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GASTROENTEROLOGY 2011;141:827–836
CLINICAL—ALIMENTARY TRACT
Val66Met in Brain-Derived Neurotrophic Factor Affects Stimulus-InducedPlasticity in the Human Pharyngeal Motor CortexVANOO JAYASEKERAN,* NEIL PENDLETON,‡ GLENN HOLLAND,* ANTONY PAYTON,§ SAMANTHA JEFFERSON,*
MILIA MICHOU,* DIPESH VASANT,* BILL OLLIER,§ MIKE HORAN,‡ JOHN ROTHWELL,� and SHAHEEN HAMDY*
*Inflammation Sciences Research Group, Manchester Academic Health Sciences Centre, University of Manchester, Manchester; ‡Clinical Gerontology, School ofranslational Medicine, Manchester Academic Health Sciences Centre, University of Manchester, Manchester; §Centre for Integrated Genomic Medical Research,
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anchester Academic Health Sciences Centre, University of Manchester, Manchester; Sobell Department of Neurophysiology, Institute of Neurology, UCL, London,United Kingdompisd
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BACKGROUND & AIMS: Polymorphisms in brain-de-ived neurotrophic factor (BDNF) can affect brain andehavioral responses. However, little is known about theffects of a single nucleotide polymorphism (SNP) inDNF, at codon 66 (the Val�Met substitution, detected inpproximately 33% of the Caucasian population) on stim-lation-induced plasticity in the cortico-bulbar system.e examined whether this SNP influenced outcomes of
ifferent forms of neurostimulation applied to the pha-yngeal motor cortex. METHODS: Thirty-eight healthyolunteers were assessed for corticobulbar excitability af-er single-pulse, transcranial magnetic stimulation of in-uced pharyngeal electromyographic responses, recordedrom a swallowed intraluminal catheter. Thereafter, vol-nteers were conditioned with pharyngeal electrical stim-lation, or 2 forms of repetitive (1 and 5 Hz) transcranialagnetic stimulation (rTMS). Repeated measurements of
haryngeal motor-evoked potentials were assessed withranscranial magnetic stimulation for as long as 1 hourfter the 3 forms of neurostimulation and correlated withNPs at codon 66 of BDNF (encoding Val or Met). RE-
SULTS: Pharyngeal electrical stimulation significantly in-creased the amplitude of motor-evoked potentials in indi-viduals with the SNP that encoded Val66, compared to thosethat encoded Met66, with a strong GENOTYPE*TIME in-teraction (F8,112 � 2.4; P � .018). By contrast, there was aignificant reduction in latencies of subjects with the SNPhat encoded Met66 after 5-Hz rTMS (F3,60 � 4.9; P � .04).n addition, the expected inhibitory effect of 1-Hz rTMS onmplitude was not observed in subjects with the SNP thatncoded Met66 in BDNF (F7,140 � 2.23; P � .035). CON-
CLUSIONS: An SNP in human BDNF at codon 66 af-fects plasticity of the pharyngeal cortex to differentforms of neurostimulation. Genetic analysis might helpselect specific forms of neurostimulation as therapeuticsfor patients with disorders such as dysphagic stroke.
Keywords: Swallowing Disorders; Nervous System; Genet-ics; Physiology.
The capacity of the nervous system to reorganize (plas-ticity) is crucial for memory and learning,1 and is
thought to form the basis for effective rehabilitation fol-
lowing neural injury.2,3 In the last decade, transcranialmagnetic stimulation (TMS) has been used extensively invarious protocols to study cortical excitability of the hu-man motor system, which is a recognized marker of suchplasticity. Neurostimulation-based experimental proto-cols have used a number of interventions, including pe-ripheral stimulation,4,5 transcranial cortical stimulation,6
or a combination of the two.7 In this context, corticallasticity of the human motor system in health can be
nfluenced either by external factors that characterize neuro-timulation paradigms (eg, type of stimulus, frequency, anduration) or intrasubject factors (eg, circadian rhythm,8 hor-
monal changes9,10 neurologically active drugs,11 and geneticolymorphisms).12,13
Brain-derived neurotrophic factor (BDNF) is a type ofneurotrophin that exerts its effect, depending on the ma-turity of the protein (precursor of mature form) or on theclass of transmembrane receptor it activates (p75 neu-rotrophin receptor or tyrosine kinase family of recep-tors).3 The versatility of BDNF is emphasized by its con-tribution to a range of adaptive neuronal responses,including long-term potentiation, long-term depression,short-term synaptic plasticity, and in homeostasis of in-trinsic neuronal excitability.14 Like other neurotrophins,BDNF is produced as a longer precursor molecule(ProBDNF), which is cleaved into mature BDNF by extra-synaptic proteases.15 The human gene responsible for
DNF synthesis is found on chromosome 11.16 A singleucleotide polymorphism of the human BDNF gene atodon 66 produces a valine to methionine substitutionVal66Met) which is found in approximately 33% of theaucasian population.17 A key study by Kleim et al,12
found that healthy subjects with the Val66Met varianthad substantially reduced changes in upper limb motormap and post-training motor-evoked potential (MEP) re-
Abbreviations used in this paper: BDNF, brain-derived neurotrophicfactor; EMG, electromyography; GABA, �-aminobutyric acid; MEP, mo-tor-evoked potential; PES, pharyngeal electrical stimulation; rTMS, re-petitive transcranial magnetic stimulation; TMS, transcranial magneticstimulation.
