Technology for targeted stimulation of specific brain region and application of technology in regulation and control of brain function

Abstract

The invention discloses a technology for targeted stimulation of a specific brain region and application of the technology in regulation and control of brain functions, and belongs to the field of animal cognitive behavior research. Techniques for targeted stimulation of specific brain regions include insulated stimulation electrodes that can generate AC voltage stimulation and a stimulation device for adjusting the AC voltage intensity and frequency. By applying the technology (ntTMS) for targeted stimulation of the specific brain region, an obvious magnetic stimulation effect can be achieved in the center of the brain region, cerebral neurons of a target region are activated, social and cognitive behaviors of animals are effectively regulated and controlled, and no obvious side effect is brought to experimental animals.

Description

Technical Field

[0001] The present invention relates to the field of research on the neural regulation of brain function, specifically the technology of regulating animal social behavior and cognitive function through targeted stimulation of the brain. Background Art

[0002] In the study of animal cognition and behavior, drug intervention or neuromodulation is often used to control animal behavior. Neuromodulation requires the use of external stimulation technologies, such as transcranial magnetic stimulation (TMS).

[0003] Transcranial magnetic stimulation (TMS), a non-invasive neuromodulation technology, uses electromagnetic fields to stimulate brain nerves. Through electromagnetic induction, tiny currents are generated to modulate the excitability of neurons in the cerebral cortex, thereby activating neurons and neural networks. Clinically, TMS for neuropsychiatric disorders has been approved by the FDA and is widely used in conditions such as depression, anxiety, and chronic pain to improve brain function and treat brain diseases.

[0004] However, existing TMS has the following problems in treating brain diseases and regulating cognitive and social behaviors:

[0005] (1) Low stimulation accuracy. The use of high magnetic field stimulation results in inaccurate stimulation position and has side effects on the experimental and treatment subjects.

[0006] (2) Low response sensitivity: Animal behavior signals are weak, and existing equipment is difficult to adjust parameters in real time.

[0007] (3) Existing TMS equipment is relatively large in size and has a large stimulation target area, making it unsuitable for scientific research on animals. Summary of the Invention

[0008] Technical problem to be solved by the invention

[0009] In view of the current lack of safe, accurate and effective technology for targeted stimulation of specific brain areas in the field of animal behavior research, this invention aims to explore the common characteristics of improving animal cognitive behavior and provide a technology and equipment for accurate and non-invasive targeted stimulation of specific brain areas.

[0010] Solutions for solving the above technical problems

[0011] The inventors found in the research that specific stimulation technology can specifically activate specific brain regions of mice without activating other brain regions. We named it non-invasive targeted TMS (ntTMS). Based on this discovery, the inventors further found that ntTMS stimulation technology can regulate the social and cognitive behavior of mice.

[0012] The present application includes the following.

[0013] 1. A technology for targeted stimulation of specific brain regions (ntTMS) comprising applying alternating voltage stimulation to the head of an animal to activate specific brain regions and neurons.

[0014] 2. The technology for targeted stimulation of specific brain regions as claimed in claim 1, comprising an insulated stimulation electrode in close contact with the scalp of the animal for generating alternating voltage. These technologies result in the insulated stimulation electrode being completely insulated from the scalp of the animal and causing no damage to the scalp of the animal; during the stimulation process, effectively avoiding the activation of brain regions and neurons near the stimulation point.

[0015] 3. The technology for targeted stimulation of specific brain regions as claimed in claims 1-2, which has been verified in model simulation and experimental processes to achieve significant magnetic stimulation effects in the center of the targeted brain region and activate brain neurons in the target region to regulate the social and cognitive behavior of animals.

[0016] 4. The technology principle as claimed in claims 1-3, a stimulation device for targeted stimulation of specific brain regions has been developed, which can achieve non-invasive targeted stimulation and the like by adjusting the stimulation intensity and frequency of alternating voltage stimulation.

