The influence of menstrual phase on synaptic plasticity induced via intermittent theta-burst stimulation

Background: Ovarian hormones influence the propensity for short-term plasticity induced by repetitive transcranial magnetic stimulation (rTMS). Estradiol appears to enhance the propensity for neural plasticity. It is currently unknown how progesterone influences short-term plasticity induced by rTMS. Objective: The present research investigates whether the luteal versus follicular phase of the menstrual cycle influence short-term plasticity induced by intermittent theta-burst stimulation (iTBS). We tested the hypothesis that iTBS would increase motor evoked potentials (MEPs) during the follicular phase. Further, we explored the effects of the luteal phase on iTBS-induced neural plasticity. Method: Twenty-nine adult females participated in a placebo-controlled study that delivered real and sham iTBS to the left primary motor cortex in separate sessions corresponding to the follicular phase (real iTBS), luteal phase (real iTBS), and a randomly selected day (sham iTBS). Outcomes included corticospinal excitability as measured by the amplitude of MEPs and short-interval intracortical inhibition (SICI) recorded from the right first dorsal interosseous muscle before and following iTBS (612 pulses). Results: MEP amplitude was increased following real iTBS during the follicular condition. No significant changes in MEP amplitude were observed during the luteal or sham visits. SICI was unchanged by iTBS irrespective of menstrual phase. Conclusion: These findings suggest women experience a variable propensity for iTBS-induced short-term plasticity across the menstrual cycle. This information is important for designing studies aiming to induce plasticity via rTMS in women.


Introduction
Synaptic plasticity pertains to alterations in synaptic efficacy that arise from the intrinsic activity of a neuron (Abraham, 2008;Abraham & Bear, 1996;Frey et al., 1995).Congregations of neurons form neural circuits that facilitate memory formation through modifications of synaptic patterns induced by circuit activity (Citri & Malenka, 2008).A well-known manifestation of synaptic plasticity is long-term potentiation (LTP), characterized by augmented synaptic potentials resulting from high-frequency stimulation of excitatory synapses or the correlation between presynaptic and post-synaptic depolarization (Martin et al., 2000).Conversely, long-term depression (LTD) represents a distinct form of synaptic plasticity that diminishes the strength and efficacy of synaptic transmissions (Wong et al., 2021).
Estradiol and progesterone impact neural activity (Inghilleri et al., 2004;Raciti et al., 2023;Smith et al., 2002).Estradiol influences learning and memory, such that increases in estradiol are correlated with better performance on spatial memory tasks (Sandstorm & Williams, 2004).Studies indicate a relationship between estradiol and glutamate (Krentzel et al.,2022), such that estradiol reduces the firing threshold for neurons and thus promotes excitability within the cortex (Grassi et al., 2010;Finocchi & Ferrari, 2011).Conversely, progesterone increases GABA A activity which reduces cortical excitability (Smith et al., 2002;Smith et al., 1999;Guennoun et al., 2015), thus leading to reductions in the amplitude of motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS).In a study conducted by Inghilleri and colleagues, 5 Hz repetitive transcranial magnetic stimulation (rTMS) was used to induce short-term plasticity in the primary motor cortex (M1) in women during the early and late follicular phase of the menstrual cycle (Inghilleri et al., 2004).Results indicated that MEP amplitude was increased following rTMS during the late follicular phase only (Inghilleri et al., 2004).
In the present study, we sought to investigate the influence of the menstrual phase on the propensity for rTMS-induced plasticity.We deviate from previous research by including a placebo control for rTMS, test women in both the follicular and luteal phase of the menstrual cycle when estrogen and progesterone peak, respectively (Reed & Carr., 2015) and use intermittent theta burst stimulation (iTBS) to induce short-term plasticity.ITBS is recognized for augmenting cortical excitability, as reflected by an increase MEP amplitude and short-interval intracortical inhibition (SICI) (Fitzgerald et al., 2006;Huang et al.,2005).In the present research we tested the hypothesis that iTBS would increase motor evoked potentials (MEPs) during the follicular phase.Additionally, we investigated the impact of the luteal phase on iTBS-induced neural plasticity.

