Immediate Effects of Preconditioning Intermittent Theta Burst Stimulation on Lower Extremity Motor Cortex Excitability in Healthy Participants

Background : Previous studies have found that inhibitory priming with continuous theta burst stimulation (cTBS) can enhance the effect of subsequent excitatory conditioning stimuli with intermittent theta burst stimulation (iTBS) in the upper limbs. However, whether this combined stimulation approach elicits a comparable compensatory response in the lower extremities remains unclear. This study aimed to investigate how cTBS preconditioning modulated the effect of iTBS on motor cortex excitability related to the lower limb in healthy individuals. Methods : Using a randomised cross-over design, a total of 25 healthy participants (19 females, mean age = 24.80 yr) were recruited to undergo three different TBS protocols (cTBS + iTBS, sham cTBS + iTBS, sham cTBS + sham iTBS) in a random order. Each TBS intervention was administered with one-week intervals. cTBS and iTBS were administered at an intensity of 80% active motor threshold (AMT) delivering a total of 600 pulses. Before intervention (T0), immediately following intervention (T1), and 20 min after intervention (T2), the corticomotor excitability was measured for the tibialis anterior muscle of participants’ non-dominant leg using a Magneuro100 stimulator and matched double-cone coil. The average amplitude of the motor-evoked potential (MEP) induced by applying 20 consecutive monopulse stimuli at an intensity of 130% resting motor threshold (RMT) was collected and analysed. Results : Compare with T0 time, the MEP amplitude (raw and normalised) at T1 and T2 showed a statistically significant increase following the cTBS + iTBS protocol ( p < 0.01), but no significant differences were observed in amplitude changes following other protocols (sham cTBS + iTBS and sham cTBS + sham iTBS) ( p > 0.05). Furthermore, no statistically significant difference was found among the three protocols at any given time point ( p > 0.05). Conclusions : Preconditioning the lower extremity motor cortex with cTBS prior to iTBS intervention can promptly enhance its excitability in healthy participants. This effect persists for a minimum duration of 20 min. Clinical


