Integrin α3 is required for high‐frequency repetitive transcranial magnetic stimulation‐induced glutamatergic synaptic transmission in mice with ischemia

Abstract Background Repetitive transcranial magnetic stimulation (rTMS) is an effective therapy in post‐stroke motor recovery. However, the underlying mechanisms of rTMS regulates long‐lasting changes with synaptic transmission and glutamate receptors function (including AMPARs or NMDARs) remains unclear. Methods Mice were received 10‐Hz rTMS treatment once daily on the third day after photothrombotic (PT) stroke for 18 days. Motor behaviors and the Western blot were used to evaluate the therapeutic efficacy of 10‐Hz rTMS in the mice with PT model. Moreover, we used wild‐type (WT) and NEX‐α3 −/− mice to further explore the 10‐Hz rTMS effect. Results We found that 10‐Hz rTMS improved the post‐stroke motor performance in the PT mice. Moreover, the levels of AMPAR, vGlut1, and integrin α3 in the peri‐infarct were significantly increased in the rTMS group. In contrast, 10‐Hz rTMS did not induce these aforementioned effects in NEX‐α3 −/− mice. The amplitude of AMPAR‐mediated miniature excitatory postsynaptic currents (EPSCs) and evoked EPSCs was increased in the WT + rTMS group, but did not change in NEX‐α3 −/− mice with rTMS. Conclusions In this study, 10‐Hz rTMS improved the glutamatergic synaptic transmission in the peri‐infract cortex through effects on integrin α3 and AMPARs, which resulted in motor function recovery after stroke.


| INTRODUC TI ON
Ischemic stroke is a major cause of disability and mortality worldwide. 1Estimates show that 70% of survivors have long-term disability, which has a serious negative impact on their daily lives. 2,3Other than traditional rehabilitation, new therapies, including nonpharmacological approaches, have gained attention to address the growing challenge of stroke and neurological disorders.
Repetitive transcranial magnetic stimulation (rTMS) is a promising therapy for patients with stroke. 4rTMS can modulate cortical excitability and increase neuronal plasticity.Specifically, high-frequency rTMS (≥5 Hz) induces facilitatory effects, whereas low-frequency rTMS (≤1 Hz) induces a reduction in synaptic efficiency. 5inical and animal investigations have both demonstrated that post-stroke motor recovery can be effectively facilitated through the application of ipsilateral high-frequency rTMS, which produces results similar to those seen with long-term potentiation (LTP). 6wever, the molecular mechanism underlying these effects remains elusive.
Glutamate transmission plays a fundamental role in LTP. 7evious studies indicated that the 10-Hz rTMS protocol could result in long-lasting structural and functional changes at excitatory synapses by activating N-methyl-D-aspartate receptors (NMDARs). 8Also, high-frequency rTMS interferes with the postsynaptic accumulation of α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs), especially for GluA1 in an animal model of stroke. 9In addition, the TMS pulses induced the voltage-sensitive current in the presynaptic compartment in an NMDAR-independent manner as revealed by recording voltage-clamp traces with glutamatergic blockers, 10 while the long-duration rTMS protocol increased the NMDAR-dependent long-term plasticity changes. 11Therefore, the complex mechanisms that mediate rTMS-induced glutamatergic synaptic plasticity remain to be elucidated.
Integrins are a class of cell adhesion receptors expressed in excitatory neurons and glial cells.They are involved in synaptic maturation and plasticity as heterodimers (αβ). 12In mammals, 18 α and 8 β subunits can generate 24 different known heterodimers (αβ)   with different binding properties and tissue distribution. 13,14Our previous studies have shown integrin α3β1 is essential for synapse stability in the late postnatal mouse brain.The loss of integrin α3 from excitatory neurons causes reduced synapse densities in adult mice. 15Integrin α3 also influences NMDAR-and AMPARmediated synaptic currents. 16,17A reduction or loss of integrin α3 significantly decreases hippocampal synaptic plasticity and LTP, resulting in working memory impairment. 18,19Above all, integrin α3 has a defined role in consolidating both structural and functional changes brought about by LTP.However, whether integrin α3 is involved in rTMS-modulated synaptic plasticity after stroke is unclear.
This study aimed to investigate the role of integrin α3 in rTMS-induced motor function recovery following stroke.First, we treated C57BL/6J mice with 10-Hz rTMS for 18 days to observe motor recovery, glutamatergic synaptic transmission, and integrin α3 expression.We found that 10-Hz rTMS could improve behavioral outcomes and increase the levels of AMPARs, glutamate transporters, and integrin α3 in the peri-infarct region.Furthermore, we used mice bearing an excitatory neuron-specific ablation of integrin α3 (NEX-α3 −/− mice) to establish a key role for integrin α3-AMPAR/NMDAR pathway in the effect of 10-Hz rTMS.Compared to WT mice where 10-Hz rTMS improved motor function and increased synaptic transmission following stroke, the same treatment did not improve outcomes in the NEX-a3 −/− mice.We also demonstrated the critical role of integrin α3 in AMPAR-mediated excitatory synaptic current and strength increase effect induced by rTMS using the whole-cell patch-clamp recording.We concluded that the protective effects of 10-Hz rTMS on post-stroke mice required the integrin α3/AMPAR pathway.

