Method for collecting discharge of different types of neuron clusters in real time

Abstract

The invention relates to the technical field of biology, and discloses a method for collecting discharge of different types of neuron clusters in real time, which comprises the following steps: S1, selecting a specific promoter: for excitatory neurons, selecting a tool virus taking CaMKIIa as the specific promoter; for the inhibitory neurons, a tool virus taking VGAT1 as a specific promoter is selected; s2, positioning and injection: positioning through a mouse brain map coordinate, drilling a hole right above the coordinate of the dentate gyrus region through a skull, and injecting a tool virus to the dentate gyrus region; s3, implanting the optical fiber: implanting the optical fiber connected with the ceramic ferrule into the virus injection hole of the tool, wherein the implantation depth is 1.95-1.98 mm; s4, fixing the optical fiber: fixing the ceramic ferrule on the target brain by using dental cement; s5, virus expression: waiting for expression of the tool virus injected in S2; and S6, performing transcranial magnetic stimulation and optical fiber recording. According to the method, real-time and long-term acquisition and monitoring of discharge activities of various types of neuron clusters can be realized under transcranial magnetic stimulation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for real-time acquisition of discharges of different types of neuron clusters. Background Art

[0002] Transcranial magnetic stimulation is a non-invasive neuromodulation technology that can generate an alternating pulsed magnetic field that penetrates the skull without attenuation, generating induced currents in the cerebral cortex, thereby affecting and changing neuronal discharge activity. In order to evaluate changes in neural excitability after transcranial magnetic stimulation, it is necessary to record the discharge activity of single neurons or neuronal clusters. Traditional electrophysiological methods include patch clamp technology, micromotor array recording technology, etc., which can all record in vitro or in vivo neural discharge activity, but these technologies are very sensitive to external signal interference during signal acquisition, especially electromagnetic signals. Therefore, traditional electrophysiological recording technology cannot eliminate the impact of transcranial magnetic stimulation magnetic field interference, and it is difficult to achieve real-time recording of neural activity under real-time stimulation of transcranial magnetic stimulation. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for real-time acquisition of discharges of different types of neuron clusters, which solves the problems in the prior art of signal interference in recording the electrical signals of neurons, difficulty in achieving selective regulation of specific types of neurons, and lack of real-time performance.

[0004] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is: a method for real-time acquisition of discharges of different types of neuron clusters, comprising the following steps:

[0005] S1, select specific promoter: for excitatory neurons, select tool virus with CaMKⅡa as specific promoter; for inhibitory neurons, select tool virus with VGAT1 as specific promoter;

[0006] S2, positioning and injection: at the coordinates of the dentate gyrus region of the target brain, the tool virus is injected into the dentate gyrus region through a burr hole in the skull;

[0007] S3, implanting optical fiber: implant the optical fiber into the hole of the skull with an implantation depth of 1.95-1.98 mm, connect the optical fiber with a ceramic ferrule and an optical fiber patch cord for connecting the equipment;

[0008] S4, fix the optical fiber: fix the ceramic ferrule on the target brain using a dental level;

[0009] S5, virus expression: wait for the tool virus injected in S2 to be expressed in the labeled neurons. The tool virus expression time is at least two weeks.

[0010] S6, Transcranial Magnetic Stimulation and Fiber Optic Recording: Using a TMS stimulation coil, magnetic stimulation is applied to the target brain, while the discharge signals of different types of neuronal populations are recorded through optical fibers.

[0011] Furthermore, the above-mentioned method for real-time acquisition of discharges of different types of neuronal clusters, S6 specifically comprises: tilting the TMS stimulation coil above the junction of the parietal bone and the optical fiber of the target brain, aligning the center point of the TMS stimulation coil with the optical fiber implantation position on the target brain, connecting the optical fiber jumper to the ceramic ferrule, connecting the end of the optical fiber jumper away from the ceramic ferrule to the optical fiber recording system, and connecting the end of the optical fiber recording system away from the optical fiber jumper to the display.