[0017] The present application has the following beneficial effects:

[0018] By using the technology for targeted stimulation of specific brain regions of mice (ntTMS) combining insulated stimulation electrodes with alternating voltage stimulation, simulation and animal experiments have proved that the magnetic field strength formed by this technology is not greater than 0.2 μT, the stimulation accuracy can reach within 0.5 mm, and significant magnetic stimulation effects can be achieved in the center of the brain region and brain neurons in the target region can be activated to effectively regulate the social and cognitive behavior of mice without causing significant side effects to the experimental mice. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The experimental operation schematic diagram and the prepared instrument of the technology of the present application are shown. Among them, A is the output current of the ntTMS stimulation device to the insulated stimulation electrode, and then the mouse brain is stimulated; B is the small ntTMS stimulation instrument that has been completed by the present application; C is the insulated stimulation electrode used by the present application.

[0020] Figure 2The resistance of the insulated stimulating electrode used in the present application and the resistance of the pathway electrode during stimulation are infinite. The present application is significantly different from the principle of the existing transcranial direct current stimulation and transcranial alternating current stimulation. The mouse head is not effectively stimulated by current, and the damage to the head neurons is reduced. Wherein, A is the basic stimulating circuit used in the present application; B is the insulated stimulating electrode used in the present application; C is the resistance of the pathway electrode when the insulated stimulating electrode voltage stimulation technology used in the present application is used.

[0021] Figure 3 The magnetic effect based on the simulation of the ntTMS technology of the present application is shown. The results show that the magnetic stimulation effect generated by the present application is small in the stimulation peripheral area, and a significant magnetic effect is formed in the central area. Wherein, A is the electromagnetic field formed by the voltage stimulation; B shows that the central magnetic field forms a gradual change, and the maximum stimulation effect is obtained in the resonance phase; C shows that the central and peripheral magnetic fields are different in size; D shows that the targeted center can generate a magnetic field of 1.4nT.

[0022] Figure 4 The effect of the ntTMS of the present application on the specific activation of the mouse hippocampus brain area of the mouse is shown, that is, only the hippocampal neurons of the mouse are activated, and the activated brain area is labeled by cFos antibody. In other brain areas, including the scalp and skull, no labeled neurons are seen, indicating that other brain areas are not affected by specific stimulation.

[0023] Figure 5 shows that the ntTMS of the present application can specifically activate multiple brain areas of the mouse, and by combining key stimulation parameters, the effect of precise stimulation can be achieved, including deep brain areas such as the hippocampus and the central canal of the midbrain, and superficial brain areas such as the prefrontal cortex, while not affecting other brain areas. These results lay the foundation for precise regulation of neuronal activity and animal behavior. Wherein, A shows that the present application can precisely stimulate relatively shallow brain areas such as the prefrontal cortex, while not activating the neurons of other brain areas; B shows that the present application can precisely stimulate deep brain areas such as the periaqueductal gray matter of the midbrain, while not activating other brain areas.

[0024] Figure 6 The ntTMS technology of the present application tests the relationship between different stimulation frequencies and the number of activated neurons. The research results show that the number of activated neurons increases with the increase of the stimulation frequency, which lays a theoretical foundation for clinical treatment.

[0025] Figure 7Figure 1 shows that ntTMS can stimulate neurons in the hippocampus. A is a schematic diagram of the experimental technique, which shows that ntTMS can stimulate neurons in the hippocampus. B shows the actual trajectory of the multi-channel electrode inserted into the hippocampus. The right-hand image shows the location of the principal neurons (PN) and interneurons (IN) in the hippocampus. C shows the firing patterns of the PN and IN neurons before and after ntTMS stimulation. The black line represents the average value, and the green and red lines represent the standard error range of all recorded neuron firing. D shows the firing frequency spectrum of the PN neurons before and after ntTMS stimulation, which shows that the firing frequency of the PN neurons increases in the high-frequency range. E shows the real-time changes in the firing frequency of all (black), PN (red), and IN (green) neurons before and after stimulation.