Ethical approval
The present study was approved by Hamilton Integrated Research Board (REB 15801).The research conformed to the standards set by the Declaration of Helsinki.Following the explanation of the study protocol, all participants provided their written informed consent prior to participation.

Participants
Thirty right-handed females were recruited to participate in the experiment.One individual participated in one session only due to scheduling conflicts and was therefore not included in further analyses.Twenty-nine individuals (22.19 ± 3.32 years) completed three sessions separated by a minimum of one week (Table 1).All participants were screened for contradictions to receiving TMS.All participants completed a menstrual cycle questionnaire to ensure a consistent menstrual cycle and were free of hormonal contraceptive use for one year prior to the study.
The projected sample size was formulated using a G*Power computed a priori analysis.This analysis was based on the significant main effect of DAY for MEPs acquired during 5 Hz rTMS in the late follicular phase (Inghilleri et al., 2004).The power was set at 0.9 and α = 0.05.

Electromyography recording
Surface electrodes (9 mm Ag-Cl) were used to record activity from the first dorsal interosseous (FDI) muscle of the right hand.grounded to the styloid process at the wrist.EMG signals were amplified x1000 and bandpass filtered between 20-2.5 kHz (Intronix Technologies Corporation Model 2024F, Bolton, Canada).An analog-digital converter was used to digitize data at 5 kHz (Power1401; Cambridge Electronics Design, Cambridge, UK), prior to being analyzed through commercial software (Signal v6.02; Cambridge Electronics Design, Cambridge, UK).

Maximum voluntary contraction
Participants completed three maximal isometric contractions of the right FDI against a stationary structure.Each contraction persisted for 5 s with 30 s rest intervals between trials.The largest EMG activity was obtained from the three trials and considered the maximum voluntary contraction (MVC) of the right FDI muscle for that participant.The level of EMG activity corresponding to 10 % of the MVC was displayed on an oscilloscope as a horizontal target line.Participants were required to match the horizontal target line by controlling a second line that displayed their FDI contraction.Active motor threshold (AMT) was obtained while having the participant maintain the FDI contraction at ~10 % MVC.

Transcranial magnetic stimulation
The hotspot of the right FDI muscle is defined as the location on the left motor cortex (M1) that, when stimulated with TMS, consistently led to the largest MEP in the muscle.This location was registered using Brainsight Neuronavigation (Rogue Research, Montreal, Canada).TMS was recorded using a 70 mm inner diameter figure-of-eight coil with a Magstim Super Rapid 2 Plus Stimulator and a 50 mm figure-of-eight coil with a Magstim 200 2 stimulator (Magstim, Whitland, UK).
Resting motor threshold (RMT) was determined as the lowest intensity required to elicit a MEP equal to or larger than 50 μv 50 % of the time in the right FDI muscle.The stimulus intensity was set to 37 % maximum Stimulator output (MSO), and twenty TMS pulses were distributed over M1, specifically the right FDI hotspot, with the stimulus intensity being adjusted after each pulse as advised by the MTAT software, TMS_MTAT_2.0 freeware (http://clinicalresearcher.org/software.htm), based on the presence or absence of an MEP on the previous trial.
AMT was quantified by the lowest intensity required to elicit a MEP>200 μv 50 % of the time, while maintaining 10 % MVC of the right FDI muscle.The stimulus intensity was set to 37 % MSO, and twenty TMS pulses were distributed over M1, specifically the right FDI hotspot, with the stimulus intensity being adjusted after each subsequent pulse as advised by the MTAT software (TMS_MTAT_2.0freeware (http://clinic alresearcher.org/software.htm)) based on the presence or absence of a silent period on the previous trial.MEPs were obtained from the right FDI muscle at rest using single pulse monophasic TMS pulses before and 10 min following iTBS.Twenty TMS pulses were delivered at three different intensities (110, 130, and 150 % RMT) in a randomized order (60 pulses total).Short-interval intracortical inhibition (SICI) was elicited by delivering 25 paired-pulse (conditioned stimuli) and 25 single pulse (unconditioned stimuli) TMS.Paired-pulses were delivered with an interstimulus interval of 2 ms between conditioning stimulus and test stimulus (Huang et al., 2005;Kujirai et al., 1993;El-Sayes et al., 2019).The conditioning stimulus was delivered at 90 % AMT (Orth et al., 2003;Wassermann et al., 2008) and the test stimulus was delivered at an intensity to elicit peak-to − peak MEP amplitude of 1 mV in the right FDI muscle at rest.
To induce synaptic plasticity, repetitive TMS (rTMS) was performed using a 90 mm outer diameter figure-of-eight coil with a Magstim Super Rapid 2 Plus Stimulator (Magstim, Whitland, UK).rTMS pulses were delivered to the hotspot of the right FDI muscle.The iTBS protocol consisted of biphasic pulses in burst of three pulses delivered at 30 Hz, in 6 Hz trains that lasted 2 s, this was followed by 8 s with no pulse delivered (Hosel & Tremblay, 2021;Jacobs et al., 2014;Fassett et al., 2017).This train was repeated for a total of 612 pulses at 80 % of AMT using a.Sham iTBS utilized a 70 mm sham iTBS coil which mimicked the sound of real iTBS, but does not deliver stimulation.

