CN112640847B - Endogenous epileptic seizure animal model and construction method thereof - Google Patents

Abstract

The invention discloses an endogenous epileptic seizure animal model, which comprises a hippocampal CA3 region which is injected into a mouse by adopting chemogenetics or optogenetics activated virus and is constructed after 3 weeks of expression, wherein the activated virus is selected from the following: a) A mixture of rAAV-CaMKIIa-hM3D (Gq) -mCherry-WPREs-pA and rAAV-VGAT1-hM3D (Gq) -mCherry-WPRE-pA, and b) a mixture of rAAV-Ef1 alpha-DIO-hCHR 2 (H134R) -EYFP-WPRE-pA and rAAV-CaMKII-CRE-WPRE-pA and AAV-VGAT 1-CRE-WPRE-pA. Compared with a multipoint injection model of a CA1 region and a VA region, the epileptic seizure animal model is more suitable for the mechanism research of epilepsy and the screening of antiepileptic drugs.

Description

Endogenous epileptic seizure animal model and construction method thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to an endogenous epileptic seizure animal model and a construction method thereof, in particular to an endogenous epileptic seizure animal model constructed by activating viruses by adopting chemical genetics or optogenetics and a construction method thereof.

Background

No obvious progress has been made in refractory epilepsy for nearly 40 years. The reason for this is that the pathogenesis is not completely clear. The previous discussion of the pathogenesis of epilepsy is based on traditional animal models of epilepsy, such as pirocarpine, hydnoceric acid or electrical stimulation, and the exogenous animal models of epilepsy are greatly different from human epileptic seizure. The brain regions involved in the seizure process or the neurons specifically involved are not known. Thus, these exogenous models of epilepsy have not been able to fully mimic human seizures. The current view is that the occurrence of epilepsy in human beings is probably related to glutamic acid and GABAergic neurons which are the main neurons in brain, and the current new drugs and physical therapies applied to anti-epilepsy treatment are almost related to the two neurons, which indicates that the two neurons are probably related to the pathogenesis of epilepsy. With the increasing application of optogenetics and chemical genetics (DREADDs) methodologies to neuroscience, a novel, universal and reliable epileptic seizure model needs to be constructed by an endogenous regulation method, so that the method is more suitable for researching epileptic pathogenesis and screening antiepileptic drugs. Is expected to become an effective measure for reversing the passive situation in the current treatment.

The disadvantages of the conventional model: the conventional ignition model has a series of problems. First, for example, pilocarpine, hydrarginic acid, and PTZ models, the use of diffusing chemicals is inherently variable, unable to pinpoint the diseased area and cell type, and difficult to distinguish the contribution of plastic mechanisms to tissue damage. Second, the experimenter has no control over the subset of cells that are activated upon ignition, making it difficult to establish causal relationships between cell types and pathological outcomes. The lack of specificity may in turn lead to a reduction in the standardized results throughout the laboratory. And after the model is ignited repeatedly, the damage of chemical substances to brain tissues is also serious, and the reliability of experimental data in part of research fields is reduced.

CN111839800 discloses a chemical genetic epilepsy persistent state disease animal model and a construction method and application thereof, the epilepsy persistent state disease animal model is a mouse brain inner nuclear group (a model I hippocampal CA1 area and a thalamus ventral precore VA area; a model II basolateral amygdala BLA area and a thalamus ventral precore VA area) brain stereotaxic virus (a chemical genetic virus rAAV-CaMKIIa-hM3D (Gq) -mChery-WPREs-pA) injection and a mouse hippocampal CA3 area electrode array embedding, after a mouse recovers for 1 week, a metabolite CNO of clozapine is injected in an abdominal cavity, so that the CNO is combined with a receptor expressed by virus to activate neurons to induce epilepsy persistent state attack, and the animal model of the epilepsy persistent state disease is obtained through behavioral observation and in-vivo multichannel local field potential recording judgment. The method for constructing the model still has many defects. First, the method requires injecting virus at multiple points (such as CA1 region, VA region, BLA region, etc.) in brain to establish status epilepticus model, but two or more nuclei are activated simultaneously, it is difficult to simultaneously define the upstream and downstream relationship of nuclei, and it is significantly limited in the study of epilepsy network. Second, the model established by injecting virus at multiple points in the brain is not conducive to the study of neurotransmitter evolution in the neural network during epileptic seizure. Thirdly, the cost of injecting virus into the brain of the same mouse at multiple points is longer, and the virus amount is relatively more. If virus injection is required to be carried out on the upstream and the downstream simultaneously, mice are easy to die in the injection process; if the injection is performed separately, the experimental time is prolonged, and the time cost is increased. And also increases the cost and cost of feeding mice. Fourthly, the model is an epileptic persistent state model, but not an epileptic model, and is suitable for the research of the epileptic persistent state field, but not suitable for the mechanism research of epilepsy and the screening of antiepileptic drugs.