[0026] Figure 8 Figure 2 shows that ntTMS can activate neurons in the brain, promote synapse formation, increase synaptic current, and not cause neuron death. A is a schematic diagram of the experimental technique, which shows that ntTMS specifically stimulates the cerebellum. B shows the neural network within the cerebellum, including the climbing fibers emanating from the inferior olive nucleus. C shows that the cerebellar cortex receives ntTMS stimulation, and the right-hand image shows the synapses (red) formed on Purkinje cells with climbing fibers. D is a statistical result showing that the number of activated Purkinje cells (PC) and interneurons (IN) significantly increases after ntTMS stimulation. E shows that the synaptic current on Purkinje cells increases after ntTMS stimulation. F shows that the expression of cleaved caspase3 in the cerebellum does not change after ntTMS stimulation and control, as detected by western blotting. Since caspase represents cell death, this result shows that ntTMS stimulation does not cause cell death.

[0027] Figure 9 Figure 3 shows that the technology can improve the cognitive behavior of mice. It was found that under specific conditions of stimulation, the new object recognition ability of mice can be enhanced. A shows the experimental scheme of giving ntTMS to mice, i.e., continuous stimulation for 5 days, and then testing spatial memory on the 6th day. B shows the location of the mice stimulated by ntTMS and the spatial memory test, which includes placing different objects in a space and then observing the memory ability of the mice for the objects. CntTMS can significantly improve the recognition ability of mice for previously remembered objects. D shows that ntTMS improves the preference of mice for previously remembered objects.

[0028] Figure 10This demonstrates that stimulating the mouse cerebellum using the technology of the present invention can improve the mice's social abilities. A represents the first phase of the three-compartment test, showing the mice's choice between an empty box and a single mouse (S1); B represents the second phase of the three-compartment test, showing the mice's choice between a single mouse (S1) and a novel mouse (S2); C shows the unstimulated mice's choice between the empty box and S1, and between S1 and S2 mice; D shows the ntTMS-stimulated mice's choice between the empty box and S1, and between S1 and S2 mice; E shows the difference between unstimulated and ntTMS-stimulated mice in their choice of the empty box and S1 mice, as well as the difference in their sniffing of the S1 mouse; F shows the difference between unstimulated and ntTMS-stimulated mice in their choice of the S1 mouse and S2 mice, as well as the difference in their sniffing of the S2 mouse. DETAILED DESCRIPTION

[0029] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are not limited to the following embodiments, but also include various alternatives and modifications within the technical scope of the present invention.

[0030] The present invention is based on the principles of wave interference and low-pass filtering of the neural membrane to achieve precise stimulation of specific brain areas. Specifically, an insulated stimulation electrode is used to generate an alternating voltage. Its design, which is completely insulated from the animal's scalp, can effectively avoid the activation of neurons in the stimulation path, thereby achieving the goal of targeted stimulation. At the same time, the alternating voltage stimulation formed on both sides of the insulated stimulation electrode produces an electrical effect through the superimposed magnetic field, selectively activating neurons in the target area. This technology can avoid the generation of current stimulation in the passing pathway, so the neurons in the pathway will not be activated. Based on this targeted stimulation technology, the present invention designs a special device that can adjust the intensity and frequency of alternating voltage stimulation. This device is used to adapt to different experimental needs through parameter optimization, and is applied to the study of animal cognition and social behavior. After verification by model simulation and animal experiments, this technology has shown significant effects in regulating animal social behavior and cognitive function, providing an effective tool for exploring the mechanism of improving cognitive behavior.

[0031] Source of experimental materials:

[0032] Experimental animals: All experimental mice were obtained from the Experimental Animal Center of Zhejiang University.

[0033] ntTMS instrument: derived from the technology of the present invention.

[0034] In vivo electrophysiological recording instrument: Apollo amplifier from Yige Biotechnology (Nanjing) Co., Ltd.

[0035] Multi-channel electrode: from Kedou Biotechnology (Suzhou) Co., Ltd.

[0036] Stereotaxic instrument: from Shenzhen Ruiwode Company.

[0037] Patch-clamp electrophysiological recordings: The patch-clamp amplifier and AD / DA controller were from Axon Corporation (USA).