© 2011 by the AGA Institute0016-5085/$36.00
doi:10.1053/j.gastro.2011.05.047
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sponse size than subjects without the Met allele followingtesting with fine motor tasks. These upper limb findingshave been further explored with functional magnetic res-onance imaging with similar results.18 Moreover, there isvidence that genetic variation in BDNF is associated witharked differences in outcomes to repetitive transcranialagnetic stimulation (rTMS) and transcranial direct cur-
ent stimulation protocols of the intact hand motor sys-em.13 Intriguingly, the direction of plastic response in
otor cortex to external stimuli is not uniform as aunction of BDNF genotype status, but appears to dependn the type of plasticity induction, with some types ofranscranial brain stimulation being more responsive inubjects with Met alleles.18
In studies of the human pharyngeal motor cortex, thereis good evidence that several neurostimulation techniquescan modulate excitability of cortico-pharyngeal pathways.Techniques such as pharyngeal electrical stimulation(PES)5,19 rTMS,20,21 paired associative stimulation,22 andranscranial direct current stimulation23 have been shown
to influence functional flexibility in the response (plastic-ity) of this system. Moreover, PES has been shown toimprove the safety of swallowing in dysphagic strokepatients when compared to sham stimulation.19 It has,however, been noticed that a proportion of healthy sub-jects and patients are nonresponders to neurostimulationin otherwise well-matched experimental conditions. Thisraises the possibility that genetic factors may be influenc-ing differences in outcomes to neurostimulation.
In this present study, we hypothesized that the BDNFVal66Met polymorphism will influence the response tocortical excitability in pharyngeal motor cortex, when con-ditioned by peripheral and cortical neurostimulation pro-tocols. A better understanding of this genetic marker hasimplications for therapeutic trials of neurostimulation indysphagia following brain injury. Thus, the aims of ourstudy were to determine if the single nucleotide polymor-phism of BDNF, Val66Met, influences the direction ofplasticity in human pharyngeal motor cortex followingPES (excitatory peripheral stimulation); 5-Hz rTMS (ex-citatory rTMS); and 1-Hz rTMS (inhibitory rTMS).
MethodsParticipantsHealthy volunteers for this study came from two
sources, volunteers from the Dyne-Steele cohort with predeter-mined BDNF genotype24 and healthy volunteers who respondedo the study advertisement. The former group were healthy olderolunteers who were part of an ongoing cognitive genetic studyndertaken by the University of Manchester, and were invited toake part in this study if they had normal swallowing function aser a validated swallowing questionnaire.25 Subjects from the
atter group provided saliva samples for analysis of BDNF ge-otype. Sixteen subjects participated in experiment 1, and 22ubjects participated in experiments 2 and 3. The volunteersere able to give written informed consent and did not meet anyf the exclusion criteria (history of epilepsy, cardiac pacemaker,
wallowing problems before the stroke, pregnancy, metal in theead or eyes, or use of medication that decreases seizure thresh-ld). The study’s ethical approval was granted by Cumbria andancashire Research Ethics Committee. Experiments were con-ucted at least 1 week apart.
ProceduresPharyngeal electromyography measurements. A
.2-mm diameter intraluminal catheter (Gaeltec Ltd, Dunvegan,sle of Skye, UK) was swallowed by subjects as described previ-usly19 (see Supplementary Material).
Single-pulse TMS. Single-pulse TMS was delivered tooth hemispheres using a Magstim 200 stimulator (The Mags-im Company, Whitland, Wales, UK) connected to a figure-of-ight coil with an outer diameter of 70 mm, which produced aaximum output of 2.2 Tesla as described by Jefferson et al21
(see Supplementary Material).PES. The bipolar ring electrodes of the pharyngeal
atheter were positioned intraluminally in the pharyngeal cavity,14 cm from the incisors or �15 cm from the nasal flare,
epending on subject preference. Pharyngeal electrical stimuli0.2-ms pulses, 280 V) were delivered at a set frequency (5 Hz),ntensity (75% of maximal tolerated), and duration (10 minutes)s reported previously (see Supplementary Material).5,19
Excitatory rTMS. A Magstim super rapid stimulatorThe Magstim Company) was used to deliver excitatory 5-HzTMS to the cortex via a figure-of-eight coil as described byefferson et al21 (see Supplementary Material).
Inhibitory rTMS. rTMS of the cortex was performedsing a Magstim Super Rapid stimulator (The Magstim Com-any) connected to a figure-of-eight coil held in an identicalrientation to single-pulse TMS. An area of focal cortical sup-ression or “virtual lesion” was then created by giving 1-HzTMS (see Supplementary Material).26
BDNF genotyping. All processes were carried out atthe Centre for Integrated Genomic Medical Research, Universityof Manchester. For volunteers from the Dyne-Steele cohort,DNA was available from previously donated blood samples,whereas for new healthy volunteers, saliva sample collection forextraction of DNA was obtained using Oragene-250 self-con-tained DNA collection kits (DNA Genotek Inc, Ontario, Canada)(see Supplementary Material).