Introduction
Transcranial magnetic stimulation (TMS) is a noninvasive neuromodulation technique [1] extensively used in clinical and functional brain research [2,3].The use of TMS to treat various disorders of the nervous system is based on the ability of repetitive stimulation protocols to induce long-lasting neuromodulation effects that persist beyond the cessation of stimulation, potentially influencing synaptic plasticity [4][5][6].Metaplasticity is a key concept within the realm of activity-dependent plasticity regulation, refers to dynamic adjustments under synaptic and/or neuronal conditions.These adjustments play a crucial role in determining the trajectory, extent, and duration of subsequent synaptic modifications [7].However, the main problem with rTMS protocols for inducing plasticity is the high level of inter-individual variability.Several factors contribute to this variation, including biological factors such as age, gender, genetics, time of day, brain state, and neuronal circuit anatomy [8,9].Notably, the level of plasticity can also be influenced by the pre-existing activity levels in the specific region targeted by the intervention [10].According to the Bienenstock-Cooper-Munro theory of bidirectional synaptic plasticity, low levels of prior synaptic activity are conducive to long-term potentiation (LTP), whereas high levels of prior synaptic activity promote long-term depression (LTD) [11].The bidirectional mechanism modifies synaptic strength, so neuronal activity is maintained within an optimal range [12].The phenomenon of homeostatic plasticity has been documented in animal studies [13,14].Substantial supporting evidence is also emerging from research conducted on the upper limb motor cortex in human subjects [15,16].Therefore, prior to rTMS intervention, non-invasive brain stimulation technology can be used to prime the activity level of synapses, thereby influ-encing subsequent effects.This preconditioning approach has been studied using various protocols of non-invasive brain stimulation combinations.The resulting modification of neuroplasticity is found to be stronger, more durable, and more stable [17].
Numerous strategies have been proposed to enhance the effectiveness of TMS, encompassing the customisation and diversification of stimulation protocols.A growing body of research is dedicated to theta burst stimulation (TBS), a type of patterned rTMS protocol [18].It necessitates shorter stimulation durations and lower intensity levels compared to conventional rTMS [19,20].Continuous TBS (cTBS) and intermittent TBS (iTBS) are two distinct stimulation protocols capable of modulating cortical excitability within a neural network.In general, cTBS decreases motor cortical excitability, whereas iTBS has a tendency to increase it [20].Compared with rTMS, iTBS effectively emulates endogenous θ rhythms and thus more efficiently induces synaptic LTP [20,21].Given that TBS is a relatively time-saving stimulation protocol, whether cTBS can be used as a preconditioning technique to modulate the motor cortex response to subsequent iTBS is interesting.Research on whether the amalgamation of cTBS and iTBS can yield more favorable results is currently inadequate.Four studies have been conducted on healthy adults using cTBS followed by iTBS.Results show that priming significantly increase the expected excitatory effect of the test protocol, specifically by targeting the upper limb primary motor cortex (M1) [22][23][24][25].These findings indicate that the inhibitory priming stimulation of cTBS may potentially stabilise or even amplify the subsequent excitatory stimulation of iTBS.The lower limb muscles play a crucial role in maintaining balance, standing, and walking [26].Many TMS studies focus on hand muscles to locate the motor cortex, but investigating lower extremity muscles is equally vital for understanding neuroplasticity.Exploring how stimulation affects neuroplasticity in lower limb muscles can illuminate cortical remodelling mechanisms.Owing to the deeper anatomical structure of the lower extremity motor cortex than the upper extremity, studies on the application of TBS to the lower extremity are limited.A study has applied TBS to the M1 of the lower limb and found that iTBS increased corticospinal excitability for up to 30 min, whereas cTBS decreased it for up to 15 min.However, no research has focused on the effects of priming iTBS with cTBS on the excitability of the lower limb motor cortex.Whether this co-stimulation pattern demonstrates a comparable trend in cases involving the lower limbs remains unclear.
Alterations in the plasticity of the M1 can be investigated by assessing motor-evoked potentials (MEPs) elicited by single-pulse TMS [27].In the present study, we hypothesised that the inhibitory preconditioning of lower limb M1 with cTBS conferred benefits for subsequent iTBS, leading to enhanced corticospinal excitability specifically related to the lower limb.This enhancement was manifested by increased in MEP amplitude.

Participants
From March 2023 to December 2023, a total of 27 participants were recruited by displaying of posters at the Wuxi Central Rehabilitation Hospital, in strict adherence to TMS safety guidelines [28].The inclusion criteria were as follows: x age over 18 years, y no central nervous system and mental disorders, z no history of drug and alcohol dependence, { no contraindications to TMS examination, and | voluntary participation and signing of informed consent.The exclusion criteria were as follow: x age <18 years, y any neurological disease; z history of epilepsy; { no serious mental or physical illness; | previous adverse reaction to TMS; } use of medication affecting cortical ex-citability; ~pregnancy; undergone lower limb surgeries; unable to evoke a response to TMS in the tibialis anterior (TA) muscle before formal testing, and poor compliance or inability to cooperate with the test.This study was approved by the Ethics Committee of Wuxi Mental Health Center/Wuxi Central Rehabilitation Hospital (no.WXMH-CIRB2023LLky044).

Study Design
This study was a self-controlled cross-over trial design.After signing informed consent, each subject underwent three different TBS protocols (cTBS + iTBS; sham cTBS + iTBS; sham cTBS + sham iTBS) in a random order.No interval existed between the cTBS preconditioning and the subsequent iTBS.Each TBS intervention was administered with one-week intervals to wash out the residual effects of the previous intervention [22,29] (Fig. 1).All procedures were conducted within the same treatment room during the designated time window (10 AM to 12 PM or 3 PM to 6 PM), ensuring a tranquil and interference-free environment in the treatment room and its surroundings.The same participants were administered different stimulus protocols at the same time of testing day with a week apart.MEP-related measurements were performed before the TBS intervention (T0), immediately after the intervention (T1), and 20 min after the intervention (T2).

