阅读说明：本技术 可塑性诱导不同时间段低频磁刺激调控突触可塑性的方法 (Method for regulating and controlling synaptic plasticity by plastic induction and low-frequency magnetic stimulation in different time periods ) 是由 郑羽 马晓旭 马卫 东磊 张康辉 田春晓 于 2019-03-01 设计创作，主要内容包括：本发明公开了一种以突触可塑性诱导为参考点,不同时间段的ELF-EMFs刺激对大鼠离体海马脑片Schaffer-CA1通路突触可塑性调控的方法。通过采用了在可塑性诱导之前、同步和之后的三种不同参数条件下的ELF-EMFs刺激模式,揭示了三种ELF-EMFs刺激模式各自对海马脑片Schaffer-CA1突触可塑性LTP/LTD的调控规律,发现了中磁刺激模式的调控效果最为明显。本发明提出了可塑性诱导不同时间段低频磁刺激调控突触可塑性的方法,有益于更深层次的认识磁刺激对大鼠海马区的生物学效应乃至对学习与记忆整个过程的调控规律,并且对临床上rTMS治疗的磁刺激模式和参数选择等均有重要借鉴意义。(The invention discloses a method for regulating and controlling synaptic plasticity of a Schaffer-CA1 passage of an isolated hippocampal brain slice of a rat by using synaptic plasticity induction as a reference point and ELF-EMFs stimulation in different time periods. By adopting ELF-EMFs stimulation modes under three different parameter conditions of before, synchronization and after plasticity induction, the regulation rule of the three ELF-EMFs stimulation modes on synaptic plasticity LTP/LTD of hippocampal brain slice Schaffer-CA1 is disclosed, and the regulation effect of the medium magnetic stimulation mode is found to be most obvious. The invention provides a method for regulating and controlling synaptic plasticity by plasticity induction and low-frequency magnetic stimulation in different time periods, which is beneficial to deeper understanding of the biological effect of magnetic stimulation on a rat hippocampal region and even the regulation and control rule of the whole process of learning and memorizing, and has important reference significance for magnetic stimulation mode, parameter selection and the like of clinical rTMS treatment.)

1. The method for regulating and controlling the synaptic plasticity by the low-frequency magnetic stimulation during different periods of plasticity induction is characterized by comprising the following specific steps of:

step 1, on the magnetic field stimulation parameters, an online magnetic field stimulation device is fixed 15 mm under a hippocampus, and can generate continuous sinusoidal magnetic fields with different frequencies and intensities by adjusting the output parameters of a signal source, wherein the magnetic field intensity range of a hippocampal region of an in-vitro brain slice is 0.5-2mT, the frequency range is 15-100Hz, and the stimulation time can be flexibly adjusted according to requirements;

step 2, in the stimulation mode of the magnetic field, the plastic induction process in the synaptic plasticity experiment is divided into: magnetic field stimulation is referred to as premagnetic stimulation before synaptic plasticity induction; synchronized with the synaptic plasticity induction process, known as mesomagnetic stimulation; after synaptic plasticity induction, called post-magnetic stimulation;

and 3, on the magnetic field stimulation protocol, a front magnetic stimulation protocol: starting a magnetic stimulation device 40 minutes before the start of synaptic plasticity induction, and closing the magnetic stimulation device 20 minutes before the start of the synaptic plasticity induction, wherein the stimulation time is 20 minutes; medium magnetic stimulation protocol: synchronously starting the magnetic stimulation device when the plasticity induction is started, and synchronously closing the stimulation device when the plasticity induction is finished, wherein the magnetic stimulation time is the same as the induction time; post-magnetic stimulation protocol: starting the magnetic stimulation device 20 minutes after the plastic induction is finished, and stopping the magnetic stimulation device 40 minutes after the plastic induction is finished, wherein the stimulation time is 20 minutes;