Experiment 1Effect of BDNF polymorphism on PES-induced
plasticity in the pharyngeal motor system. Subjects(n �16, 7 male, 9 female; mean age, 78 � 4 years) sat in aomfortable, reclining chair. A disposable cap was positionedver the head and taped down to minimize movement. Theharyngeal catheter was inserted transnasally. The cranial vertexas identified and marked on the scalp. The pharyngeal motor
hot spot” was determined by discharging the magnetic stimu-ator at suprathreshold intensities over both cortices to identifyhe hemispheric site evoking the greatest pharyngeal response;his site was marked on the scalp. The pharyngeal motor thresh-ld for this hemisphere was determined using single-pulse TMS.
Following baseline recordings of MEPs from the pharyngealnd hand motor cortex using single-pulse TMS as describedreviously, the maximum-tolerated PES intensity was deter-ined using the methods described here. This was followed by
pplication of PES to the participants using optimal parameterss described by Fraser et al.5 After 10 minutes of PES, electro-
myography (EMG) amplitudes were reassessed again at 120%
motor threshold, with 10 single-pulse TMS stimuli performed
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for both transcranial sites (pharynx and hand) immediately, andat 30 and 60 minutes following PES.
Experiment 2Effect of BDNF polymorphism on excitatory (5 Hz)
rTMS-induced plasticity in the pharyngeal motor system.Subjects (n � 22, 7 male, 15 female; mean age, 60 � 20 years)underwent identical experimental set up as in protocol 1 andbaseline recordings from the pharyngeal and hand motor corti-cal hot spots were obtained. Because the magnetic output differsbetween the single-pulse and rapid stimulators, the hand motorthreshold was then re-measured with the rTMS system at thehand hot spot. This value was used to define the intensity ofrTMS to be delivered so that paradigms complied with currentsafety guidelines.27 Following this, subjects received excitatory
-Hz rTMS over the pharyngeal site as described earlier. EMGmplitudes were reassessed post-intervention at 120% motorhreshold, with 10 single-pulse TMS stimuli performed at eachf the 2 transcranial sites immediately, and at 30 and 60 minutesollowing 5-Hz rTMS.
Experiment 3Effect of BDNF polymorphism on inhibitory (1-Hz)
rTMS (“Virtual Lesion”) induced plasticity in the pharyn-geal motor system. Subjects (n � 22, 7 male, 15 female; meanage, 58 � 23 years) underwent identical experimental set up asin protocol 1, and baseline recordings from the pharyngeal andhand motor cortical hot spots were obtained. Following this,subjects received inhibitory 1-Hz rTMS over the pharyngeal siteas described earlier. EMG amplitudes were reassessed post-inter-vention at 120% motor threshold, with 10 single-pulse TMSstimuli performed at each of the 2 transcranial sites immediatelyand at 30 and 60 minutes following 1-Hz rTMS.
Data AnalysisThe latencies and amplitudes of individual MEPs in each
group of 10 EMG traces (for each muscle group and intensity)were determined and then averaged. Data were then normalizedto baseline and expressed in the results as a percentage changefrom baseline. Graphs show data as percentage change frombaseline and in all figures, error bars refer to standard error ofthe mean.
Statistical MethodsStatistical analyses were performed on normalized data
using SPSS 15.0 (SPSS Inc, Chicago, IL). Log transformed peak-�peak amplitudes of the mean MEPs and untransformed meanlatencies of each subject were analyzed using a general linear
Table 1. Baseline Parameters of Subjects in all 3 Experiment
Genotype
Study 1: PES
Val/Val Val/Met t Test V
o. of subjects 9 7ge (y), mean � SEM 78.1 � 1.4 77.9 � 1.7 0.91 59.ale/female, n 5/4 2/5Stimulation intensity,mean � SEM
62.1 � 4.4 55.3 � 3.2 0.27 69.
aseline MEP (�V),mean � SEM
79.7 � 16.3 73.1 � 13.8 0.76 67.
NOTE. No significant differences were found between groups at baseline
model repeated measures analysis of variance (ANOVA) withwithin-subject factor of TIME (before and after intervention)and between-subjects factor of GENOTYPE (Val/Val and non-Val/Val). P values of � .05 were taken as a measure of statisticalsignificance, and data are expressed as mean (�standard error ofmean) unless stated otherwise.
ResultsAge and sex distribution, baseline recordings for
stimulation intensity of single-pulse TMS and MEP forboth Val/Val and non-Val/Val subjects for the 3 experi-ments are shown in Table 1. In our study population,given the low prevalence of the Met66Met genotype, allnon-Val/Val subjects were Val66Met heterozygotes.
Effects of Genetic Polymorphism on PlasticityInduced by PESPharyngeal MEP traces evoked from the stronger
pharyngeal hemisphere from 2 representative subjects areshown in Figures 1A and B. There were no differences insensory and pain thresholds or stimulation intensity forPES between BDNF groups (Supplementary Table 1).When amplitude data from the 2 groups were combined,repeated-measures ANOVA showed that there was anoverall strong trend toward an effect of TIME on pharyn-geal excitability (F3,24 � 2.65, P � .072). The application of
ES produced an expected increase in MEP amplitude inhe pharyngeal motor cortex in the Val/Val group with aignificant TIME*GENOTYPE interaction on 2-way re-eated measures ANOVA (F8, 112 � 2.4, P � .018), seeigure 1C. This was due to the significant increase in theEPs after PES in the Val/Val group (P � .004, paired t
est) but not in the Val/Met group. On the contrary, noignificant difference in MEP latencies between groupsere found following PES (F4,54 � 0.66, P � .62) (Figure
1D and Table 2). There was no significant effect of inter-vention on the excitability of colocalized hand motorcortex (F8, 112 � 0.532, P � .830).