Therefore, the research of epilepsy mechanism and the screening of antiepileptic drugs are facilitated; the upstream and downstream relation of the nucleus is more convenient to be clear and definite, and the epileptic network is beneficial to be clear; meanwhile, the method is simpler and more convenient to operate, and the survival rate of the model animal is ensured.

Disclosure of Invention

The invention aims to provide an endogenous epileptic seizure animal model constructed by activating viruses by adopting chemical genetics or optogenetics and a construction method thereof. The animal model has the following advantages: first, it can stimulate neurons relatively precisely in time, as with classical PTZ and like models. The experimental animal mortality is low, the use amount of animals in an experimental group can be reduced, and the same mouse can be compared before and after the experimental process, so that the experimental data is more reliable. Second, it can manipulate the activity of specific neuronal populations and can study the pathogenesis of epilepsy by exciting different types of neurons. Thirdly, no stimulation artefacts are produced, so that the spatiotemporal dynamics of neuronal activity during stimulation can be studied electrophysiologically; does not cause obvious brain injury or glial reaction. Fourthly, compared with the prior intracerebral multipoint virus injection model, the brain activation area range is smaller, and the propagation path and direction of the epilepsy can be more clearly positioned, namely the network research of the epilepsy is most ideal. Fifth, the activation of brain regions is clear without the intervention of exogenous damaging chemicals, and this model has unique advantages in the field of neurotransmitter evolution before, during and after epileptic seizures. Sixth, compared to the previous model of multiple injection of virus, the injection of virus is reduced by half, the injection time cost is reduced by half, and the virus cost is also reduced by half. And the injection of the upstream and downstream nucleoplasm viruses can be simultaneously carried out, so that the time cost and the cost in the mouse feeding process can be saved. Seventh, it can be used for screening antiepileptic drugs. Eighth, optogenetic validation, further determining temporal specificity.

The following embodiments are provided to achieve the object of the present invention.

In one embodiment, the endogenous animal model of seizure according to the present invention comprises injecting a chemically genetically or optogenetically activated virus into the mouse hippocampal CA3 region, the activated virus being established after 3 weeks of expression, the activated virus being selected from the group consisting of:

a) A mixture (or called mixed virus) of rAAV-CaMKIIa-hM3D (Gq) -mChery-WPREs-pA (titer 5.04E + 12vg/mL) and rAAV-VGAT1-hM3D (Gq) -mChery-WPRE-pA (titer 5.01E + 12vg/mL), and

b) rAAV-Ef1 alpha-DIO-hCR 2 (H134R) -EYFP-WPRE-pA (titer 5.48E + 12vg/mL) and rAAV-CaMKII-CRE-WPRE-pA (titer 5.79E + 12vg/mL)

And AAV-VGAT1-CRE-WPRE-pA (titer 4.73E + 12vg/mL).

Preferably, the endogenous animal model of epileptic seizure of the invention is characterized in that: the volume ratio of the virus rAAV-CaMKIIa-hM3D (Gq) -mCherry-WPREs-pA to the virus rAAV-VGAT1-hM3D (Gq) -mCherry-WPRE-pA in the mixed virus of the a) is (1-8): 1, more preferably (6-8): 1.

the volume ratio of the virus rAAV-Ef1 alpha-DIO-hCHR 2 (H134R) -EYFP-WPRE-pA to the virus rAAV-CaMKII-CRE-WPRE-pA and the virus AAV-VGAT1-CRE-WPRE-pA is 7:6:1.

the animal model of epileptic seizure of the present invention further comprises a method for igniting epileptic seizure by using CNO or light stimulation.

The invention also aims to provide a method for constructing an endogenous epileptic seizure animal model, which is characterized by comprising the following steps of adopting optogenetic or chemogenetic activated viruses to construct the endogenous epileptic seizure animal model:

1) Selecting a wild type C57BL/6 mouse of more than 8 weeks old, removing hair after anesthesia, shearing off scalp after disinfection, exposing bregma, drilling holes by taking bregma as a central point, pricking meninges, and exposing brain tissues;

2) Injecting activated virus into the CA3 region of the hippocampus, suturing scalp after disinfection, maintaining the body temperature of the mouse at about 36 ℃, and closing the mouse into a cage for breeding;

3) Completing the construction of an endogenous epileptic seizure animal model after the activated virus is expressed for 3 weeks in a CA3 region;

4) Optionally, further comprising using CNO injection or light ignition epileptic seizure to judge whether the model constructed in step 3) was successful,

wherein the activating virus is selected from the group consisting of:

a) A mixture (or called mixed virus) of rAAV-CaMKIIa-hM3D (Gq) -mCherry-WPREs-pA (titer 5.04E + 12vg/mL) and rAAV-VGAT1-hM3D (Gq) -mCherry-WPRE-pA (titer 5.01E + 12vg/mL), and

b) A mixture (or called mixed virus) of rAAV-Ef1 alpha-DIO-hCR 2 (H134R) -EYFP-WPRE-pA (titer 5.48E + 12vg/mL) and rAAV-CaMKII-CRE-WPRE-pA (titer 5.79E + 12vg/mL) and AAV-VGAT1-CRE-WPRE-pA (titer 4.73E + 12vg/mL).