| Animals
All procedures were approved by the Fudan University Animal

| Photothrombotic stroke model
Mice were deeply anesthetized with sodium pentobarbital (50 mg/ kg) intraperitoneally before the surgery, and a stereotactic apparatus was used to place animals.Photothrombotic (PT) stroke was induced in the left sensorimotor cortex (coordinates: 2.5-1.5 mm rostral to caudal and 0-4 mm lateral to the medial). 22Briefly, the hair was removed before the initial incision, and the skull was exposed.
The mice were given 0.1% Rose Bengal (0.01 mL/g body weight,) by intravenous injection.Subsequently, a green laser beam (532 nm, ~50 mW intensity at the sample) was stereotactically positioned onto the target left sensorimotor cortex for 10 min.

| Repetitive transcranial magnetic stimulation (rTMS) treatment
A magnetic stimulator (CCY-II, Yiruide Medical Instrument Co., Ltd., Wuhan, China) was used to stimulate conscious mice on the third day after PT.Then, 10-Hz repetitive transcranial magnetic stimulation (rTMS) at 35% maximum stimulator output (600 pulses per day) was administered for 18 consecutive days.During the treatment, the mouse heads were placed at the center of the circular coil.

| Behavioral assessments
The rotarod test and the ladder rung walking test were used in this study.Mice were brought to the testing room for habituation 30 min prior to each test.All assessors were blinded to the group allocation.

| Rotarod test
The rotarod test was used to evaluate the coordination, balance, and motor performance of the mice. 23The test was performed on the 14th and 21st days post-PT surgery.On the experimental day, the mice were placed on an accelerating rotating rod at a speed of 5-40 rpm for 5 min.The latencies to falling from the rod were recorded for each mouse, and the mean of the two fall latency values was used for data analysis.All animals received 3 days of training before the surgery.

| Ladder rung walking test
The ladder rung walking task was used to assess limb placement and limb coordination of the mice with stroke. 24The apparatus comprised two plexiglass side walls linked by inserting metal rungs (length: 100 cm, width: 5 cm, 30 cm above the ground).The test was conducted on the 14th and 21st days post-PT after the pre-surgery training sessions.Any deep paw slips, slight paw slips, replacement, and complete misses were scored as errors, and the forelimb slip rate was calculated as ratios of errors to total steps.

| Brain slice preparation and electrophysiological recording
Seven-week-old male mice were anesthetized with isoflurane and

| Statistical analysis
Electrophysiology raw-trace offline analysis was performed using Igor 6.2 (WaveMetrics).EPSCs and mEPSCs were analyzed using Igor procedures, and incorrect mEPSC events were removed.Data were expressed as the mean ± standard error of the mean (SEM) and analyzed with Prism 8 software.The normal distribution of continuous data was detected by the Shapiro-Wilk test.One-way analysis of variance (ANOVA) (one factor) or two-way ANOVA (two factors) followed by Tukey's multiple comparison test was used to compare multiple groups.A p value <0.05 indicated a statistically significant difference.