[0012] Furthermore, in the above method for real-time acquisition of discharges of different types of neuronal clusters, the coordinates of the dentate gyrus region of the target brain in S2 are: 1.7 mm posterior to the anterior bregma, 1.2 mm to the left and right sides of the raphe, and 2 mm in depth.

[0013] Furthermore, in the above method for real-time acquisition of discharges of different types of neuron clusters, the size of the optical fiber in S3 is: 200 μm in diameter, 3 mm in length; and the diameter of the ceramic ferrule is 1.25 mm.

[0014] Furthermore, in the above method for real-time acquisition of discharges of different types of neuronal clusters, the stimulation coil for small animals is a circular coil with an outer diameter of 56 mm and an inner diameter of 14 mm; the maximum magnetic field output intensity of the coil is 3.6T.

[0015] The beneficial effects of the present invention are as follows: the dentate gyrus of the hippocampus plays an irreplaceable role in cognitive function. TMS regulates changes in the excitability of neurons related to the dentate gyrus, which is an important target and research direction for TMS to improve cognitive dysfunction. The neural discharge activities of different types of neurons such as excitatory and inhibitory neurons in the dentate gyrus jointly affect the changes in neural excitability. Therefore, under the real-time stimulation of TMS, it is particularly important to achieve real-time recording and collection of neural discharge activities of excitatory neurons and inhibitory neurons in the dentate gyrus. The present invention comprehensively utilizes neuron in situ labeling technology, neuron calcium ion imaging-optical fiber recording technology, and TMS neural regulation technology, based on the idea of ​​photoelectric signal conversion (using the optical signal of neuron discharge activity to characterize the electrical signal of neuron discharge activity) to avoid TMS magnetic field interference, and establishes a detection method for the real-time response of in vivo excitatory and inhibitory neuron clusters in the bilateral dentate gyrus brain area under the action of TMS. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of real-time stimulation and fiber optic recording;

[0017] Figure 2 Schematic diagram of specific labeling for different types of neurons;

[0018] Figure 3 Recording flow charts for optical fibers;

[0019] Among them: 1. Target brain; 2. Optical fiber; 3. Ceramic ferrule; 4. Optical fiber jumper; 5. Excitatory neuron marker; 6. TMS stimulation coil; 7. Optical fiber recording system; 8. Display; 9. Inhibitory neuron marker. DETAILED DESCRIPTION

[0020] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0021] In the resting state, the intracellular calcium concentration of most neurons is maintained at 50-100nM. When neurons are active, the intracellular calcium concentration can rise 10-100 times. The increased calcium ions are essential for the exocytosis and release of synaptic vesicles containing neurotransmitters. Therefore, the activity of neurons is closely related to the calcium concentration inside them. When neurons discharge, a short peak of calcium concentration is generated. There is a strict correspondence between calcium concentration and neural activity.

[0022] The principle of neuronal calcium imaging is based on the strict correspondence between calcium ion concentration and neural activity. The calcium ion concentration in neurons is expressed as fluorescence intensity through calcium ion indicators.

[0023] Calcium-sensitive fluorescent protein (GCaMP) is composed of enhanced green fluorescent protein (EGFP), calmodulin (CaM) and calmodulin-binding peptide M13. After calmodulin (CaM) binds to calcium ions, it interacts with M13 to cause changes in the spatial structure of GFP and emit green fluorescence. GCaMP can observe a certain type of neurons that are specifically marked among thousands of cells, as well as their discharge patterns and regularities, so as to further explore various intrinsic neural mechanisms. Calcium-sensitive fluorescent protein neural specific markers such as Figure 3 In order to express the target gene in neurons, an adeno-associated virus vector (AAV) is used to package the target gene, and the AAV tool virus is delivered to a specific brain region by intracranial injection to achieve specific labeling of a specific type of neurons.

[0024] GCaMP can well characterize neuronal activity through changes in fluorescence signal intensity, among which GCaMP6 has high time sensitivity and fluorescence signal noise ratio. The method of expressing GCaMP into neurons, stimulating the fluorescence of GCaMP through optical fiber 2 and monitoring and recording the fluorescence signal intensity in real time is called optical fiber 2 recording.