Preferably, in the above construction method of the present invention, the activated virus is a virus, wherein the mixture of group a) has a volume ratio of rAAV-CaMKIIa-hM3D (Gq) -mCherry-WPREs-pA to rAAV-VGAT1-hM3D (Gq) -mCherry-WPRE-pA virus of (1-8): 1, more preferably (6-8): 1.

preferably, in the above construction method of the present invention, the activated virus is a virus, wherein the volume ratio of the mixture rAAV-Ef1 α -DIO-hCHR2 (H134R) -EYFP-WPRE-pA in group b) to rAAV-CaMKII-CRE-WPRE-pA and AAV-VGAT1-CRE-WPRE-pA viruses is 7:6:1.

preferably, in the construction method of the present invention, the weight of the mouse is 20g to 33g, the injection dose of the chemogenetically activated virus is 200nl to 300nl, and the injection dose of the optogenetically activated virus is 200nl.

In the above construction method of the present invention, the evaluation includes:

1) Observing and constructing animal model abdominal cavity injection CNO or light stimulation ignition epileptic seizure ethology;

2) Post-seizure behaviours and in vivo channel field potentials were recorded.

The epileptic seizure animal model of the invention is constructed by exciting GABAergic neurons and glutamatergic neurons simultaneously in the CA3 region by a method of optogenetics or chemogenetics (the chemogenetics mixes viruses according to the proportion of 6-8, the optogenetics mixes viruses according to the proportion of 7.

The construction method of the epileptic seizure animal model comprises the following steps:

1) Model construction

A. Activated virus injection by chemogenetics, rAAV-CaMKIIa-hM3D (Gq) -mCherry-WPREs-pA (titer about 5.04E + 12vg/mL) and rAAV-VGAT1-hM3D (Gq) -mCherry-WPRE-pA (titer about 5.01E + 12vg/mL) are optimally (6-8): after mixing at a ratio of 1, 200nl to 300-nl were mixed and injected into CA3 region virus for 3 weeks after expression, and CNO test was performed.

B. In the optogenetic virus injection, rAAV-Ef1 alpha-DIO-hCR 2 (H134R) -EYFP-WPRE-pA (the titer is about 5.48E + 12vg/mL) is mixed with rAAV-CaMKII-CRE-WPRE-pA (the titer is about 5.79E + 12vg/mL) and AAV-VGAT1-CRE-WPRE-pA (the titer is about 4.73E + 12vg/mL) in a mixing volume ratio of 7:6:1, 200nl of the virus mixture was finally injected at CA 3.

2) Observation of behavioral science of CNO (carbon monoxide injection) injected into abdominal cavity and epilepsy ignited by light stimulation

After 3 weeks of expression of the chemical genetic virus, CNO (2 mg/kg) was intraperitoneally injected to ignite the seizures. The behavior was observed for 3 hours. And at the same time mortality was compared to the traditional PTZ model.

3) Ethology and in vivo multichannel field potential recording after CNO ignition epileptic seizure

The CNO-ignited mouse is internally provided with electrodes, and the CNO ignition is carried out after 1 week, and simultaneously the field potential of the mouse epileptic seizure is recorded by an in-vivo multi-channel field potential recording system.

4) Long-term follow-up of chemogenetic ignition models

The stability of CNO-ignited mice was known by repeated multiple ignitions over a long period of time.

Drawings

FIG. 1 is a diagram showing the observation result of the behavior of the animal model of epilepsy of the present invention after ignition of epileptic seizure by CNO intraperitoneal injection;

FIG. 2 is a graph of the behavioral observations of the animal model of the invention after light stimulation to ignite a seizure;

FIG. 3 is a graph of behavioral observations after 7 weeks after reignition of seizures with CNO;

FIG. 4 models field potential and spectral flux after 7 weeks of reinitiation of seizures with CNO;

FIG. 5 is a graph of the appearance of viral infection in fluorescence detection of an animal model of the invention;

fig. 6 is a graph of behavioral observations after repeated multiple rounds of seizure.

Detailed Description

The following examples are merely representative for further understanding and illustrating the spirit of the present invention, and are not intended to limit the scope of the present invention in any way.