[0038] Anymaze software: originated from the American company Stoeling.

[0039] SDS-PAGE electrophoresis apparatus: from Bio-Rad.

[0040] Main reagents:

[0041] cFos, DAPI, caspase3, calbindin and vGluT2 antibodies were purchased from Merck Chemical Company, USA.

[0042] like Figure 1 As shown, the experimental setup based on the present invention includes an ntTMS instrument, insulated stimulation electrodes, and connecting wires. During the experiment, researchers can adjust the parameters of the AC voltage and obtain instantaneous output signals by connecting current for real-time feedback. The insulated stimulation electrodes are adhered to the animal's scalp with non-conductive glue. The hair on the animal's scalp is shaved without damaging the scalp.

[0043] like Figure 2 As shown, the stimulation of the ntTMS instrument adopts a bidirectional voltage circuit output. The insulated stimulation electrodes adopt a double-sided non-conductive ring design, which is close to the two sides of the mouse scalp and completely insulated from the mouse scalp. According to the atlas of mouse brain structure (AllenBrain Map), the spatial position of the target brain area is calculated, and the position of the insulated stimulation electrode is obtained. The electrode is then adhered to the corresponding scalp position. Depending on the target brain area to be stimulated, the intensity and frequency of the two AC voltages are adjusted to obtain an interfering low-frequency signal to achieve precise stimulation of the target brain area. The output signal of the ntTMS was tested with a multimeter, and it was found that the output resistance of the ntTMS instrument was infinite, and its output current was zero.

[0044] like Figure 3 As shown, the simulation software Ansys Maxwell was used for geometric modeling. A voltage electric field source was selected to generate electromagnetic waves. The finite element method, suitable for accurately modeling complex structures, was used to calculate the superposition effect in the spatial domain. To improve accuracy, the magnetic field grid analysis accuracy was set to 1μm, and the boundary conditions were absorbing. During the simulation, the field effect was optimized by adjusting the size of the stimulus source and the antenna structure. Furthermore, the accuracy of the simulation results was verified by comparing them with experimental data or theoretical solutions.

[0045] Simulation results: The diameter of the insulating stimulating electrode designed in the application is not greater than 0.3 mm, and the minimum magnetic effect generated in the target area is not greater than 0.2 μT.

[0046] Example 1: Precise stimulation in the brain of a mouse is realized by using an ntTMS prototype

[0047] The experimental results show that, in combination with key parameters, the target brain area can be precisely stimulated without affecting other brain areas, the scalp and the skull, and the stimulation precision can reach 500 μm.

[0048] As shown in Figure 4 , the insulating stimulating electrode is pasted on the scalp of a 2-month-old mouse, aiming to stimulate the hippocampus. The stimulation frequency is 100 Hz, and the stimulation time is 1 minute. Then, the mouse brain is immediately removed, brain slices are made at a distance of 20 μm, and brain staining is performed by using cFos and DAPI antibodies. cFos can mark activated neurons, and DAPI can mark all nerve cells by marking cell nuclei.

[0049] By observing the whole brain slices, it is found that activated neurons can be clearly found in the hippocampus on one side, but no activated neurons are found in the hippocampus on the opposite side, other brain areas and the scalp area where the electrode is pasted, and the mouse scalp surface is very complete. The above results show that the ntTMS can precisely activate hippocampal neurons (indicated by arrows in the dashed box), but does not affect other brain areas. At the same time, by counting multiple brain slices, it is found that from the nose side to the tail side of the mouse, the diameter of the brain area where activated neurons exist is 500 μm, indicating that the stimulation precision of the ntTMS can reach 500 μm.

[0050] As shown in Figure 5A and 5B , two target stimulation experiments for different target brain areas are performed. The insulating stimulating electrode is pasted on the scalp of a 2-month-old mouse, aiming to stimulate the prefrontal cortex (PFC) Figure 5A ) and the periaqueductal gray (PAG) Figure 5B ) of the brain, respectively. The stimulation frequency is set to 100 Hz, and the stimulation time is 1 minute. After the target stimulation is completed, the mouse brain is immediately removed, brain slices are made at a distance of 20 μm, and brain staining is performed by using cFos and DAPI antibodies.

