely to penetrate deeply into brain tissue. Most studies have suggested that TMS can only penetrate to between 25 and 30 mm beneath the scalp. Given that the first structure beneath the coil would be the cerebellum, it seems likely that most of the field would be dissipated by this organ. Secondly, the figure of eight coil used in our study does not generate a highly focused, high field stimulus that one might need for brainstem stimulation. Nonetheless, stimulating afferent inputs to swallowing centers could provoke a short latency reflex response, although this appears to be very different in morphology and latency from direct trigeminal reflexes. However, Meyer et al., showed that stimulation of the posterior fossa in patients with complete hemi-cerebellar agenesis and cerebellar infarction was still able to modulate cortical motor responses originating contralaterally to the site of stimulation.29 In addition, Meyer et al. showed that scalp muscles at a distance from the stimulation site can also be concomitantly activated with TMS over the posterior fossa, thus persuading the author to propose the possibility of concurrent stimulation of the brainstem or cranial nerves such as the trigeminal nerve. Taking this into account, we decided to carefully evaluate the contribution of the trigeminal input in resulting PMEPs.

Magnetic stimulation of a trigeminal nerve branch produced PMEP morphology and latency similar to that described earlier by Hamdy et al.20 These responses are different from the short latency or elementary reflexes described in animal studies to afferent stimulation.30 The MEP amplitude from late pharyngeal responses to cranial nerve stimulation was lower in this study compared with that found by Hamdy et al., but this is probably explained by the lower stimulation intensity used during our protocol (110% in this study vs 120% stimulator output by Hamdy et al.). In view of the proximity of the brainstem and cranial nerve pathways to the cerebellum, it might be expected that TMS over the posterior fossa could also stimulate some extra-cerebellar structures. It is thus reassuring that pharyngeal MEP amplitude, latency, and morphology elicited from stimulation of the cerebellum was clearly different from that produced by the stimulation of the trigeminal nerve.

There are of course, limitations on using trigeminal nerve stimulation to reflect brainstem involvement during cerebellar stimulation, namely the probability of different circuitries and mechanisms. However, even after considering these limitations, this study appears to suggest that TMS stimulation of the posterior fossa is not likely to primarily induce brainstem stimulation and hence effects on pharyngeal excitation. Indeed, only invasive monitoring of brainstem activity can confidently exclude the direct involvement of the brainstem in modulating the swallow motor pathways during TMS of the cerebellum.

Preconditioning with cerebellar stimulation facilitates pharyngeal responses

We found that cerebellar stimulation was facilitatory to the pharyngeal motor evoked responses and that the maximal facilitation of pharyngeal motor cortex following preconditioning with cerebellar stimulation occurred at ISIs of between 50 and 200 ms. Moreover, there was concomitant shortening of latency by cerebellar stimulation at equivalent ISIs. Added to this, when the effects of trigeminal stimulation were subtracted from that of cerebellar stimulation, we still observed a potent effect of cerebellar stimulation in facilitating pharyngeal motor responses. Contrary to other studies 19,26,27 of the hand motor cortex, our study showed that cerebellar stimulation was facilitatory to the pharyngeal motor cortex. Therefore, why should cerebellar stimulation be excitatory to the swallowing system, but inhibitory to the hand system?

One possible reason may reflect methodological differences. For example, in the hand studies, only a relatively short range of ISIs (3–20 ms) was tested. Hence, it is conceivable that investigation of longer ISIs may reveal a different outcome. In fact, this was supported by Daskalakis et al. who showed that cerebellar stimulation can both excite and inhibit neurons in the human motor cortex.31 However, a more plausible explanation may relate to the likelihood of the different neural network connecting the cerebellum to the swallow muscles in comparison with hand muscle. In hand muscle, for example, studies of cortical activity of areas representative of hand in primates 32 and retrograde transneuronal viral studies in primates 33 both suggest that deep cerebellar nuclei such as the dentate nuclei have disynaptic excitatory pathways to the motor cortex via the ventral thalamus.28 In addition, stimulation of the cerebellum activates Purkinje cells in the cerebellar cortex, which in turn exert an inhibitory effect on deep cerebellar nuclei that are the source of cerebellar outflow neurons.17 Pinto et al. also showed that conditioning stimulation of the cerebellum had no influence on hand MEP latency, while inhibiting the motor cortex,28 thus suggesting that the cerebellum-hand neutral network is unlikely to receive significant modulation by brainstem nuclei. This is contrary to findings of our study, which found significant shortening of latency of pharyngeal MEP, thus highlighting the likelihood of the cerebellar-pharynx network, possibly involving brainstem interneurons. Taken together, the above evidence suggests that the involvement of the brainstem and its pattern generator in the swallowing neural network may explain the contrasting effects of cerebellar stimulation when comparing the swallow and hand systems.