Blind
The design of this experiment was single-blind, and only the subjects were blinded, so the subjects did not know which intervention they were receiving each time.Furthermore, the RMT and motor cortex excitability of all participants were evaluated using TMS by an impartial physical therapist who was blinded to the randomisation.

Measurement of RMT
During the experiment, the assessment was delivered by a physical therapist with specialist training in TMS, us-ing a Magneuro100 stimulator (VISHEE Medical Technology Co., Ltd., Nanjing, Jiangsu, China) and its matching double-cone coil (YCZ001).The participant did not engage in any exercise or physical activity prior to the measure-ment.Participants were in a quiet and awake state, maintaining an upright posture while being relaxed, and wearing a positioning cap (designed according to the international 10-20 electroencephalographic system) on their heads.After skin preparation, a pair of Ag-AgCl surface-adhesive recording electrode were placed on the subject's tibialis anterior (TA) muscle belly, and the ground electrode was placed on the lateral ankle.The electromyography signals were amplified with a gain of 1000×, filtered with a bandpass of 20-500 Hz, and sampled at 2000 Hz using the wireless portable motor evoked potential MEP detection module.The TMS coil was tangentially placed on the surface of the skull, with the coil handle positioned perpendicular to the scalp.Biphasic single magnetic pulses were generated at least every 5 s [30] to stimulate the M1 area contralateral to the recorded TA muscle around the Cz point [31].MEPs were recorded utilising the TMS system and evaluated using the corresponding software.The hot spot was identified as the most easily excitable location, consistently yielding large MEP amplitudes at a relatively low TMS output intensity.After identifying the hot spot, it was marked on the cap with a pen to keep it fixed.Subsequently, the output intensity gradually decreased (Fig. 1).The RMT was determined by using the minimum magnetic stimulation intensity that resulted in a MEP amplitude greater than 50 µV for at least 5 out of 10 stimuli.The active motor threshold (AMT) was defined as the minimum stimulus intensity that elicited a MEP amplitude of at least 200 µV from at least 5 out of 10 stimuli on a slightly contracted target muscle [32,33], and generally, the RMT exceeded the AMT.

Assessment of Motor Cortex Excitability
The corticomotor excitability was measured for the TA muscle of the participants' non-dominant leg at three time points, namely, before stimulation (T0), after stimulation (T1), and 20 min post-stimulation (T2).The participants' dominant leg was determined as the one they would use to kick a ball [34].The average peak-to-peak MEP amplitude induced by the application of 20 consecutive monopulse magnetic stimuli at 130% RMT intensity was collected and analysed [35].MEP amplitude represented the net changes in excitability and inhibition of corticospinal pathway [36].It reflected the physiological and pathological changes of the corticospinal tract and intracortical loop [37].To minimise individual differences, the MEP amplitude before and after each of the three TBS interventions was normalised.Normalised MEPs were calculated by dividing the raw MEP amplitude by the mean MEP at T0 time point in the same intervention group and presenting the result as a percentage [35].

TBS Intervention
The TBS intervention was conducted using the aforementioned magnetic stimulator and its accompanying double cone coil.The stimulation target was the "hot spot" of the M1 region for the lower limb.For cTBS, three-pulse bursts were delivered at 50 Hz and repeated at 5 Hz, with 200 consecutive clusters without intermittent stimulation, resulting in a total of 600 pulses lasting approximately 40 s.As for iTBS, it involved delivering three-pulse bursts at 50 Hz and repeating them at 5 Hz in consecutive short trains of 10 bursts (lasting for about 2 s) every 10 s for a total of 20 cycles, resulting in a total of approximately 192 s and delivering around 600 pulses [20].The stimulation intensity was set to 80% AMT [20].
The intervention targeted the same areas and utilised identical stimulus parameters for sham and real stimuli.The sham stimulation coil was designed to mimic the real stimulation coil, with participants being able to hear the machine's sound, yet no magnetic pulse was emitted.