[0051] Experimental results: By observing the whole brain slices, it is found that whether the prefrontal cortex (shallow layer of the brain) is stimulated or the periaqueductal gray area (deep layer of the brain) is stimulated, activated neurons can be found, but no activated neurons are found in brain areas other than the target brain area and the scalp area where the electrode is pasted, and the mouse brain is very complete.

[0052] The above experimental results show that ntTMS can accurately activate neurons in the superficial or deep brain. Figure 5A or Figure 5B The activated neurons (indicated by red color) can be clearly seen in the magnified box. In both experiments, it was found by statistically analyzing multiple brain sections that the diameter of the brain region with activated neurons was about 500 μm from the nose side to the tail side of the mouse.

[0053] Example 2: Exploring the effect of ntTMS stimulation parameters on the number of activated neurons

[0054] The uneven distribution of neurons and nuclear clusters in the brain poses high requirements for the stimulation parameters of electromagnetic field stimulation. In this embodiment, the ntTMS instrument is used to test the stimulation parameters for multiple brain regions.

[0055] As shown in Figure 6 , the results show that the number of activated neurons is significantly correlated with the stimulation frequency. At the same time, the stimulation frequency and stimulation time for effectively activating different brain regions are also different. These results lay a theoretical foundation for future clinical treatment.

[0056] Example 3: Exploring the mechanism of ntTMS regulating brain function

[0057] Using a stereotaxic instrument, multi-channel electrodes were implanted in 6 pairs (2-month-old) mice, with the electrode tip in the DG region of the hippocampus. Then the mice were returned to the cage for at least 3 weeks of recovery. The hair on the scalp was removed, and the insulated stimulating electrodes were pasted on the corresponding scalp position. A 1-minute high-frequency ntTMS stimulation was given, and the discharge frequency of the targeted neurons was detected before and after the stimulation to determine whether ntTMS changes the discharge ability of neurons. According to the different waveforms of neuron discharge, neurons were divided into two categories: principal neurons (PN) and interneurons (IN). The full power spectrum of hippocampal neuron discharge frequency was recorded for a period of time, and the power spectrum changes before and after stimulation were compared. In this embodiment, in vivo electrophysiological recording is used to compare the effect of ntTMS stimulation on the activity of neurons in the target region in real time.

[0058] Experimental results: as shown in Figure 7 , it can be found that ntTMS does not change the discharge waveform of PN and IN, but it can change the discharge frequency of PN and IN, and this change returns to the original initial level after about 10 minutes. At the same time, ntTMS stimulation can significantly affect the power spectrum of hippocampal neuron discharge, mainly in the high-power gamma band.

[0059] Example 4: Exploring the basic mechanism of ntTMS regulating neuron activity

[0060] The scalp hair of six pairs of mice (2 months old) was shaved, and insulated stimulating electrodes were attached to the scalp location targeting the cerebellar cortex. High-frequency ntTMS stimulation was applied for 1 minute. Then, a variety of experiments were used to detect the stimulatory effect of ntTMS on the cerebellum, including staining synapses in cerebellar slices, patch clamp electrophysiological recording of synaptic currents in cerebellar slices, and western blot detection of caspase3 activity expression, to test whether ntTMS affects the number of neuronal synapses, whether it affects the synaptic currents of Purkinje cells, and whether it causes neuronal death.

[0061] The results are as follows Figure 8 As shown in Figure 2, when ntTMS is targeted to the cerebellum, the stimulation can activate Purkinje cells, which is manifested by an increase in the number of Purkinje cell synapses ( Figure 8 C and D in the figure). Whole-cell recordings were made on Purkinje cells, and then electrical stimulation was given to the molecular layer of the cerebellum to record the synaptic currents of Purkinje cells. The results showed that when the same intensity of stimulation was given, the synaptic currents of Purkinje cells increased significantly after ntTMS stimulation ( Figure 8 E), confirming the aforementioned increase in synaptic number. These results suggest that ntTMS activates cerebellar Purkinje cells and causes structural changes. Western blot results showed that the expression of caspase-3, a marker of cell death, remained unchanged ( Figure 8 F), indicating that ntTMS did not cause cell death and ntTMS stimulation was safe.