Experimental Design
Individuals participated in three sessions corresponding to the follicular phase (real iTBS), luteal phase (real iTBS), and a randomly selected visit (sham iTBS).All individuals were pseudo-randomized to attend their first session during the follicular phase (n = 10), luteal phase (n = 10) and a randomly selected visit (n = 9).During each visit (real or sham) stimulation was delivered over left M1 at the hotspot corresponding to the FDI muscle.All participants were naïve to rTMS.Individuals were anonymized to the type of stimulation, real iTBS or sham iTBS received.The follicular phase visit was conducted on day 8 ± 1 (real iTBS; mid-follicular phase) of the menstrual cycle when estradiol levels are highest (Reed & Carr, 2015).The luteal phase visit was conducted during day 21 ± 2 (real iTBS; mid-luteal phase) when progesterone levels are highest (Reed & Carr, 2015).To determine the onset of the luteal phase, participants were provided with take-home BFH Ovulation tests (Fairhaven Health Bellingham WA, USA).Each participant received these take-home tests during their follicular phase.Beginning at day 9, participants were instructed to use one test each day until they received a positive reading.Once a positive ovulation test reading was detected, the participant was scheduled for a session that occurred ~7 days later.
During all visits, TMS was used to record measures of AMT, RMT, MEPs, and SICI before (T0) and 10 min after (T1) real or sham iTBS delivery.

Statistical analyses
All MEP data was visually inspected, and trials contaminated with EMG activity exceeding 50 μV 100 ms prior to the stimulus artefact were discarded (Turco et al., 2019;Rehsi et al., 2023;Ramdeo et al., 2023).The magnitude of SICI was expressed as a ratio of the conditioned MEP amplitude to the unconditioned MEP amplitude.MEP data was expressed as the average of all MEP intensities (60 trials) collected at baseline and 10 mins post iTBS.To calculate the area of the recruitment curve the averaged amplitude of the MEP for each intensity was summed across all intensities for each individual.Normality was assessed using Shapiro-Wilks Test.If the data was not normally distributed a square root transformation was applied.Significance was set to α ≤ 0.05.
To assess baseline differences between the follicular and luteal phases for AMT, RMT, SICI and MEPs, a two-tailed paired samples t-test was performed for each dependent measure.
For MEPs, two-way ANOVA using within-subject factors CONDITION (Follicular, Luteal, Sham) and TIME (T0, T1) was performed.The same analyses were performed for SICI.Further, a one-tailed paired samples ttest was conducted to test the hypothesis that iTBS would increase motor evoked potentials (MEPs) during the follicular phase.
Reliability statistics were performed on the sham MEP data to assess the measurement error that occurred from repeat testing of an individual.Absolute reliability was determined for MEPs acquired before and following the sham condition using the SEMeas values (SEMeas = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ ̅ MeanSquared error √ ) (Schambra et al., 2015).SEMeas values greater than 10 % indicate large amounts of measurement error (Schambra et al., 2015).Furthermore, the SEMeas was used to determine the SDC group (SDC group = SEMeas x 1.96 x √2) √n ) where n is the sample size (Button et al., 2013).