Effects of Genetic Polymorphism on CorticalExcitability Induced by Excitatory rTMSPharyngeal MEP traces evoked from the stronger
pharyngeal hemisphere from 2 representative subjects areshown in Figures 2A and B. When amplitude data from
Study 2: 5 Hz rTMS Study 3: 1 Hz rTMS
al Val/Met t Test Val/Val Val/Met t Test
7 16 65.6 59.7 � 7.3 0.97 55.5 � 6.0 64.5 � 8.7 0.43
1 2/5 7/9 2/43.6 73.7 � 5.4 0.49 60.6 � 6.1 58.5 � 6.2 0.83
9.0 78.8 � 15.6 0.50 83.1 � 12.3 49.6 � 5.2 0.12
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Figure 1. In panel A, shown are examples of typical traces of averaged MEPs recorded before and following PES in a Val/Val subject, and in panel, shown are a Val/Met subject recorded from “dominant” pharyngeal motor cortices. Percentage change in pharyngeal motor cortical excitability ofharyngeal motor cortex at baseline and then immediately and at 30-minute intervals following PES (mean � standard error of the mean) are shown
n panels C and D. The gray shaded arrow represents the application of 10 minutes PES. In panel C, following PES, amplitude of MEPs increaseds expected in the Val/Val group (�), whereas there was no increase in amplitude in the Val/Met group (‘) as shown by a TIME*GENOTYPE
interaction (�P � .018). In panel D, although there was trend to prolongation of latency in the Val/Met group (‘), no between group differences over
time on ANOVA were found.
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the 2 groups were combined, repeated-measures ANOVAshowed that 5-Hz rTMS produced an overall significantincrease in response size, with an effect of TIME onpharyngeal responses (F2.4, 50.6 � 7.4, P � .001). Although
oth Val/Val and Val/Met groups showed an increase themplitude of pharyngeal MEP over 60 minutes (FigureC), there was no between-group differences on 2-wayepeated-measures ANOVA (F3, 60 � 0.26, P � .853). By
contrast, when comparing MEP latencies, there was sig-nificant TIME*GENOTYPE interaction using 2-wayANOVA (F3, 60 � 4.9, P � .04). This was due to theignificant decrease in the latencies in the Val/Met groupP � .001, paired t test) following 5-Hz rTMS but not in
the Val/Val group as shown in Figure 2D and Table 3.There was, however, no significant effect of interventionon the excitability of colocalized hand motor cortex(F3, 60 � 1.837, P � .150).
Effects of Genetic Polymorphism on CorticalExcitability Induced by Inhibitory rTMSPharyngeal MEP traces evoked from the stronger
pharyngeal hemisphere from 2 representative subjects areshown in Figures 3A and B. When amplitude data fromhe 2 groups were combined, repeated-measures ANOVAhowed no overall inhibitory effect of 1-Hz rTMS (F3, 63 �.8, P � .154). However, application of 1-Hz rTMS overharyngeal motor cortex produced an expected reductionf pharyngeal MEP amplitude in the Val/Val group (Fig-re 3C), which was abolished in the Val/Met group ashown by the failure to undergo inhibition to 1-Hz rTMS.wo-way repeated-measures ANOVA of MEP amplitude
ound a significant TIME*GENOTYPE interactionF7,140 � 2.23, P � .035) (Figure 3C). This was due to aignificant reduction in MEP amplitude following 1-HzTMS in the Val/Val group (P � .036; paired t test) but not
in the Val/Met group. There was a trend toward a short-ening in latency over time for Val/Met group in associa-tion with a lengthening of latency in the Val/Val group(Figure 3D and Table 4). However, this difference in la-tency between groups was not significant (F8, 45 � 0.834,
� .482). As before, there were no significant rTMSassociated changes in the hand motor cortex on analysisof hand MEP (F1, 20 � 0.5, P � .476).
DiscussionThis study aimed to define the influence of the
BDNFVal66Met polymorphism on functional flexibility/plasticity in the intact human pharyngeal motor cortex.
Table 2. Latencies (ms) for Pharyngeal and Thenar MEPs Afte
Genotype Baseline
Pharynx Val/Val 9.3 � 0.4Val/Met 9.7 � 0.8
Hand Val/Val 22.1 � 1.7Val/Met 22.0 � 2.0
NOTE. Values are mean � SEM.
Pharyngeal motor cortex was stimulated by transcranial
magnetic stimulation that excited a polysynaptic descend-ing pathway or pathways that activated motor neurons inthe nucleus ambiguus leading to the recruitment of mo-tor units in the pharyngeal muscles. The findings of thisstudy suggest that the pharyngeal motor cortex’s responseto different plasticity inducing protocols is directly asso-ciated with this BDNF polymorphism. This implies thatgenetic variation in the population may predetermine thelikelihood of an individual’s responsiveness to specificneurostimulatory paradigms and is in keeping with theliterature in limb motor cortex.12,13,18 If such associationsare reflected in clinical states, BDNF profiling may help todirect patients to optimal plasticity-promoting protocols.Moreover, our choice of an older population in theseexperiments adds more credence to the findings for futuredisease populations such as stroke. The findings of thisstudy have important implications for neurorehabilita-tion and merit further investigation.
