Brain stimulation, as an important research tool for neural regulation, is of significant importance in understanding, improving, protecting, and simulating the brain. It has important potential and application prospects in the treatment of brain diseases and enhancing brain function.

Methods for improving and enhancing human motor abilities through neural electrical stimulation have received widespread attention in the field of sports science. Traditional transcranial electrical stimulation can alter the excitability of brain neurons and enhance the body’s motor abilities. However, traditional transcranial electrical stimulation is a general stimulation and cannot precisely regulate specific brain regions responsible for motor control and regulation, as well as deep brain nuclei.

In 2017, a research team led by Boyden from the Massachusetts Institute of Technology proposed temporal interference (TI) brain stimulation technology as a new non-invasive deep brain stimulation technology in the journal Cell. TI technology involves the interaction of two sets of high-frequency and slightly different sinusoidal alternating current fields. Due to the frequency difference between the two sinusoidal alternating current fields, a superimposed electric field is generated, forming a low-frequency envelope wave. The low-frequency envelope wave is sufficient to drive neural activity, and neurons can be activated at the selected focal point without affecting neighboring or overlapping areas, thus enabling specific stimulation of certain locations without affecting other irrelevant brain areas, providing the possibility for precise control of the brain. The primary motor cortex (M1) is a key brain region involved in motor planning, execution, control, and consolidation of human and animal motor skills. Traditional transcranial alternating current stimulation (tACS) can enhance motor skills, but it is unclear whether TI stimulation has an impact on mouse motor skills, whether it has advantages over tACS, and the mechanism by which TI stimulation enhances mouse motor skills.

Recently, a team led by Liu Yu and Wang Xiaohui from Shanghai University of Sport published an article in the journal Brain Stimulation titled Temporally interfering electric fields brain stimulation in primary motor cortex of mice promotes motor skill through enhancing neuroplasticity. The study confirmed that TI can achieve non-invasive stimulation of the M1 area in mice, and that TI stimulation at an envelope wave frequency of 20 Hz once a day for 20 minutes, continuously for 7 days, significantly improves the motor skills of mice. Compared with traditional tACS, TI stimulation can better enhance the motor skills of mice. Further research found that its mechanism of action is related to regulating neurotransmitter metabolism, increasing synaptic-related protein expression, promoting neurotransmitter release, increasing dendritic spine density, enhancing synaptic vesicle number, and synaptic post-dense material thickness, ultimately enhancing the excitability and plasticity of neurons. This is the first report on the promotion of mouse motor skills and its mechanism by TI stimulation.

To evaluate the motor skills of mice, the authors used classic single-pellet grasping training and testing. The success rate was used to assess the completion of the single-pellet grasping task: the ratio of successful grasps (grabbing food and delivering it to the mouth) to the total number of attempts, expressed as a percentage. When the success rate of mice in grasping food reached 40%, it was considered that the mice had mastered the task. The results showed that giving mice left M1 area TI stimulation for 20 minutes each day for 7 consecutive days significantly improved the success rate, speed, and preferred forelimb percentage of mice in completing the single-pellet grasping task. As a positive control, tACS given to the M1 area of mice for 20 minutes also significantly increased the success rate of mice in completing the single-pellet grasping task, but to a lesser extent than TI; in addition, tACS had no significant effect on other indicators of the single-pellet grasping task, suggesting that TI is more effective in enhancing the motor skills of mice compared to the positive control tACS.

To investigate the relationship between stimulation regulation and neurotransmitter metabolism, the authors used metabolomics techniques to detect changes in neurotransmitter content in the M1 area of mice, and explored the effects of TI and tACS stimulation on neurotransmitter metabolism through various statistical analyses and visualization methods (Figure 1, original Table 1). The results showed that compared to the TI-Sham group, the DA, Glu, and other 11 neurotransmitter contents were significantly higher in the TI-20 min group, while compared to the tACS-Sham group, the DA, Glu, and other 8 neurotransmitter contents were higher in the tACS-20 min group; and the increase in TI was greater than tACS, suggesting that both TI and tACS stimulation can improve mouse motor skills, which is related to the significant increase in Glu, DA, and other neurotransmitter contents in the M1 area.