Results
Twenty-nine participants completed all three visits.Baseline data (T0) demonstrated no significant differences in AMT, RMT, SICI and MEPs between phases of the menstrual cycle (Table 2).
Baseline sham condition data was divided into sham sessions conducted during the follicular condition (n = 13) and those conducted during the luteal condition (n = 16).The data reveals no differences in the baseline measurements between the sham-follicular and sham-luteal conditions (Table 3).

Discussion
In the present study, MEPs and SICI were measured before and following iTBS during the mid-follicular and luteal phases.We observed a statistically significant increase in corticospinal excitability during the follicular condition only.Furthermore, iTBS did not alter SICI in any condition.We discuss these findings and their possible neural  mechanisms below.iTBS delivered during the follicular phase resulted in MEP facilitation.Subtracting the experimental error (SDC group ~ 5 %), MEPs were facilitated by approximately ~ 17 % following iTBS in the follicular condition.One previous study demonstrated facilitation of MEPs following 5 Hz rTMS delivered in the late (Day 14) but not early follicular phase (Inghilleri et al., 2004).The mechanism underlying MEP facilitation may be attributed to elevated levels of estradiol which increase corticospinal excitability (Finocchi & Ferrari, 2011;Woolley & McEwen, 1990;Gazzaley et al.,1996).Estradiol increases the number of spines on the apical dendrites in the hippocampal CA1 pyramidal neurons, thus increasing the production of mRNA for NMDA receptor subunits (Woolley et al., 1990).The greater density of NMDA receptors increases Ca 2+ entry to promote LTP (Finocchi & Ferrari, 2011).Importantly it is the combination of increased estradiol and the delivery of iTBS that leads to MEP facilitation.Increased estradiol on its own is insufficient to alter corticospinal excitability since MEPs were not different between the follicular and luteal conditions at baseline.Collectively, these data suggest the influence of estradiol is likely mediated through NMDA receptors that are stimulated during highfrequency stimulation.
This study is also the first to test the response to iTBS during the luteal phase and revealed no significant MEP modulation.The lack of short-term iTBS induced plasticity may be the result of elevated levels of progesterone that increases GABA A activity (Finocchi & Ferrari, 2011).Progesterone modulates GABA A via a progesterone metabolite, allopregnanolone, which enhances GABA A mediated chloride conductance, leading to inhibitory effects on neural activity (Wilson, 1996;Barth et al., 2015).With the activation of GABA A receptors, LTP induction is blocked (Evans & Viola-McCabe, 1996).Progesterone has also been shown to decrease the number of dendritic spines in the CA1 area, which is responsible for the production of NMDA receptors correlated with LTP responses (Finocchi & Ferrari, 2011).
GABA mediated SICI was unchanged following real and sham iTBS similar to previous reports (Brownjohn et al., 2014;Doeltgen & Ridding, 2011;Huang et al., 2010;Lopez-Alonso et al., 2014;Zamir et al., 2012) and in contrast to others (Huang et al., 2005;Murakami et al., 2008).Further, at baseline, SICI was not different between the follicular and luteal phases.Previous research demonstrated greater inhibition in the luteal vs follicular phase when ISIs of 2,3,4, and 5 ms were combined (Smith et al., 1999).In another study, paired-pulse conditioned MEPs were averaged for ISIs of 2-10 ms and demonstrated differences between the early and late follicular phases but no significant differences between the follicular and luteal phases (Smith et al., 2002).Averaging MEPs for ISIs 2-10 ms, a third study observed differences between the control group and the PMS group such PMS demonstrated facilitation of the conditioned MEP (Smith et al., 2003).However, in the aforementioned studies, SICI was not specifically isolated as ISIs greater than 2 ms were included in the averaged data.Importantly, SICI is proposed to be contaminated by short-interval-intracortical facilitation at ISIs greater than 2 ms (Peurala et al., 2008).In the present study, we specifically assess SICI using a 2 ms ISI and although it appears that inhibition is greater in the luteal phase (Fig. 2), there is substantial variability in SICI response which is not different between the follicular and luteal phase.Indeed, the repeatability of SICI within subjects is poor (Nielsen et al., 2021), and this may preclude the opportunity to expose differences between the follicular and luteal phase using this measure.In addition, in women who experience premenstrual syndrome (PMS), there is evidence of reduced serum GABA during the luteal phase (Halbreich et al., 1996) and facilitation of the MEP compared to non-PMS controls.In the present study, we did not assess PMS and are unable to determine whether serum GABA levels contribute to the lack of SICI differences between the two phases.Measures of RMT and AMT at baseline were not different across phases of the menstrual cycle.Previous studies also demonstrate no differences in RMT (Hattemer et al., 2007) and AMT across the phases of the menstrual cycle (Inghilleri et al., 2004;Smith et al., 2002;El-Sayes et al., 2019).