| rTMS promoted motor recovery after a photothrombotic stroke
The ladder rung walking and rotarod tests were used to evaluate the motor functional recovery at different time points after PT to explore the therapeutic effects of 10-Hz rTMS on post-stroke mice (Figure 1A).In the ladder rung walking test (Figure 1B,C), a higher forelimb slip rate was observed on the 14th day after PT compared with that in the sham group (sham vs PT, p < 0.0001; sham vs PT + rTMS, p = 0.036).However, rTMS treatment decreased the forelimb slip rate (PT vs PT + rTMS, p = 0.016).Similarly, on the 21st day, the PT group (0.52 ± 0.02%) also exhibited more slips in the ladder rung walking test compared with the sham group (0.27 ± 0.02%), whereas the PT + rTMS groups showed fewer slips (0.38 ± 0.03%) (sham vs PT, p < 0.0001; sham vs PT + rTMS, p = 0.025; PT vs PT + rTMS, p = 0.006).
Additionally, in the rotarod test (Figure 1D,E), both PT and PT + rTMS results showed motor deficiency compared with that in the sham group on the 14th day post-PT (sham vs PT, p < 0.0001; sham vs PT + rTMS, p = 0.003).However, motor deficiency only occurred in the PT group on the 21st day (sham vs PT, p = 0.0015).
The PT + rTMS group showed a robust recovery, with no significant differences compared with the sham group (sham vs PT + rTMS,

| Integrin α 3 deletion inhibited the motor recovery effect of rTMS in post-stroke mice
We analyzed the motor recovery in the WT + PT, NEX-α3 −/− + PT, WT + PT + rTMS, and NEX-α3 −/− + PT + rTMS groups on the 14th and 21st days post-stroke to further explore the role of integrin α3 in the motor recovery effect of rTMS in post-stroke mice (Figure 3A).