step 4, applying a pre-magnetic stimulation protocol, using 9 magnetic stimulation parameters: the magnetic field frequency is 15Hz (the magnetic field intensity is 0.5, 1, 2mT), 50Hz (the magnetic field intensity is 0.5, 1, 2mT), 100Hz (the magnetic field intensity is 0.5, 1, 2mT), a field potential electrophysiological acquisition system is adopted to record a field potential signal of a Schafer-CA 1 channel of the isolated hippocampus of the SD rat, the influence of the pre-magnetic stimulation protocol on the synaptic plasticity LTP is analyzed through the amplitude of fEPSP, and the inhibition effect of the magnetic stimulation parameters of 15Hz/2mT on the synaptic plasticity LTP is confirmed to be most obvious;

step 5, applying three magnetic stimulation protocols of front magnetic, middle magnetic and rear magnetic, using 15Hz/2mT magnetic stimulation parameters, recording a field potential signal of a Schafer-CA 1 channel of an SD rat in vitro hippocampus brain slice by using a field potential electrophysiological acquisition system, analyzing the influence of the front magnetic stimulation protocol on synaptic plasticity LTP/LTD through the amplitude of fEPSP, and confirming that the three magnetic stimulation protocols can generate inhibition effect on the synaptic plasticity LTP/LTD, wherein the inhibition effect of the middle magnetic stimulation protocol is most obvious;

and 6, summarizing regulation rules of the three magnetic field stimulation protocols on synaptic plasticity through a frequency response function on the basis of the completion of the steps.

2. The method for modulating synaptic plasticity by low-frequency magnetic stimulation according to claim 1, wherein the synaptic plasticity induction frequency is 1Hz, 5Hz, 20Hz, and 100Hz, and the magnetic stimulation time and the plasticity induction time in the medium-frequency magnetic stimulation protocol are the same, and are 15, 3, 0.75, and 1.0667 minutes.

Technical Field

The invention discloses regulation rules of three continuous sinusoidal ELF-EMFs stimulation modes with different parameters on synaptic plasticity LTP/LTD by taking an acute separated SD rat hippocampal Schafer-CA 1 pathway as a research object, is beneficial to deeper understanding of the regulation rules of magnetic stimulation on synaptic plasticity, has important reference significance on magnetic stimulation modes, parameter selection and the like of clinical rTMS treatment, and belongs to the fields of electronic information technology, biomedical engineering and the like.

Background

In modern society, Extremely low frequency electromagnetic field (ELF-EMFs) devices are widely used, and long-term exposure can have potential risks for memory and cognitive functions of human beings, and ELF-EMFs stimulation is also an effective treatment means for neurodegenerative diseases such as alzheimer disease, but the action mechanism of the ELF-EMFs stimulation is not clear. Synaptic plasticity refers to changes in the morphology and function of synapses, manifested by increased or decreased synaptic transmission efficiency due to repeated activity of synapses, such as increased and inhibited long-term synapses, which are cellular mechanisms for learning and memory. The formation of synaptic plasticity has a very complex process, whose molecular mechanisms involve two glutamate receptors, NMDA and AMPA. When the Schaffer collateral branch of the presynaptic afferent fiber is stimulated at high or low frequencies, the neurotransmitter glutamate is released from the presynaptic membrane into the synaptic cleft, and Ca is prevented after a strong depolarization²⁺Mg in the incoming NMDA receptor channel²⁺Remove and allow a large amount of Ca²⁺Entry into the postsynaptic membrane resulting in postsynaptic Ca²⁺The increase in concentration and activation of protein kinases improves ionic conduction and thus enhances synapse-forming LTP. While only partial substitution of Mg on the postsynaptic membrane occurs when a weaker level of depolarization occurs²⁺Resulting in less Ca²⁺Lower intracellular Ca into postsynaptic neurons²⁺Concentration and protein kinase activation will inhibit synapse-forming LTD. Since synaptic plasticity plays an important role in many higher brain functions, such as learning and memory, synaptic plasticity has become the most advanced and productive research field in learning and memory in recent years.