Hence, a key question is, at what level do the conditioning pulses of the cerebellum facilitate cortico-pharyngeal MEPs (cortex, brainstem or both)? Intriguingly, apart from animal studies,32,34,35 there have been no studies in humans that have investigated the motor output tracts from the cerebellum to help answer this question. Thus, one explanation for this question may come from some key principles in neurophysiology.36 If a brainstem motor neuron is activated by TMS, its discharge threshold will fall, with consequent shortening of the MEP latency to cortical stimulation by temporal summation of the excitatory postsynaptic potentials. Conversely, if the cortex is excited while the brainstem motor nucleus is at resting levels of activation, this will result in an increase in the amplitude of cortically evoked response without a large effect on latency.

Our study demonstrated that the application of cerebellar preconditioning increased the amplitude of PMEPs, while greatly shortening its latency, suggesting that modulation was occurring within brainstem circuitry. However, in the absence of direct recordings to confirm brainstem and cortical activity, these assumptions must remain speculative, and do not exclude a direct or indirect cortical effect.

It is interesting to note that with cerebellar preconditioning, significant reduction in latency of cortically induced pharyngeal MEP begins to occur at 20 ms ISI, just prior to the increase in amplitude that begin at 50 ms ISI. This may reflect the evolving sequence of events triggered by cerebellar stimulation as indicated by a fall in threshold of the brainstem motor neuron (reduction in latency) followed by excitation of the pharyngeal motor cortex (increase in amplitude). Alternatively, the facilitation effect may be somehow linked to the time taken for peripheral inputs to reach the cortex. For example, evidence from the swallow literature suggests that pharyngeal motor cortex is critically responsive to stimulation applied at a frequency of 5 Hz, which translates to trains of stimuli with 200 ms ISIs.37 Moreover, electrical stimuli to the pharynx seem optimally effective for inducing excitability in the pharyngeal motor cortex at 5 Hz.38 Further evaluation by the same group with paired pulse techniques also confirmed that maximal facilitation of TMS evoked PMEPs occurred when pharyngeal electrical stimulation was followed by cortical stimulation at ISIs between 50 and 100 ms (in a test range of 10–100 ms).39 Additional support of this significant ISI comes from the application of repetitive TMS at 5 Hz, which was found to be optimal at inducing long-term facilitation of the swallow motor cortex.37,40 This current paired pulse study may also help us postulate that using high frequency rTMS (between 5 and 20 Hz) to stimulate the cerebellum may be effective at inducing longer term excitation in the swallowing network. However, future studies will be required to confirm this assertion.

In conclusion, our findings suggest that magnetic stimulation of the cerebellum can evoke motor responses within the pharynx. Secondly, the application of stimulation to the cerebellum as a conditioning stimulus is facilitatory to the swallow motor pathway, this process being time dependent. In particular, these observations provide parameters suitable for future studies designed to explore manipulations to the swallow motor network following cerebellar stimulation. These findings may contribute to the application of neurostimulation to the cerebellum for the purpose of exploiting recovery mechanisms in dysphagia following brain injury.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. Conflicts of interest
  10. Study sponsor
  11. Author contributions
  12. References

Images were courtesy of Medical Imaging Department, Salford Royal Foundation Hospital. The authors thank Rupert Oliver (Speciality Registrar in Neurology, UCL, London), Satish Mistry and Emilia Michou (University of Manchester) for helping in the design of the study.

Grant support: The authors acknowledge support from the Medical Research Council (G0400979)

Study sponsor

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. Conflicts of interest
  10. Study sponsor
  11. Author contributions
  12. References

The study was sponsored by the University of Manchester, UK, which did not have a role in the study design, in the collection, analysis or interpretation of data.

Author contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. Conflicts of interest
  10. Study sponsor
  11. Author contributions
  12. References

VJ wrote the paper and performed the studies, JR helped in conceptualization and in data interpretation. SH conceptualized the study, helped with data interpretation and helped write the paper.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. Conflicts of interest
  10. Study sponsor
  11. Author contributions
  12. References