| DISCUSS ION
This study has revealed that a course of 10-Hz rTMS treatment spanning 18 days not only ameliorated motor recovery in poststroke mice but also instigated an enhancement in glutamatergic synaptic transmission, alongside elevated levels of AMPARs, vGlut1, and integrin α3.Notably, these changes were paralleled by augmented AMPAR-mediated currents, as observed through assessments of both AMPAR-mediated mEPSCs and eEPSCs.
Intriguingly, no discernible disparity in post-stroke motor recovery emerged between mice subjected to rTMS and those without it, particularly evident in cases involving NEX-α3 −/− mice.From these observations, we deduced that the positive impact of 10-Hz rTMS on motor recovery among post-stroke mice potentially hinges upon the regulation of the integrin α3/AMPAR signaling pathway.Different from the earlier studies that rTMS promotes synaptic plasticity by upregulating AMPARs post-stroke, 26 our findings provide direct evidence that integrin α3 is a critical factor in rTMS-mediated AMPAR upregulation.This suggests that the direct modulation of integrin α3 may offer novel avenues to mitigate stroke-related dysfunction, via the regulation of glutamatergic neurotransmission.
In this study, we found that the induced photothrombosis triggered an increase in the expression of GAD65, coupled with a concomitant decrease in the expression of the glutamatergic transporter vGlut1 within the peri-infarct cortex.In contrast, administration of 10-Hz rTMS resulted in notable elevations in the levels of vGlut1, GluA1, and GluA2/3/4.Although there was a slight increment in the levels of GluN2A and GluN2B following 10-Hz rTMS, these distinctions did not attain statistical significance.These changes in protein expression are likely tied to a reduction in cortical excitability post-stroke.In addition, the level of integrin α3 in the PT group was elevated compared to the sham group; however, it remained notably lower than the levels observed in the rTMS group.Considering that integrin α3 plays a role in the cellular membrane localization of AMPARs, we hypothesized that this relationship might follow a dose-dependent pattern.The heightened integrin α3 expression in the PT group could potentially signify a compensatory response, albeit insufficient to induce significant changes in AMPARs at this stage.Nevertheless, further investigations are imperative to elucidate this divergence.
Aligned with the alterations in the glutamatergic signaling components, a discernible rise in the amplitude of AMPAR-mediated mEPSCs and eEPSCs emerged in post-stroke mice subjected to rTMS.These findings harmonize with existing evidence that high-frequency rTMS heightens GluA1 levels and promotes the insertion of AMPARs into synapses.However, prior investigations have positioned NMDARs and AMPARs as the principal receptors underpinning rTMS effects, 27 the involvement of NMDARs in rTMS-induced neuronal plasticity remains a possibility.Yet, the amplitudes of NMDAR-mediated mEPSCs and eEPSCs remained unperturbed in our results.This hints at a preference for 10-Hz rTMS to primarily activate rapidly and briefly opening AMPARs, which could interact with NMDARs characterized by slower depolarizing currents.Thus, more extensive rTMS protocols could illuminate the role of NMDA receptor-mediated long-term plasticity in future inquiries. 28,29ilding upon our prior previous studies highlighting integrin α3's role in synaptic maturation, plasticity, and neuronal stability, 15 we found an upsurge in integrin α3 expression within the peri-infarct cortex following a stroke event.Furthermore, we have established that integrin α3 plays a pivotal role in rTMS-induced functional recovery.Notably, the beneficial effects of rTMS on motor recovery were nullified with the loss of the integrin α3 gene in excitatory neurons, accompanied by an absence of rTMS impact on AMPARmediated currents and GluA1 and GluA2/3/4 clustering in NEX-α3 −/− mice.Considering that rTMS could modulate the neuronal activity in the ipsilateral hemisphere, it is plausible for integrin α3, pivotal in cell adhesion and migration, to transduce activity-dependent signals leading to AMPAR and vGlut1 recruitment and insertion.Furthermore, since the integrin α3-regulated Arg kinase coordinates the maturation of presynaptic and postsynaptic compartments within a subset of hippocampal synapses, 30 this process aligns with the significance of LTP-like plasticity in the plastic changes induced by rTMS.Consequently, it is conceivable that integrin α3's response to rTMS encompasses multifaceted pathways.However, the precise interplay between AMPAR activation and integrin α3-mediated signaling in rTMS-induced post-stroke recovery necessitates further investigation.Some limitations were existed in this study.Since the integrin α3 is an accomplice of integrin β1, the integrin α3β1 ligands exert influence on neuronal development, structure, regeneration, maintenance, and plasticity. 31,32Nonetheless, our study did not explore the role of integrin β1 in rTMS-induced motor recovery post-stroke.
Furthermore, during the course of this research, we concurrently pursued two investigations linked to integrin α3, involving distinct cohorts of mice subjected to behavioral tests and protein assessments across various time points.The omission of time-variable analyses could potentially limit our ability to fully grasp the evolving behavioral changes following stroke in different groups.Lastly, the intricate interactions between extracellular matrix (ECM) proteins 33 and integrins post-ischemic stroke need more investigation.For instance, β1 integrin in conjunction with laminin provides scaffolds, guiding neural migration toward injury sites post-stroke. 34Our prior study demonstrated the functional interaction between laminin α5 and integrin α3 in regulating dendritic spine density and animal behavior. 35The broader impact of integrin-extracellular matrix remodeling following rTMS treatment in stroke remains a fascinating area for exploration, considering its potential significance for neurogenesis and angiogenesis.

| CON CLUS IONS
Our study showed that 10-Hz rTMS promoted the glutamatergic synaptic transmission after stroke.Additionally, we found that integrin α3 is critical for AMPAR-dependent motor functional improvement induced by 10-Hz rTMS.These results might highlight integrin α3 as a potential novel therapeutic target for treating stroke.