Statistical Analyses
Data analysis was performed using SPSS22.0(IBM SPSS statistics, Chicago, IL, USA) for statistical analysis and GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) for visualisation purposes.The normality of data distribution was assessed using the Shapiro-Wilk test.Demographic data were described using the mean ± standard deviation (continuous variables by Analysis of Variance (ANOVA) test) and counts (categorical variables by chi-square test).Generalised estimating equations were used to estimate the interaction (protocol × timepoint) and main effect of MEP amplitudes (raw and normalised).When the interaction effect was significant, we conducted a separate effect analysis, followed by pairwise comparisons using the Bonferroni correction method.Results are presented as the mean and 95% confidence intervals.We also observed the individual response to different protocols, categorising each participant as either responsive or non-responsive depending on the average amplitude µ (expressed as a percentage relative to the baseline) at each time point within each experimental protocol.Those classified as facilitators had a µ > 110% of the baseline, whereas those classified as inhibitors had a µ < 90%, and those classified as non-responders had 90% < µ < 110% [38][39][40].Responses were compared for rates of facilitation and inhibition across different protocols and time points through the use of a Friedman test for comparison.A two-tailed test was used, considering p-values less than or equal to 0.05 as indicative of a statistically significant difference.

Results
A total of 27 healthy young subjects were enrolled in the study.Two participants withdrew during the pro-gram; one experienced a headache after cTBS, which resolved spontaneously the following day, whereas the other reported an uncomfortable sensation of pressure at the stimulation site that subsequently resolved on its own.Ultimately, 25 participants fulfilled all of the intervention pro-  tocols (mean age = 24.80 years; 6 males).Table 1 presents the demographic data of the subjects.No significant differences were observed among the intervention protocols regarding RMT (F = 0.27, p = 0.764).
As results shown in Table 2 and Fig. 2, the analysis of normalized MEP amplitudes revealed a significant interaction effect between the protocol and time point (Wald x 2 = 14.90, p = 0.005), as well as a significant effect of time (Wald x 2 = 8.87, p = 0.012).Post hoc analyses revealed that participants who received the cTBS + iTBS intervention demonstrated significantly greater improvement at T1 (p = 0.001) and T2 (p = 0.003) than those at T0 time point (Table 3).However, no significant differences were observed between the other two interventions at any time point (p > 0.05).Furthermore, no significant differences were observed in pairwise comparisons among the three groups at either the T1 or T2 time points (p > 0.05).The results obtained from analysing raw MEP also yielded similar findings to those obtained from analysing normalised MEP (refer to Tables 2,3 and Fig. 2).
Based on the average amplitude µ at each time point in every protocol (expressed as a percentage of the baseline), participants were categorised into responders and non-responders.Among the responders, facilitators were identified when µ exceeded 110% of the baseline, whereas inhibitors were identified when µ fell below 90%.The resulting proportions of subjects in each category are illustrated in Fig. 3.The proportion of facilitators ranged from 20% to 64%, whereas the proportion of inhibitors varied between 12% and 32%.As shown in Table 4, a significant difference was observed in the responder rate between the different stimulation protocols at T1 (x 2 = 6.74, p = 0.034) and T2 (x 2 = 7.84, p = 0.020) (Table 4).Pairwise comparison revealed that the difference between the cTBS + iTBS intervention and sham intervention at T1 (p = 0.071) and T2 (p = 0.085) was marginally significant [41].However, this disparity was not observed in any other pairwise comparisons.