Limitations
One limitation is that serum concentration of estrogen and progesterone were not obtained.Although the absolute magnitude of hormones was not quantified, this study delineated between phases of the  menstrual cycle based on the timing of ovulation.Further, the present study used a 30 Hz iTBS protocol used elsewhere (Hosel & Tremblay, 2021;Jacobs et al., 2014;Fassett et al., 2017) which is less frequently explored compared to 50 Hz iTBS.Last, dependent measures included only MEPs and SICI and other measures may have exposed iTBS-induced changes during the luteal phase.

Future directions
Our research demonstrates that women experience iTBS-induced plasticity that is influenced by the phase of the menstrual cycle.The present study tested women who were naturally cycling aged 18-30 years old.Future research should aim to explore how the use of oral contraceptives influences iTBS-induced plasticity.In addition, a subsequent study should aim to explore iTBS in aging women (pre-menopausal and post-menopausal).
In the present study we demonstrate that women experience a variable propensity for short-term plasticity between the two phases of the menstrual cycle.We confirm previous research which demonstrates the response to rTMS-induced plasticity is only present during the follicular phase (Inghilleri et al., 2004).In addition, our research is the first to demonstrate that iTBS-induced plasticity does not occur during the luteal phase.These results should be considered in basic and clinical neuroscience research aiming to induce short-term plasticity in women using non-invasive brain stimulation.

Fig. 1 .
Fig. 1. A. Group-averaged MEP amplitudes (with standard error) at baseline (T0) and T1.Follicular condition (real iTBS) demonstrates increases in the MEP amplitude.Real iTBS in the luteal phase and sham iTBS did not significantly alter MEPs.*Asterisk indicate a significance of p = 0.016.Fig. 1B.Group-averaged MEP Area (with standard error) at baseline (T0) and T1.Follicular condition (real iTBS) demonstrates increases in MEP Area.Real iTBS in the luteal phase and sham iTBS did not significantly alter MEP Area.*Asterisk indicate a significance of p = 0.016.

Fig. 2 .
Fig. 2. Group-averaged (with standard error) SICI at T0 and T1.No significant changes in SICI were observed following iTBS in the follicular and luteal condition, and following sham iTBS.

Table 1
Menstrual History Data.Reported as mean ± standard deviation.

Table 2
Data is reported as mean ± standard deviation for each time point within each menstrual condition.%RMT: resting motor threshold; AMT: active motor threshold, %MSO: maximum stimulator output, SICI: short-interval intracortical inhibition; MEP: motor evoked potential at 110, 130, 150 % of RMT.

Table 3
Data is reported as mean ± standard deviation for each time point within the sham condition.%RMT: resting motor threshold; AMT: active motor threshold, %MSO: maximum stimulator output, SICI: short-interval intracortical inhibition; MEP: motor evoked potential at 110, 130, 150% of RMT.