The main means of magnetic stimulation to synapse plasticity study at present comprises long-term magnetic stimulation exposure during rat living period and magnetic stimulation in the isolated hippocampal brain slice incubation period, and then changes of magnetic stimulation to synapse plasticity activity are reflected by recording fEPSP. The influence of the cumulative effect of the magnetic stimulation on the synaptic plasticity can be reflected by adopting a long-term magnetic stimulation exposure method, but the method hinders the research on the influence of the magnetic stimulation on the learning and the memory, because the synaptic plasticity experiment process on the in vitro hippocampal brain slice is the learning and memory process, and the research purpose can be realized only by carrying out the magnetic stimulation on the in vitro hippocampal brain slice before the synaptic plasticity experiment is started under the prior art condition, so that the development of the research on the regulation and control process of the magnetic stimulation on the different stages of the synaptic plasticity of the hippocampal brain slice is restricted to a certain extent.

Disclosure of Invention

The invention aims to provide a method for regulating and controlling synapse plasticity of a Schaffer-CA1 passage of a rat in vitro hippocampal brain slice by low-frequency low-intensity continuous sinusoidal magnetic field stimulation, so as to overcome the defect that the development of experimental research on synapse plasticity by magnetic stimulation is restricted to a certain extent by a research method that magnetic stimulation can be only carried out before synapse plasticity under the prior art, thereby promoting the basic research on the influence of magnetic stimulation on learning and memory, further disclosing the action mechanism of the magnetic stimulation and providing necessary objective basis for the application of the magnetic stimulation in the subjects of pharmacology, neurophysiology, clinical medicine and the like.

The technical scheme of the invention is as follows:

the invention uses an ELF-EMFs generating device self-made by a laboratory, can generate sine uninterrupted ELF-EMFs, is applied to an experiment for recording the potential of the neuroelectrophysiological field, and determines that the device can generate the ELF-EMFs required by the experiment through COMSOL simulation and actual measurement of a Tesla meter. And then measuring fEPSP signals on an acute separated rat hippocampal brain slice Schafer-CA 1 pathway by applying an ELF-EMFs stimulation protocol, analyzing the influence on LTP, and finally determining an experimental parameter which has the largest influence on LTP in 9 ELF-EMFs parameters with different frequencies and different intensities as the basis of subsequent experiments. On the basis, the influence of the ELF-EMFs on synaptic plasticity before plasticity induction (pre-magnetic stimulation protocol), synchronous with plasticity induction (middle magnetic stimulation protocol) and after plasticity induction (post-magnetic stimulation protocol) is further elucidated, which is helpful for directly understanding the role of the ELF-EMFs in learning and memory-related processes.

The invention provides a method for regulating and controlling synapse plasticity by plasticity induction and low-frequency magnetic stimulation at different time periods, which comprises the following specific steps:

step 1, on the magnetic field stimulation parameters, an online magnetic field stimulation device is fixed 15 mm under a hippocampus, and can generate continuous sinusoidal magnetic fields with different frequencies and intensities by adjusting the output parameters of a signal source, wherein the magnetic field intensity range of a hippocampal region of an in-vitro brain slice is 0.5-2mT, the frequency range is 15-100Hz, and the stimulation time can be flexibly adjusted according to requirements;

step 2, in the stimulation mode of the magnetic field, the plastic induction process in the synaptic plasticity experiment is divided into: magnetic field stimulation is referred to as premagnetic stimulation before synaptic plasticity induction; synchronized with the synaptic plasticity induction process, known as mesomagnetic stimulation; after induction of synaptic plasticity, it is called post-magnetic stimulation.

And 3, on the magnetic field stimulation protocol, a front magnetic stimulation protocol: starting a magnetic stimulation device 40 minutes before the start of synaptic plasticity induction, and closing the magnetic stimulation device 20 minutes before the start of the synaptic plasticity induction, wherein the stimulation time is 20 minutes; medium magnetic stimulation protocol: synchronously starting the magnetic stimulation device when the plasticity induction is started, and synchronously closing the stimulation device when the plasticity induction is finished, wherein the magnetic stimulation time is the same as the induction time; post-magnetic stimulation protocol: and (3) starting the magnetic stimulation device 20 minutes after the plastic induction is finished, and closing the magnetic stimulation device 40 minutes after the plastic induction is finished, wherein the stimulation time is 20 minutes.