Mechanisms of Excitatory Plasticity in HumanPharyngeal Motor CortexIn experiment 1, the after-effects of PES on the
pharyngeal motor system were examined. Peripheral elec-trical stimulation to the pharynx via intraluminal stimu-lating catheter for 10 minutes can induce changes inmotor cortical excitability that outlasts the stimulus for30 to 120 minutes.5,28 These studies have also indicatedhat the excitability of the pharyngeal motor pathwaysollowing PES is most likely to be driven at the level of theortex as opposed to the brainstem, as evidenced by lackf brainstem reflexes or changes to latency of pharyngealEPs. Previously, Fraser et al showed that in nongeno-
yped healthy subjects, PES accentuated cortical excitabil-ty as evidenced by an increase in the amplitude of MEPs.5
However in that study, about 25% of healthy subjects anddysphagic stroke patients did not produce the expectedfacilitation of cortical motor responses following the ap-plication of PES.19 It could be hypothesized that geneticvariations may contribute to that observation. The cur-rent study showed that in a mixed group of Val andnon-Val genotyped subjects, the overall effect of PES wasexcitatory, albeit reduced, but there was clear PES facili-tation of the pharyngeal motor system in individuals withVal/Val genotype with a sustained increase in MEP. Onthe contrary, PES had much less effect on the pharyngealmotor system of individuals that carried the Met allele ofthe BDNF gene. This contrast between groups was notrelated to any baseline differences in pharyngeal motor or
ES Across Each Time Point
Immediate 30 min 60 min
9.8 � 0.4 9.3 � 0.4 9.5 � 0.310.0 � 0.5 10.1 � 0.5 9.4 � 0.322.6 � 1.7 23.0 � 0.5 22.3 � 1.022.0 � 1.1 22.3 � 0.9 22.3 � 1.0
r P
sensory thresholds, or pharyngeal stimulation intensities
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Figure 2. In panel A, shown are examples of typical traces of averaged MEPs recorded before and following 5-Hz rTMS in a Val/Val subject, andin panel B, a Val/Met subject recorded from “dominant” pharyngeal motor cortices. Percentage change in pharyngeal motor cortical excitability inpharyngeal motor cortex at baseline and then immediately and at 30-minute intervals following 5-Hz rTMS (mean � standard error of the mean) areshown in panels C and D. The gray-shaded arrow represents the application of 5-Hz rTMS. In panel C, the amplitude of MEP increased over timeollowing 5-Hz rTMS for both BDNF groups, while a visible increase was noted in the Val/Mat group (‘), there was no between-group differences inhanges to amplitude (P � .853) post�5-Hz rTMS. In panel D, following 5-Hz rTMS, the latency of pharyngeal MEPs decreased in the Val/Met group‘), while an increase in latency was noted in the Val/Val group (�). This TIME*GENOTYPE interaction for latency on 2-way repeated measures
ANOVA was significantly different between groups (�P � .04).
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(Table 2). Moreover, there was no effect of PES on latencyof pharyngeal MEP.
In experiment 2, the relevance of BDNF polymorphismon the effects of 5 Hz of rTMS was studied. In healthysubjects, 5-Hz rTMS on the pharyngeal motor cortex isknown to increase the size of MEP amplitude with little orno change in response latency.29 In the current study, theamplitudes of pharyngeal MEPs across the combinedgroups showed a significant increase in excitability, withno clear intergroup differences. In contrast to the findingsfound with PES, there was a significant reduction inlatency of pharyngeal MEP for the non-Val/Val groupcompared to the Val/Val group. It is noteworthy that thelatency differences between genetic groups in the currentstudy could not be attributable to baseline differences incortical excitability or stimulation intensities of 5-HzrTMS. Recent work by Gow et al suggested that 5-HzrTMS exerted its effect by increasing the excitability ofintracortical neurons, rather than through subcorticalmechanisms.29 It is intriguing therefore that this studyappears to suggest that facilitation of motor neurons atthe bulbar level may be an important factor in producingthe after-effects of rTMS, given the observation of short-ening of latency in the Val/Met group. In addition to bothVal/Val and Val/Met groups displaying a “net” increase inMEP amplitude to rTMS, the reduction in latency couldimply that motor neurons are more excitable in the non-Val/Val group and nearer threshold. The factors thatmight account for this observation remain speculative,however, at least 2 possible explanations exist. First, theTMS responses in the Val/Met group following 5-HzrTMS may be recruiting alternative or additional motorneural circuitry that elicits plasticity mechanisms at thesubcortical level, a phenomenon not seen with the Val/Valgroup. Indeed there is evidence that the Met allele isassociated with decreased hippocampal volume30 andother variations in human cortical and subcortical mor-phology,31 thus supporting this possibility. Secondly, thepresence of the Met allele may alter functionality of theBDNF protein, which in turn may have an effect onsynaptic plasticity in brainstem neurons. This latter hy-pothesis is discussed further.
