| Purpose In recent years,methods to improve and enhance human motion ability through electrical nerve stimulation have received widespread attention in the field of sports science.Traditional transcranial electrical stimulation(TES)can change the excitability of brain neurons and enhance the body’s motor ability.However,traditional TES belongs to pan stimulation and can not precisely regulate specific areas of the brain and deep nuclei responsible for performing motor control and regulation.In 2017,the Grossman team proposed temporally interference(TI)electric field technology in the journal Cell,as a new non-invasive deep brain stimulation technology that can achieve targeted and precise regulation of brain regions.TI technology utilizes the characteristics of neurons that are unresponsive to high-frequency stimuli(>1000 Hz).Through the interaction of two sets of high-frequency and slightly different sinusoidal AC electric fields(such as 2000 Hz and 2010 Hz),low-frequency envelope waves(such as △ f =10 Hz)are formed.The low-frequency envelope waves are sufficient to drive the activities of the mind meridian without affecting other unrelated brain regions,providing the possibility of achieving precise regulation of the brain.At present,the research on TI technology is mainly in the exploratory stage,and there are few relevant research reports on TI technology improving sports performance.Our team’s previous research work explored the effects of TI stimulation of the primary motor cortex(M1)of mice for seven consecutive days under different dose-effect conditions(△ f =10 Hz,20 Hz)on their motor abilities(strength,balance,coordination,etc.).It was preliminarily determined that TI stimulation of the M1 region for seven consecutive days(once a day,10 minutes each time,△ f =20 Hz)can effectively enhance their motor abilities such as strength,balance,endurance,coordination,etc.Motor skills,also known as "action skills," refer to the ability of the human body to master and effectively complete specialized actions during movement.As a key brain region that regulates motor control and motor skill formation in humans and animals,M1 region plays an important role in mastering and consolidating motor skills.Based on the previous team’s relevant stimulation parameters,this study continued to explore the effects of TI stimulation of the M1 region of mice under different time-effective conditions(stimulation time: 10 min,20 min)on motor skills,and compared it with traditional transcranial alternating current stimulation(t ACS),so as to explore the potential advantages of TI stimulation as a new non-invasive deep brain stimulation.Further,in order to clarify the mechanism of TI stimulation in improving motor skills,this study utilizes technical means such as metabolomics,western blot,and multichannel optical fiber recording systems to explore the effects of TI stimulation on neurotransmitter metabolism and release,and synaptic plasticity.This will provide data support for the application of TI stimulation to human research,and providing a new non-invasive practical strategy for deep brain stimulation in the field of sports science.MethodsFive-week-old male C57BL/6J mice were selected for training in the single-pellet reaching task,and the mice that mastered the single-pellet reaching task were randomly divided into the TI-stimulated primary motor cortex sham stimulation group(TI-Sham group),the TI-stimulated primary motor cortex duration 10 min stimulation group(TI-10 min group),the TI-stimulated primary motor cortex duration 20 min stimulation group(TI-20 min group),and t ACS primary motor cortex sham stimulation group(t ACS-Sham group),t ACS primary motor cortex duration 10 min stimulation group(t ACS-10 min group),and t ACS primary motor cortex duration 20 min stimulation group(t ACS-20 min group).Improvements in motor skills of mice were detected after TI and t ACS stimulation,such as success rate,success speed,loss rate,failure rate and percentage of preferred forelimbs in completing single-pellet reaching task.After it was clear that TI and t ACS stimulation could improve the motor skills of mice,the effective intervention conditions of TI and t ACS(stimulation duration of 20 min)were selected for the study.Including the following experiments:(1)The metabolic changes of neurotransmitters in M1 region were detected using metabonomic techniques;(2)Western blot was used to detect the expression of synaptic plasticity related proteins and receptors,including postsynaptic densities-95(PSD-95)and synaptophysin(SYN),N-methyl-D-aspartate receptor(NMDAR)α-Amino-3-hydroxy-5methyl-4isoxazole receptor(AMPAR),brain-derived neurotrophic factor(BDNF),and c AMP-response element binding protein(CREB);(3)Multichannel optical fibers record the release of calcium ions and neurotransmitters in M1 region;(4)Sparse markers were used to detect the density of dendritic spines in neurons;(5)Transmission electron microscopy was used to detect the microstructure of synapses;(6)The muscle glycogen content and muscle mass of the triceps brachii and deep finger flexors of the forelimbs were examined.The above experiments