[0025] like Figure 1 As shown, this embodiment provides a method for real-time acquisition of discharges of different types of neuronal clusters. This embodiment uses a mouse brain as the target brain 1 for surgery.

[0026] Neuron-specific labeling and Fiber Optic 2 implantation:

[0027] For excitatory neurons, the tool virus rAAV-CaMKⅡa-GCaMP6m with CaMKⅡa as the specific promoter was selected. For inhibitory neurons, the tool virus rAAV-VGAT1-GCaMP6m with VGAT1 as the specific promoter was selected. The coordinates of the dentate gyrus brain area were selected as follows: 1.7mm posterior to the anterior fontanelle, 1.2mm to the left and right sides of the raphe, and 2mm in depth. The size of the optical fiber 2 is: 200μm in diameter, 3mm in length, and the diameter of the ceramic ferrule 3 is 1.25mm. The surgical procedure is divided into the following 6 stages:

[0028] Pre-anesthesia: Place the mouse in the pre-induction box of the anesthesia machine and introduce isoflurane with a concentration of 3% and a flow rate of 0.5 L / min to anesthetize the mouse.

[0029] Head fixation and window opening: The anesthetized mouse was transferred to a brain stereotaxic instrument and fixed with ear bars. Isoflurane with a concentration of 1.5% and a flow rate of 0.5L / min was continuously administered through a mask and anesthesia was maintained throughout the operation. After disinfecting the mouse scalp, a window the size of a little finger cover was opened at the back center of the top of the head with surgical scissors. The connective tissue and periosteum on the surface of the mouse skull were cleaned with hydrogen peroxide, saline, etc. until there was no bleeding and the bone sutures were clear. A thin layer of light-curing glue was applied on the surface of the skull to protect the skull from drying out during the operation.

[0030] Skull leveling and virus injection: Fix the injection needle that has been inhaled with the virus in advance on the digital display three-dimensional manipulator. Move the injection needle tip to the top of the mouse skull bregma under a stereo microscope and slowly lower it until it just touches the bregma. Take the bregma as the origin, measure the depth value of the site 2mm away from the left and right, and adjust the height knobs on the left and right sides until the depth difference between the two sides does not exceed 0.03mm. Also take the bregma as the origin, adjust the height difference between the bregma and the posterior bregma to no more than 0.03mm. After the skull is leveled, move the injection needle to the coordinate site of the DG area for positioning, and then use a handheld skull drill to drill a hole at the positioning point, and open the skull without penetrating the dura mater. Take the meninges under the skull as the zero point, and slowly insert the injection needle to a depth of 2mm. Slowly inject 200nL of virus into the DG area at a speed of 30nL / min.

[0031] Implantation of optical fiber 2: Figure 2 As shown, the ceramic ferrule 3 is fixed on the support rod, the end of the optical fiber 2 of the ceramic ferrule 3 is moved above the needle path, and the optical fiber 2 is slowly implanted into the mouse brain with the meninges under the skull as the zero point, and the implantation depth is 1.95-1.98mm. Two optical fibers 2 are implanted, and the ends of the two optical fibers 2 are inhibitory neuron markers 9 and excitatory neuron markers 5, respectively.

[0032] Fiber 2 fixation: After fiber 2 implantation is completed, the fiber 2 ferrule is sealed on the mouse head with a dental level.

[0033] Surgery completed: After dental cement sealing is completed, remove the mouse and wait for it to wake up. Keep it in a single cage. The virus expression period is generally two weeks, and fiber optic 2 recording can be performed after two weeks.

[0034] Transcranial magnetic stimulation and fiber optic 2 recording:

[0035] The experimental equipment used was a CCY-I transcranial magnetic stimulator and a stimulation coil for small animals. The coil was a Y064 circular coil with an outer diameter of 56 mm, an inner diameter of 14 mm, and a maximum magnetic field output intensity of 3.6 T. Before the stimulation was implemented, the motor threshold of the mouse was first measured. In order to avoid interference with the results caused by animal limb twitching caused by stimulation with an intensity higher than the motor threshold, a stimulation intensity of 80% of the motor threshold was finally selected.