Pharmacological studies of excitation in the brainstemswallowing circuitry have demonstrated that the applica-tion of �-aminobutyric acid (GABA)�enhancing musci-
ol to the nucleus tractus solitarii of anesthetized ratseversibly inhibited pharyngeal swallows evoked by either
Table 3. Latencies (ms) for Pharyngeal and Hand MEPs After
Genotype Baseline
Pharynx Val/Val 9.2 � 0.3Val/Met 9.7 � 0.4
Hand Val/Val 21.1 � 0.4Val/Met 22.5 � 0.5
NOTE. Values are mean � SEM.*P � .05, ANOVA.
lectrical or chemical stimulation of the nucleus tractus M
olitarii.32 This suggested that GABA neurons exert atonic inhibition of the brainstem swallowing pattern gen-erator. By contrast, Bariohay et al33 investigated how var-ous drugs (eg, BDNF, GABA, GABA agonists, and GABAntagonists) affected the rhythmic swallowing of rats elic-ted by electrical stimulation of the superior laryngealerve. Microinjection into the nucleus tractus solitariiomponent of the swallowing network with BDNF pro-uced a rapid dose-dependent inhibition of rhythmicwallowing. Moreover, co-injection with bicuculline aABAA receptor antagonist removed this inhibitory effectf BDNF on rat’s swallowing. Intriguingly, they also dem-nstrated that electrical stimulation of the superior laryn-eal nerve reduced BDNF protein within the dorsal vagalomplex. Taken together, these observations suggests thatDNF plays a pivotal role in synaptic plasticity of lower-
evel swallowing motor circuitry and, depending on itsunctional properties, may exert an inhibitory effect onhe brainstem swallowing pathways secondary to the typef single nucleotide polymorphism present. In addition,s GABA and BDNF appear to work in synergy at the levelf the brainstem swallowing center, this may provide alausible explanation for the reduction of latency of pha-yngeal MEP (reflecting an increase in brainstem excitabil-ty) in the Val/Met group following 5-Hz rTMS (andossibly other forms of neurostimulation) where the cor-esponding BDNF being generated might be less inhibi-ory. Why this process is less/not noticeable in the PESnd inhibitory rTMS paradigms is unclear. One mightpeculate that the latter 2 inputs recruit different/addi-ional lower-level circuitry in the pharyngeal system,hich is less susceptible to local BDNF effects. Of rele-
ance both PES and 1Hz rTMS were more effective inal/Val subjects, possibly implying that the local BDNFffects (if any) are weaker.
Mechanisms of Inhibitory Plasticity in HumanPharyngeal Motor CortexIn experiment 3, the effects of 1-Hz rTMS on the
pharyngeal motor cortex (a virtual lesion) on healthysubjects carrying the Val66Val and Val66Met forms ofBDNF were studied. In nongenotyped healthy subjects,1-Hz rTMS is inhibitory to the pharyngeal motor cor-tex.21,26 Jefferson et al21 demonstrated that application ofhe virtual lesion is associated with an increase in intra-ortical inhibition and reciprocal decrease in intracorticalacilitation. The current study found that the presence of
z rTMS Across Each Time Point
Immediate 30 min 60 min
9.6 � 0.3 9.8 � 0.4 9.6 � 0.48.4 � 0.4* 8.5 � 0.5* 8.7 � 0.5*
21.7 � 0.4 21.6 � 0.4 21.3 � 0.422.4 � 0.7 22.5 � 0.0 22.2 � 0.7
5 H
et allele negated the influence of 1 Hz on the pharyngeal
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Figure 3. In panel A, shown are examples of typical traces of averaged MEPs recorded before and following 1-Hz rTMS in a Val/Val subject, andin panel B, a Val/Met subject recorded from “dominant” pharyngeal motor cortices. Percentage change in pharyngeal motor cortical excitability of thehemisphere with the strongest pharyngeal projection at baseline and then immediately and at 30-minute intervals following 1-Hz rTMS (mean �standard error of the mean) are shown in panels C and D. The gray-shaded arrow represents the application of 1-Hz rTMS. In panel C, following 1-HzrTMS, there was expected decrease in amplitude of MEPs in the Val/Val group (�). By contrast, there was an increase in amplitude of MEPs in theVal/Met group (‘). There was significant difference in amplitude between groups post�1-Hz rTMS when TIME*GENOTYPE interaction on ANOVAwas evaluated (�P � .035). In panel D, there was a suggestion of shortening of latency in Val/Met group (‘) with reciprocal increase in latency in
al/Val group (�). However, there was no statistical difference in latency.
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motor cortex. As such, no consistent reduction of corticalexcitability occurred in the Val/Met group in response tothe normally inhibitory paradigm of 1-Hz rTMS. Thereason why it was not possible to induce cortical inhibi-tion in healthy subjects with Met allele in the currentstudy is of interest. Indeed, it can be argued that BDNFpolymorphisms may influence the inter-relationship be-tween GABA and glutamate-mediated pathways, makingit more difficult to produce inhibitory changes inVal66Met subjects. Perhaps a more detailed understand-ing of the genetic basis of cortical plasticity of humanswallowing motor pathways is required to address severalunanswered questions. For example, do polymorphismsin BDNF influence neuroplasticity via synaptic mecha-nisms or changes in neural network morphology? In orderto explore this further, future studies using plasticity-inducing protocols should be complemented with func-tional magnetic resonance imaging and magnetic reso-nance spectroscopy to assess the effects of BDNFpolymorphism on the human motor system.