AUTH O R CO NTR I B UTI O N S
Yi Wu, Xiao Xiao, Junfa Wu, Nianhong Wang, and Qun Zhang designed the study and supervised the entire project.Anthony J.

E 1 | 5 of 12 LIU
Effects of 10-Hz rTMS on post-stroke motor function recovery.(A) Time points of behavioral tests and surgery in C57/6 J mice.Behavioral training was started from the 3rd day before PT surgery, whereas motor assessments were conducted on the 14th and the 21st days after the PT stroke.rTMS was given from the 3rd to the 21st days post-stroke.The mice were sacrificed on the 21st day after PT for Western blot.(B, C) Percentage of forelimb slips in the ladder rung walking test on the 14th and the 21st days after stroke in different groups.(D, E) Latency to fall off the rotarod on the 14th and the 21st days after stroke in different groups.Data are expressed as the mean ± SEM (n = 7-15).*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.et al. p > 0.05).The PT + rTMS group showed better performance on both the 14th (p = 0.02) and 21st (p = 0.03) days compared with the PT group.

F I G U R E 2
Effect of rTMS treatment on glutamatergic receptor and transporter levels in the peri-infract cortex of mice with stroke.(A) Schematic representation of the tissue used for Western blot.(B) Representative blots of vGlut1 and GAD65 in different groups.(C, D) Quantitative analysis of vGlut1 and GAD65 levels.(E) Representative blot of GluA1 and GluA2/3/4 in different groups.(F,G) Quantitative analysis of GluA1 and GluA2/3/4 levels.(H) Representative blot of GluN2A and GluN2B in different groups.(I, J) Quantitative analysis of GluN2A and GluN2B levels.(K) Representative blot of integrin α3 in different groups.(L) Quantitative analysis of integrin α3 levels (n = 3-6).*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.F I G U R E 3 Effects of rTMS on motor recovery in WT and NEX-α3 −/− mice after PT. (A) Experimental process in WT and NEX-α3 −/− mice.Behavioral training was started from the 3rd day before PT, whereas the motor assessments were conducted on the 14th and 21st days after PT stroke.The rTMS was conducted from the 3rd day to the 21st day post-stroke.The mice were sacrificed on the 21st day after PT for electrophysiological recording and Western blot.(B, C) Percentage of forelimb slips in the ladder rung walking test in different groups.(D, E) Latency to fall off the rotarod in different groups (n = 6-18).Data are expressed as the mean ± SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.F I G U R E 4 Effects of rTMS treatment on glutamatergic receptor and transporter levels in WT and NEX-α3 −/− mice after PT. (A) Representative blot of vGlut1 and GAD65 in different groups.(B, C) Quantitative analysis of vGlut1 and GAD65 levels.(D) Representative blot of GluA1 and GluA2/3/4 in different groups.(E, F) Quantitative analysis of GluA1 and GluA2/3/4 levels.(G) Representative blot of GluN2A and GluN2B in different groups.(H, I) Quantitative analysis of GluN2A and GluN2B levels.(J) Representative blot of integrin α3 in different groups.(K) Quantitative analysis of integrin α3 levels.Data are expressed as the mean ± SEM (n = 4-8).*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.F I G U R E 5 Effects of rTMS treatment on mEPSCs in WT and NEX-α3 −/− mice after PT. (A) Representative traces of the AMPAR-mEPSCs in different groups.(B) Quantitative analysis of the amplitude of AMPAR-mEPSCs.(C) Quantitative analysis of the frequency of AMPAR-mEPSCs.Data are expressed as the mean ± SEM (n = 18-22).*p ≤ 0.05.F I G U R E 6 Effects of rTMS treatment on the evoked EPSCs in WT and NEX-α3 −/− mice after PT. (A) Representative traces of AMPAR and NMDAR currents in different groups.(B, C) Statistical analysis of the amplitude of AMPAR-eEPSCs and NMDAR-eEPSCs.Data are expressed as the mean ± SEM (n = 8-25).***p ≤ 0.001; ****p ≤ 0.0001.