[0036] According to the set stimulation scheme, stimulation is performed after setting the stimulation parameters. When applying magnetic stimulation to mice that have undergone optical fiber 2 implantation surgery, the small animal stimulation coil is attached to the top of the junction between the parietal bone and the optical fiber 2 at a slightly inclined angle with the parietal bone, and the center point of the coil is aligned with the implantation position of the optical fiber 2. While stimulating, the optical fiber jumper 4 is connected to the ceramic ferrule 3 on the mouse's head and the device respectively, so as to realize the collection of neural activity signals while stimulating.

[0037] This method can avoid TMS electromagnetic interference and achieve simultaneous recording and acquisition of neural discharge activities of different types of neurons such as excitatory and inhibitory neurons in the dentate gyrus under real-time TMS stimulation. This method can achieve real-time, long-term monitoring of the discharge activities of neuronal clusters. The optical fiber 2 recording has the advantages of high resolution, low noise and strong anti-interference ability, and can capture subtle changes in neural activity. At the same time, the precise design of the implantation depth and size of the optical fiber 2, as well as the stable connection between the ceramic ferrule 3 and the optical fiber jumper 4, ensure the stability and reliability of signal transmission. This real-time, long-term monitoring method makes long-term and continuous neuroscience research possible, while also reducing the pain and risks of experimental animals.

Claims

1. A method for real-time acquisition of discharges of different types of neuron clusters, characterized in that: The following steps are involved: S1, select specific promoter: for excitatory neurons, select tool virus with CaMKⅡa as specific promoter; for inhibitory neurons, select tool virus with VGAT1 as specific promoter; S2, positioning and injection: at the coordinates of the dentate gyrus region of the target brain (1), the tool virus is injected into the dentate gyrus region through a burr hole in the skull; S3, implanting optical fiber: implanting the optical fiber (2) into the hole of the skull, with the implantation depth being 1.95-1.98 mm, wherein the optical fiber (2) is connected to a ceramic ferrule (3) and an optical fiber jumper (4) for connecting the device; S4, fix the optical fiber: fix the ceramic ferrule (3) on the target brain (1) using a dental level; S5, virus expression: wait for the tool virus injected in S2 to be expressed on the labeled neurons. The tool virus expression time is at least two weeks; S6, Transcranial magnetic stimulation and fiber optic recording: A TMS stimulation coil (6) is used to apply magnetic stimulation to the target brain (1), while the discharge signals of different types of neuronal populations are recorded through optical fibers (2).

2. The method for real-time acquisition of discharges of different types of neuron clusters according to claim 1, characterized in that: The S6 specifically comprises: the TMS stimulation coil (6) is tiltedly arranged above the junction of the parietal bone and the optical fiber (2) of the target brain (1), the center point of the TMS stimulation coil (6) is aligned with the implantation position of the optical fiber (2) on the target brain (1), the optical fiber jumper (4) is connected to the ceramic ferrule (3), the end of the optical fiber jumper (4) away from the ceramic ferrule (3) is connected to the optical fiber recording system (7), and the end of the optical fiber recording system (7) away from the optical fiber jumper (4) is connected to the display (8).

3. The method for real-time acquisition of discharges of different types of neuron clusters according to claim 1, characterized in that: The coordinates of the dentate gyrus region of the target brain (1) in S2 are: 1.7 mm posterior to the anterior bregma, 1.2 mm to the left and right of the raphe, and 2 mm in depth.

4. The method for real-time acquisition of discharges of different types of neuron clusters according to claim 1, characterized in that: The dimensions of the optical fiber (2) in S3 are: diameter 200 μm, length 3 mm; the diameter of the ceramic ferrule (3) is 1.25 mm.

5. The method for real-time acquisition of discharges of different types of neuron clusters according to claim 1, characterized in that: The stimulation coil for small animals is a circular coil with an outer diameter of 56 mm and an inner diameter of 14 mm. The maximum magnetic field output intensity of the coil is 3.6 T.

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