ConclusionsWe have shown that a common polymorphism of
BDNF can exert contrasting effects on neurophysiologicaloutcomes in experimentally induced plasticity paradigmsin the intact human pharyngeal motor cortex with theimplication that genotype profiling may improve bespokerehabilitative tools for individual patients.
Supplementary Material
Note: To access the supplementary materialaccompanying this article, visit the online version ofGastroenterology at www.gastrojournal.org, and at doi:10.1053/j.gastro.2011.05.047.
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Table 4. Latencies (ms) for Pharyngeal and Thenar MEPs Afte
Genotype Baseline
Pharynx Val/Val 9.5 � 0.4Val/Met 11.6 � 1.7
Hand Val/Val 22.4 � 0.4Val/Met 21.5 � 1.1
NOTE. Values are mean � SEM.
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Hz rTMS Across Each Time Point
Immediate 30 min 60 min
10.9 � 0.5 10.4 � 0.6 9.9 � 0.510.9 � 1.5 10.4 � 1.1 9.9 � 0.922.3 � 0.4 22.4 � 0.4 22.4 � 0.421.3 � 1.5 20.8 � 2.1 19.2 � 2.3
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Received December 30, 2010. Accepted May 26, 2011.
Reprint requestsAddress requests for reprints to: Shaheen Hamdy, MD, PhD,
Inflammation Sciences Research Group, University of Manchester,Clinical Sciences Building, Salford Royal Hospital, Salford, M6 8HD,England. e-mail: [email protected]; fax: �44 161
06 4364.
cknowledgmentsThe authors would like to thank staff in CIGMR and the volunteers
rom the Dyne-Steele cohort.
onflicts of interestThe authors disclose no conflicts.
undingGrant support received from the Action Medical Research
Reference: A/P/1091); Biotechnology and Biological Sciencesesearch Council (BBSRC) (Reference BB.F02244101.1). The studyas sponsored by the University of Manchester, UK, which did notave a role in the study design, in the collection, analysis, or
nterpretation of data.
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September 2011 BDNF POLYMORPHISM AND PHARYNGEAL CORTEX 836.e1
Supplementary Material and Methods
Power CalculationThe ratio of subjects with Val/Val to non-Val/Val
group in the UK population is approximately 4:1. Inorder to achieve a power of 80% and to see a 50% differ-ence in the excitability of the brain in response to tech-niques of neurostimulation between the 2 genetic groups(cf, Kleim et al12), a sample size calculation using Statashowed that if an � value of .05 is used, a minimum of 10Val/Val individuals and 5 non-Val/Val individuals wasrequired.
Pharyngeal EMG MeasurementsA 3.2-mm diameter intraluminal catheter (Gaeltec
Ltd, Dunvegan, Isle of Skye, UK) was swallowed by sub-jects either transnasally or transorally, depending on sub-ject preference. The catheter houses a pair of bipolarplatinum ring electrodes that are positioned in the phar-ynx (�14�15 cm aboral from the incisors or nasal flare)to record EMG traces. The catheter was connected via apreamplifier and interface to a personal computer thatrecorded the traces using “Signal” Application Program(Cambridge Electronic Design Ltd, Cambridge, UK). Anearth lead was connected to a skin electrode sited over theupper portion of one of the sternocleidomastoid musclesof the neck. The catheter was connected via a preampli-fier (CED; Cambridge Electronic Design Ltd), amplifier(CED 1902), and interface (CED 1401) to a personalcomputer, which allowed real-time visualization and re-cording of the traces using the Signal Application Pro-gram (CED). This had filters set at 200 Hz to 2 kHz andallowed a sampling rate of 4�8 kHz. Response signalswere processed through a 50/60-Hz noise eliminator(HumBug; Quest Scientific, North Vancouver, British Co-lumbia, Canada) to remove any unwanted electrical in-terference. Analysis of the amplitude and latencies of thetraces was conducted using Signal program.
Single-pulse TMSSingle-pulse TMS was delivered to both hemi-
spheres using a Magstim 200 stimulator (The MagstimCompany, Whitland, Wales, UK) connected to a figure-of-eight coil with an outer diameter of 70 mm, whichproduced a maximum output of 2.2 Tesla. The cranialvertex was marked on a thin cloth cap placed over thescalp and the magnetic stimulator discharged over bothcortices to identify the site evoking the greatest pharyn-geal response on each hemisphere (ie, hot spot). The siteover either hemispheric motor strip that evoked thegreatest pharyngeal response was termed the dominantpharyngeal motor cortex hot spot. This hot spot wasmarked on the scalp. At this site, the motor thresholdwas identified using single pulses of stimulation toachieve MEPs of at least 20 �V on 50% of occasions. MEP
mplitude was assessed by giving TMS at 20% of stimu-
ator output above motor threshold, with 10 stimuliiven over the dominant pharyngeal motor hot spot asdentified previously. As a control, hand electrodes werelaced on the hand opposite the side of the brain evokinghe largest pharyngeal response and then the optimal sitend motor threshold for adjacent hand motor cortextimulation was identified using the technique describedere. MEP amplitude for the hand was assessed as for theharyngeal motor cortex.