were conducted to elucidate the mechanism of action of TI in enhancing motor skills in mice.Results1.Criteria for the assessment of learned motor skills in mice: the change in body weight of mice during the single-pellet reaching task training was stable at more than90% of the baseline level,meeting the requirements for weight control during training;the number of mice completing the task successfully reached more than 20 and the proportion of the preferred forelimb(right limb)completing the task reached more than70% by day 5 of the shaping training phase,meeting the criteria for the end of shaping training,and the single-pellet reaching task training could be carried out after day 6.On day 7 of the single-pellet reaching task phase,the success rate of mice completing the task was more than 40%,which met the end criteria for single-pellet reaching task training,and the mice were considered to have learned the motor skills.2.Effects of TI and t ACS stimulation of M1 region on the motor skills of mice:after 7 consecutive days of TI stimulation of the left M1 region of mice,there was no statistical difference between the TI-10 min group and the TI-Sham group(p>0.05).The success rate,failure rate,success speed and percentage of preferred forelimbs of mice completing the single-pellet reaching task in the TI-20 min group were significantly different compared to the TI-Sham group(p<0.01).The success rate,success speed and percentage of preferred forelimbs in the TI-20 min group were significantly higher and the failure rate in the TI-20 min group was significantly lower.After 7 consecutive days of t ACS stimulation in the left M1 region,there was no statistical difference between the t ACS-10 min group and the t ACS-Sham group(p>0.05).The success rate and failure rate of mice in the t ACS-20 min group in completing the single-pellet reaching task were significantly different from those in the t ACS-Sham group(p<0.05).The success rate of completing the task was significantly higher and the failure rate of completing the task was significantly lower in the t ACS-20 min group.There was no statistically significant difference(p>0.05)in the loss rate,success rate and percentage of preferred forelimbs in the t ACS-20 min group compared to the t ACS-Sham group.This indicates that both TI stimulation and t ACS can improve motor skills,and the degree of improvement TI is better than t ACS.3.Effects of TI and t ACS stimulation of M1 region on neurotransmitter metabolism: TI stimulation for 7 consecutive days(once a day,20 minutes each time)significantly increased the content of 11 neurotransmitters such as DA and Glu(p<0.05,p<0.01);Continuous 7 days of t ACS stimulation(once a day,20 minutes each time)significantly increased the content of 8 neurotransmitters such as DA and Glu(p<0.05,p<0.01).This indicates that TI stimulation affects more types of neurotransmitters than t ACS,and the increase is also greater than t ACS.4.Effects of TI and t ACS stimulation of M1 region on the neuronal excitability:both TI and t ACS stimulation for 7 consecutive days(once a day,20 minutes each time)significantly enhanced the release of calcium,glutamate,and dopamine(p<0.01).The increase in calcium and glutamic acid release caused by TI stimulation was greater than that caused by t ACS.5.Effects of TI and t ACS stimulation of M1 region on synaptic plasticity-related proteins: the expression levels of PSD-95,SYN,NMDAR,AMPAR,BDNF,and CREB proteins were significantly increased by stimulation with TI and t ACS for 7consecutive days(once a day,20 minutes each time)(all p<0.01).The increase in synaptic plasmin expression induced by TI stimulation was greater than that induced by t ACS.6.Effects of TI and t ACS stimulation of M1 region on dendritic spine and synaptic microstructure: 7 days of TI and t ACS stimulation(once a day,20 minutes each time)significantly increased dendritic spine density(p<0.01),the number of synaptic vesicles and the thickness of postsynaptic dense material(p<0.01).TI stimulation improved dendritic spine density and synaptic morphological microstructure more than t ACS.7.Effects of TI and t ACS stimulation of M1 region on muscle mass and muscle glycogen content: 7 days TI and t ACS stimulation(once a day,20 minutes each time)had no significant impact on muscle glycogen content(p>0.05),nor did they have a significant impact on muscle quality(p>0.05).Conclusions1.Stimulating the M1 region with TI and t ACS for 7 consecutive days(once a day,20 minutes each time)can effectively enhance motor skills in mice,and the effect of TI stimulation is better than that of t ACS.2.The mechanisms of stimulating M1 region with TI and t ACS to enhance motor skills in mice is related to its regulation of neurotransmitter metabolism,enhancement of neuronal excitability and excitatory transmission,promoted expression of synaptic plasticity related proteins,and enhancement of neuronal dendritic spine density and synaptic microstructure.The mechanisms by which TI acts better than t ACS is also related to its stronger regulatory effect on the above factors. |
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