Step 4, applying a pre-magnetic stimulation protocol, using 9 magnetic stimulation parameters: magnetic field frequency 15Hz (magnetic field)

The strength is 0.5, 1, 2mT), 50Hz (the magnetic field strength is 0.5, 1, 2mT), 100Hz (the magnetic field strength is 0.5, 1, 2mT), a field potential electrophysiological acquisition system is adopted to record a field potential signal of a Schafer-CA 1 channel of the isolated hippocampal brain slice of the SD rat, the influence of the pre-magnetic stimulation protocol on the synaptic plasticity LTP is analyzed through the amplitude of fEPSP, and the inhibition effect of the magnetic stimulation parameter of 15Hz/2mT on the synaptic plasticity LTP is confirmed to be the most obvious.

And 5, applying three magnetic stimulation protocols of front magnetic, middle magnetic and rear magnetic, recording a field potential signal of a Schafer-CA 1 channel of an isolated hippocampal brain slice of an SD rat by using a 15Hz/2mT magnetic stimulation parameter and adopting a field potential electrophysiological acquisition system, analyzing the influence of the front magnetic stimulation protocol on synaptic plasticity LTP/LTD through the amplitude of fEPSP, and confirming that the three magnetic stimulation protocols can generate an inhibition effect on the synaptic plasticity LTP/LTD, wherein the inhibition effect of the middle magnetic stimulation protocol is most obvious.

And 6, summarizing regulation rules of the three magnetic field stimulation protocols on synaptic plasticity through a frequency response function on the basis of the completion of the steps.

The invention has the advantages and beneficial effects that:

the ELF-EMFs stimulation is an effective treatment means for neurodegeneration such as memory and cognition at present, but the action mechanism of the ELF-EMFs stimulation is not clear, the method for regulating and controlling synapse plasticity by plasticity induction and low-frequency magnetic stimulation at different time periods is provided, the result of the method is beneficial to deeper understanding of the biological effect of the magnetic stimulation on the rat hippocampus and the regulation and control rule of the whole learning and memory process, and the method has important reference significance for the magnetic stimulation mode, parameter selection and the like of clinical rTMS treatment.

Drawings

FIG. 1 is a graph of experimental results of pre-ELF-EMFs magnetic stimulation patterns on synaptic plasticity. (A) ELF-EMFs were stimulated for 20min before baseline recording, with a plasticity induction frequency of 1 Hz; (B) ELF-EMFs were stimulated for 20min before baseline recording, with a plasticity induction frequency of 5 Hz; (C) ELF-EMFs were stimulated for 20min before baseline recording, with a plasticity induction frequency of 20 Hz; (D) ELF-EMFs were stimulated for 20min before baseline recording with a plasticity induction frequency of 100 Hz.

FIG. 2 is a graph of experimental results of magnetic stimulation patterns on synaptic plasticity in ELF-EMFs. (A) ELF-EMFs and plasticity induction are synchronously stimulated for 15min, and the plasticity induction frequency is 1 Hz; (B) ELF-EMFs and plasticity induction are synchronously stimulated for 3min, and the plasticity induction frequency is 5 Hz; (C) ELF-EMFs and plasticity induction are synchronously stimulated for 0.75min, and the plasticity induction frequency is 20 Hz; (D) the ELF-EMFs and plasticity induction are synchronously stimulated for 1.0667min, and the plasticity induction frequency is 100 Hz.

FIG. 3 is a graph of experimental results of magnetic stimulation patterns after ELF-EMFs on synaptic plasticity. (A) Stimulating ELF-EMFs for 20min in the recording process after plasticity induction, wherein the plasticity induction frequency is 1 Hz; (B) stimulating ELF-EMFs for 20min in the recording process after plasticity induction, wherein the plasticity induction frequency is 5 Hz; (C) stimulating ELF-EMFs for 20min in the recording process after plasticity induction, wherein the plasticity induction frequency is 20 Hz; (D) ELF-EMFs was stimulated for 20min in the recording process after plasticity induction, plasticity

The induction frequency was 100 Hz.

FIG. 4 FRF functions of the ELF-EMFs in pre-, mid-and post-magnetic stimulation modes.

The following further describes embodiments of the present invention by way of example with reference to the accompanying drawings.

Detailed Description