Discussion
We investigated the immediate effects of cTBS preconditioning iTBS on the excitability of the motor cortex of the lower limbs in healthy subjects, as measured by MEP amplitude.The stimulation protocols used in this study were deemed safe and well tolerated by the participants.No serious adverse events such as seizures occurred during the study.Previous preconditioning studies with TBS have primarily focused on the motor cortex of the upper limb and have neglected its application to the lower limb.This discrepancy may be attributed to the anatomical location of the lower limb motor cortex, which is in a deeper region within the longitudinal fissure of the brain and is less amenable to  stimulation using an eight-figure coil [42,43].Our previous research has proven that the double-cone coil can stimulate the lower limb motor cortex to induce a more stable MEP amplitude [31].In the current work, we positioned the coil over the M1 of the lower limb to investigate the effects of preconditioning with cTBS prior to administering iTBS.The findings demonstrated that prior application of cTBS preconditioning before iTBS can significantly enhance the amplitude of MEP in healthy individuals.Furthermore, the alteration in cortical excitability profile induced by this specific stimulation protocol persisted for an extended duration, exceeding 20 min.
Researchers have focused on the effects of inhibitory priming and excitatory test protocols through various techniques, such as inhibitory priming including cathode transcranial direct current stimulation (c-tDCS) and cTBS on healthy subjects.A meta-analysis by Hassanzahraee et al. [16] has shown that c-tDCS-rTMS protocols not significantly boost the expected excitatory effect of the test protocol [44,45], but cTBS-iTBS protocols significantly intensify the excitatory effects of the test iTBS protocol [22][23][24][25].Researchers have also observed positive effects from the c-tTDS-iTBS protocol.Dai et al. [35] used high-definition c-tDCS combined with iTBS and found that the MEP amplitude of the priming iTBS group shows significant changes at almost all time points, lasting 5-30 min.Similarly, the excitatory after-effects of c-tDCS-iTBS increase by 12% in comparison with iTBS [46].The inhibitory priming appears to have reduced the historical neuronal activity, shifting the modification threshold in favour of LTP-like effects and intensifying the excitatory effect of the test protocols.The mechanism of synaptic homeostasis plasticity can help explain the role of inhibitory preconditioning [27,44].Synaptic homeostatic plasticity is believed to regulate neural activity within a specific physiological range and guarantee the stability of neural network function [15,47,48].This mechanism also serves as the foundation for our intervention aimed at altering the functional state of the motor cortex through cTBS preconditioning, which in turn impacts subsequent iTBS-induced changes in cortical plasticity.Furthermore, in contrast to prior research on combined interventions involving tDCS or rTMS with TBS, we opted for cTBS as the priming technique for iTBS owing to its shorter overall intervention duration.
The only statistically significant improvement in MEP amplitude was observed at T1 and T2 in the cTBS + iTBS intervention group compared with the baseline assessment at T0.This finding was consistent with a previous one on the upper limb study [22], which showed interventions involving the combination of iTBS + iTBS and cTBS + iTBS (70% RMT, left upper limb M1, 600 pulse) resulted in significant increases in MEP amplitude.However, the MEP amplitude elicited by single cTBS + iTBS was found to be greater than both iTBS + iTBS (p = 0.001) and sham (p < 0.001) intervention.Mastroeni et al. [25] showed that preconditioning iTBS with cTBS (80% AMT, left upper limb M1, 600 pulse) significantly enhances MEP amplitudes.Conversely, preconditioning iTBS with iTBS leads to less robust facilitation of MEP.Murakami et al. [23] demonstrated that priming of cTBS primed iTBS (80% AMT, left upper limb M1, 600 pulse) enhances the input-output curves of MEP amplitude compared with non-primed iTBS.Their result showed that cTBS enhanced the excitatory effect of iTBS, resulting in increased excitability of motor cortex.Notably, no statistically significant difference existed between iTBS and baseline, in contrast with prior research where iTBS obviously affected the MEP amplitude [20,25,49,50].However, several other studies have also found no significant alteration in MEP amplitude following single iTBS intervention [24,38,51,52].No consensus has been reached on whether cortical excitability can be enhanced after single iTBS stimulation because our findings are both similar and contrary to previous research.One possible explanation for this phenomenon is that, as reported in a study previous (n > 50), significant variability exists among individual responses to iTBS intervention (80% AMT), which may have nullified any net difference at the group level [53].Another study involving 15 healthy participants shows that single iTBS (70% RMT) does not significantly increase MEP amplitude, and less than half of the sample responds to iTBS as a facilitator [52].This finding is similar to ours, where only 48% of subjects showed excitatory effects on iTBS.Player et al. [24] demonstrated that more than half of the participants (80% AMT) responded to iTBS with increased post-stimulation MEP amplitudes, whereas the remaining participants experienced significant decreases or no significant changes.Another study has suggested that the individual susceptibility to iTBS after stroke was influenced by interindividual differences in the motor network connectivity of the lesioned hemisphere [54].Another factor contributing to this phenomenon might be associated with variations in stimulus protocols between our study and previous research.Firstly, the effect of stimulation interval on plasticity following repeated blocks of iTBS.In contrast to previous studies that have used a 2 min interval [24], a 10 min interval [22] or a 15 min interval [23,52], no time interval was used between each stimulation block in our study.Our study further utilized a stimulus intensity of 80% AMT, whereas previous studies have used 70% RMT [22,49,52,55].Notably, the absolute pulse output may vary depending on the TMS device [56].Our stimulation target also focused on the M1 area of the lower limb, whereas previous studies commonly target the M1 of hand.Therefore, the excitatory effects of iTBS vary depending on the stimulus parameters and individual differences.However, our results showed that cTBS stimulation prior to iTBS can effectively stabilise its excitatory effects on motor cortex, increasing the proportion of facilitators by up to 64%.This finding indicated that preconditioning with cTBS prior to iTBS was a particularly valuable regimen for enhancing the excitability of the lower extremity motor cortex.
Although iTBS remains under investigation for its potential benefits in lower limb recovery, this study demonstrated the sustained efficacy of our combined stimulation protocol lasting up to 20 min.The cumulative trend of immediate effects was also highlighted.Siebner et al. [27] reported that preconditioning with tDCS increased upper extremity corticospinal excitability for at least 20 min.However, some studies have examined longer post-stimulus effects, such as 30 [35] or 60 min [22].Clinical treatment studies have demonstrated that compared with therapy alone, a virtual reality-based cycling training lasting for 60 min and conducted 10 min following iTBS stimulation or a 45-min upper limb physical therapy immediately after iTBS stimulation can significantly enhance motor function and daily-life participation in stroke patients [57,58].Similarly, Zhang et al. [59] demonstrated on stroke patients that robot-assisted training, which commences approximately 5 min after completion of priming (cTBS + iTBS) and iTBS sessions, results in significantly higher improvements in Fugl-Meyer assessment scores than those of the sham group.Therefore, our findings may offer valuable insights for individuals with lower limb motor disorders undergoing TBS-based rehabilitation because the 20 min timeframe of heightened cortical excitability allows ample opportunity for patients to engage in additional physical training.
This study represents the initial investigation into the application of cTBS preconditioning prior to iTBS on lower limbs for potential therapeutic purposes.However, certain limitations associated with this study need to be acknowledged.First, the inclusion of participants who were relatively young and in good health may limit the generalisability of the findings to patients.Future research should strive to gather data using diverse protocols, encompassing neurophysiological techniques such as TEPs [60] and behavioural measurements [61], to more deeply understanding this phenomenon.Secondly, we did not account for the potential placebo effect induced by sham stimulation.Thus, future endeavours should prioritise high-quality randomised controlled trials to validate the therapeutic efficacy of this combined stimulation parameter in individuals with motor impairment.

Conclusions
Preconditioning with cTBS enhanced iTBS-induced cortical plasticity in the M1 area of the lower limb, with a duration of after-effect lasting at least 20 min.This stimulation technique holds promise as a novel therapeutic strategy for future clinical applications of TMS in improving lower limb motor function and thus warrants further investigation.

Fig. 1 .
Fig. 1.Study design and methods.(A) TBS protocols and timing of the MEP assessment at each visit.Each subject underwent three different TBS protocols at different time points in a random order.(B) Illustration of TBS intervention.The coil handle was perpendicular to the scalp, and it was positioned in the anterior-posterior (AP) direction to generate a current along the long axis of the two-wing intersection.This resulted in a current aligned in the posterior-anterior (PA) direction within the cortex of the lower limb.(C) TBS program.(D) A sample of the real tibialis anterior (TA) MEP recording.RMT, rest motor threshold; MEP, motor-evoked potential; cTBS, continuous theta burst stimulation; iTBS, intermittent theta burst stimulation; T0, before intervention; T1, Immediately after intervention; T2, 20 min after intervention.