PESThe bipolar ring electrodes of the pharyngeal
catheter were positioned intraluminally in the pharyngealcavity, �14 cm from the incisors or �15 cm from thenasal flare, depending on subject preference. The maxi-mum tolerated PES intensity was predetermined fromeach participant’s first perceived sensation and painthreshold (the point when the pharyngeal sensation be-came uncomfortable), which were calculated from anaverage of 3 trials. Electrical stimulation of the pharynxwas carried out using this catheter, described here, whichwas connected to an electrical stimulator (Model DS7;Digitimer, Welwyn-Garden City, Herts, UK) via a triggergenerator (Digitimer model DL2). Pharyngeal electricalstimuli (0.2-ms pulses, 280 V) was delivered at a setfrequency (5 Hz), intensity (75% of maximal tolerated),and duration (10 minutes).
Excitatory rTMSA Magstim super rapid stimulator (The Magstim
Company) was used to deliver excitatory rTMS via afigure-of-eight coil. Single pulses from the rapid stimu-lator were used to identify the site evoking the greatesthand muscle response on the dominant pharyngealhemisphere and the resting motor threshold at this sitewas determined using single pulses of TMS to achieveMEPs of at least 50 �V on 50% of occasions; these handhresholds were used in order to comply with currentafety guidelines.27 For rTMS, the previously determined
optimal stimulation parameters for excitatory rTMS werethen used (a frequency of 5 Hz, an intensity 90% ofresting hand motor threshold and a train of 250 pulses,in 5 blocks of 50 pulses with a 10-second pause betweenblocks).
Inhibitory rTMSA Magstim super rapid stimulator (Magstim
Company) delivered trains of stimuli via a figure-of-eightcoil and run through the “Magstim Rapid Session” com-puter software. The virtual lesion was induced by provid-ing 1-Hz rTMS at 120% of pharyngeal resting motorthreshold and limited to a maximum of 100% of stimu-lator output, for a duration of 10 minutes over thedominant pharyngeal motor cortex (the cortex contain-ing the site evoking the greatest pharyngeal response
when tested with single-pulse TMS).
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BDNF GenotypingFor blood DNA, HapMap (www.hapmap.org) was
used to select the single nucleotide polymorphisms forVal66Val and Val66Met. Amplification by polymerasechain reactions (PCR) was carried out on PTC-225 PeltierThermal Cyclers (MJ Research, Waltham, MA) in 384-wellmicrotiter plates using 10 ng genomic DNA with finalreaction volume of 10 �L. Genotyping was performed
sing Sequenome technology (Sequenome Inc, Hamburg,ermany). The Nautilus Laboratory Information Man-
gement System (Thermo Electron Corporation, Altrin-ham, England, UK) was used for sample management andracking. For saliva DNA, standard operating proceduresere used to extract and purify the salivary DNA. BDNFas genotyped using Applied Biosystems Assays-by-De-and kit (Applied Biosystems, Warrington, Cheshire, Eng-
and, UK). Genotyping was carried out using a reactionixture containing 2.5 �L of DNA Probe Master (Rocheiagnostics, West Sussex, UK), 20 ng genomic DNA in a 5-
�L reaction volume, comprising 2 �L DNA, 0.125 �L Assayix (40�) and 0.375 �L nuclease-free water. The PCR
conditions were 95°C for 10 min then 50 cycles of 95°C for10 s and 60°C for 30 s. The reaction was allowed to run and
the PCR products were electrophoresed. The sample re- rporter fluorescence was measured using a Roche Lightcycler480 genotyping real-time PCR platform (Roche Diagnos-tics, West Sussex, England, UK).
Effects of PES and 5-Hz rTMS in Met/MetBDNF HomozygotesMet/Met homozygotes represent �3�4% of the
uropean population and are therefore a relatively un-ommon genotype to find and experiment on. In ournitial cohort of subjects, we did not have any Met/Metubjects. However, post-hoc, we identified 2 Met/Metomozygotes from a larger cohort of study participantsho were not included in the original data set. Both
ubjects (1 male aged 22 years, 1 female aged 30 years)ere studied with 2 of the neurostimulation paradigms,ES and 5-Hz rTMS, at least 1 week apart using the exactame protocols to those described in experiments 2 and. The averaged results are shown in Supplementaryigure 1 (as percent change from baseline [mean � stan-ard error of mean]) for each of the 2 interventions inharyngeal motor cortex. It can be seen from these datahat as predicted by the Val/Met heterozygote observa-ions, Met/Met subjects are highly responsive to 5-Hz
TMS and not responsive to PES.
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Supplementary Table 1. Stimulation and SensoryPerception of Subjects ReceivingPES
Genotypea
t TestVal/Val Val/Met
Sensory threshold (mA) 8.2 � 1.3 8.7 � 1.4 0.81Pain threshold (mA) 12.8 � 1.8 14.4 � 1.7 0.54Stimulation intensity (mA) 11.1 � 1.5 12.9 � 1.7 0.45
aValues are mean � standard error of the mean.
Supplementary Figure 1. Plot of cortical excitability in the pharyngeal projection presented as percentage change from baseline in BDNFMet66Met homozygote subjects (n � 2). Data presented as mean � standard error of the mean. It can be seen that in the Met/Met participants, PES
oes not produce an increase in excitability, while 5-Hz rTMS produces a clear rise in excitability, both being predicted by the Val/Met heterozygote
henotypic responses.