Exploring the Effects of TI and tACS Stimulation on Different Metabolic Pathways of Metabolites through KEGG Metabolic Pathway Maps, it was found that TI and tACS Stimulation can increase the content of DA neurotransmitters, regulate the PKA signaling pathway, promote the expression of CREB and BDNF proteins. TI and tACS stimulation can increase the content of Glu and other neurotransmitters, regulate NMDAR and AMPAR sites, and promote the expression of synaptic plasticity-related proteins such as CREB and BDNF (Figure 2). In addition, TI and tACS stimulation can increase the content of neurotransmitters such as 5-HT, its precursors and metabolites, as well as increase levels of Spd, Spm, His, Hist, and other neurotransmitters, promote calcium release, and thereby increase neuronal excitability.

Combining metabolomics test results selecting Glu and DA as important excitatory neurotransmitter probes , using an in vivo multichannel fiber photometry system (using fiber photometry system produced by RWD company ) detected the release of calcium ions, glutamate, and dopamine neurotransmitters in the M1 area, confirming the effects of TI and tACS stimulation on neuronal excitability (calcium ion release) and the release of excitatory neurotransmitters DA and Glu, with the magnitude of change in TI stimulation being higher than tACS stimulation (Figure 3), indicating that TI stimulation in the M1 area enhances mouse motor skills by increasing the release of excitatory neurotransmitters and enhancing neuronal excitability.

In order to study the effects of TI and tACS stimulation on synaptic plasticity, the authors used Western blot method to detect the expression levels of several synaptic plasticity-related proteins such as PSD-95, SYN, NMDAR, AMPAR, and BDNF. It was found that both TI stimulation and tACS stimulation could increase the levels of PSD-95, BDNF, and CREB proteins in the M1 area of mice, with TI stimulation showing a greater increase in magnitude (Figure 4). In addition, laser confocal imaging results showed that continuous 7 days of TI and tACS stimulation significantly increased the number of synaptic vesicles and PSD thickness in the M1 area (Figure 5). These results suggest that the effects of TI and tACS stimulation on enhancing mouse motor skills in the M1 area may be achieved through increasing the levels of synaptic plasticity-related proteins and enhancing synaptic plasticity.

In conclusion, TI stimulation can improve mouse motor skills, and the effect of TI stimulation on enhancing motor skills is superior to the positive control tACS stimulation. This effect of TI stimulation is related to its enhancement of neuronal excitability and synaptic plasticity, as well as improvement in synaptic microstructure.

Research Method Highlights

This work first confirmed that continuous 7-day TI stimulation (once a day, 10 minutes each time, ∆f=20Hz) in the M1 area can effectively enhance the mouse’s strength, balance, endurance, coordination, and other motor abilities. The mechanism of action is related to its enhancement of neuronal excitability and synaptic plasticity, as well as improvement in synaptic microstructure. Experimental techniques used in the research include temporal interference stimulation, transcranial alternating current stimulation, multichannel fiber photometry system, metabolomics, Western blot, laser confocal, Liquid Chromatography-Mass Spectrometry, and animal behavioral evaluation. Among them, the multichannel fiber photometry system was purchased from RWD Life Science Co., Ltd. RWD has been deeply involved in the field of life science research for 22 years, dedicated to providing reliable solutions and services to customers. In this study, researchers used the fiber photometry system produced by RWD, which provided support for the smooth progress of the experiment. In addition, RWD can also provide a complete solution for the neural circuit research involved in this study. As of now, RWD’s products and services have covered more than 100 countries and regions worldwide, serving over 700 hospitals, 1000 research institutions, and 6000 higher education institutions, helping global researchers publish over 14500 SCI articles and gaining wide industry recognition.

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