[0062] Example 5: Exploring the regulatory effects of ntTMS on the emotions and cognitive behaviors of mice

[0063] This example uses targeted stimulation of the hippocampus as an example, administering 100 Hz high-frequency stimulation once a day for 5 consecutive days for 10 minutes each time, to compare the spatial object recognition memory of three pairs of mice.

[0064] After five days of continuous stimulation, spatial memory was tested on the sixth day. In the novel object recognition test, mice were introduced to a room for 10 minutes of free exploration. After 30 minutes, two cylindrical blocks were placed diagonally in the room, and the mice were then allowed to explore for another 10 minutes. Finally, one of the cylinders was replaced with a square block, and the mice were allowed to explore the room again for another 10 minutes.

[0065] During the experiment, Anymaze software was used to record the time the mice interacted with the cylinder and the square.

[0066] The experimental results are as follows Figure 9 As shown in the results, ntTMS stimulation of the hippocampus can effectively restore the spatial object recognition ability of AD mice. Compared with mice that did not receive ntTMS stimulation, the ability of mice that received ntTMS stimulation to recognize existing memory objects was significantly improved ( Figure 9 C and D in the figure); the mice that received ntTMS stimulation also had a significantly improved ability to recognize new memory objects ( Figure 9 These results indicate that ntTMS can modulate the spatial position cognition ability of mice.

[0067] Example 6: Exploring the regulatory effects of ntTMS on social behavior in mice

[0068] The stimulation target area in this embodiment is the cerebellum.

[0069] ntTMS targeted the Crus1 region of the cerebellum, delivering 100 Hz high-frequency stimulation once daily for 5 consecutive days. After 5 consecutive days of stimulation, three pairs of mice were compared in a three-compartment social behavior test between unstimulated and ntTMS-treated mice.

[0070] In the three-chamber social interaction test, the experimental mouse was placed in the central chamber, and then the S1 mouse was placed in the central chamber, where the experimental mouse freely explored all three chambers. Then, the S2 mouse was placed in the central chamber, where the experimental mouse again explored all three chambers.

[0071] During the experiment, the time the experimental mice stayed in each chamber was recorded, and the ratio of (S1-E) to (S1+E) was used as the preference index, and the ratio of (S2-S1) to (S2+S1) was used as the preference index (S2-S1).

[0072] The experimental results are as follows Figure 10 As shown, targeted stimulation of Crus1 in the cerebellum can significantly improve the social ability of mice, mainly reflected in the improvement of their ability to interact with S2 mice. These results show that ntTMS targeted modulation of the cerebellum can regulate its neuronal activity, thereby improving the social ability of mice.

[0073] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited by these specific embodiments. It is apparent to those skilled in the art that various modifications and variations can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the technical concepts and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A technology for targeted stimulation of specific brain areas (ntTMS), characterized by: It involves applying AC voltage stimulation to the animal's head to form a superimposed magnetic field to produce an interference effect, thereby activating specific brain areas and neurons of the animal.

2. The technology for targeted stimulation of specific brain regions according to claim 1, characterized in that: It also includes an insulating stimulation electrode that is in close contact with the animal's scalp and is used to generate an AC voltage. At the same time, the insulating stimulation electrode is completely insulated from the animal's scalp, effectively avoiding activation of brain areas and neurons near the stimulation point during the AC voltage stimulation process.

3. The technology for targeted stimulation of specific brain regions according to claims 1-2, characterized in that: It also includes a stimulation device that targets deep brain stimulation and is used to regulate the stimulation intensity, frequency, etc. of AC voltage stimulation.

4. An application of the technology of targeted stimulation of specific brain areas as claimed in any one of claims 1 to 3 in regulating animal social behavior and cognitive function.

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