Example 1 construction of seizure actor model

Wild type C57BL/6 mice are selected for the experiment, the male and female are not limited, the age is more than 8 weeks, and the weight is 20g to 33g. The chemicogenetic and optogenetic virus injection coordinates were identical, the CA3 region (coordinates: AP-2.3mm ML, -2.5mm DV, -2.7 mm. All viruses used were commercially available.

The specific experimental process is as follows:

1. brain stereotaxic virus injection, electrode array and optical fiber embedding

1.1 Virus injection:

1.1.1 anaesthesia and fixation mice were weighed, anaesthetised by intraperitoneal injection with 0.8% pentobarbital and then fixed in a stereotactic frame.

1.1.2 exposing bregma, coordinate positioning and drilling

The hair on the top of the mouse head is cut off, the scalp in the operation area is disinfected by iodophor and 75% alcohol, the scalp is cut along the deformed line, the bregma is fully exposed, the microsyringe is connected to the injection arm of the microinjection pump, and the skull plane is leveled with the bregma as the central point. Relevant nuclear coordinates were determined using a Paxinos mouse brain map. The brain membrane is marked by a marking pen for positioning, then a skull drill is used for carefully drilling holes at the marked points to expose the brain membrane without bleeding, and the fine needle tip is used for breaking the brain membrane to fully expose the brain tissue.

1.1.3 Virus injections (all viruses purchased from Wuhan Shumi Co., ltd.)

Experimental mice were divided into 7 groups, and mixtures with different volume ratios (total 5 groups of mixed virus experimental groups) were prepared from the chemogenetic virus rAAV-CaMKIIa-hM3D (Gq) -mChery-WPREs-pA (abbreviated as CaMKIIa-hM3D (Gq) -mChery with titer of 5.04E + 12vg/mL) and rAAV-VGAT1-hM3D (Gq) -mChery-WPRE-pA (abbreviated as VGAT1-hM3D (Gq) -mChery with titer of 5.01E + 12vg/mL), as shown in Table 1. In addition, the chemogenetic virus VGAT1-hM3D (Gq) -mCherry (titer 5.01E + 12vg/mL) and rAAV-CAMKII-EGFP-WPRE-pA (abbreviated as CAMKII-EGFP, titer 5.15E + 12vg/mL) are prepared into a mixture (total 2 groups of mixed viruses) according to different volume ratios, and the mixture is used as a virus control group and is specifically shown in the following table.

TABLE 1 preparation of virus experimental groups

TABLE 2 Virus control group Table

Chemogenetics-activated virus injection

The mixed viruses of the above 5 experimental groups and 2 control groups were injected into the CA3 region of 7 mice, respectively, as follows:

experimental groups:

200nl of each mixed virus was aspirated by a 5. Mu.l microsyringe, and the mixture was injected into the CA3 region of the hippocampus of mice of 5 groups of experimental groups at a rate of 20nl/min, with the needle left for 10min, and the tip of the needle was slowly pulled out. The scalp was sutured after 75% alcohol disinfection. After operation, the animals were placed on a heating pad to maintain their core body temperature at 36 ℃ and housed in their home cages for breeding after they were awake.

Optogenetic virus injection

Experimental groups:

first, rAAV-Ef1 alpha-DIO-hCR 2 (H134R) -EYFP-WPRE-pA (titer 5.48E + 12vg/mL) and rAAV-CaMKII-CRE-WPRE-pA (titer about 5.79E + 12vg/mL) or AAV-VGAT1-CRE-WPRE-pA (titer about 4.73E + 12vg/mL) are mixed according to the proportion of 1. Secondly, ((DIO-hCHR 2 (H134R) -EYFP + VGAT 1-CRE): (DIO-hCHR 2 (H134R) -EYFP + CaMKII-CRE)) was also mixed in the ratio of 1. Then, 200nl of the virus mixture was aspirated by a microsyringe, and 200nl of the virus was injected into each CA 3.

Control group:

400nl of the control virus was aspirated by a 5. Mu.l microsyringe and injected at a rate of 200nl/min into the DG + CA3 regions of the hippocampus of the mice in the control group. Injecting 200nl into a DG area, and reserving a needle for 10min; after the needle tip was slowly pulled out, the remaining 200nl of virus was injected into the CA3 region in the same manner, the needle was left for 10min, and the needle tip was slowly pulled out. After the operation, the operation is carried out.

2. Embedded electrode and optical fiber

(1) In mice injected with chemogenetic virus, the virus was expressed for 3 weeks and the CNO-ignited mice were subjected to the process of implantation of the embedded composite electrode. The anesthesia, fixation, bregma exposure and positioning are all 1.1.1 to 1.1.3. A2X 1.4X 6mm screw was attached to the cranium above the forehead as a reference electrode and a 2X 1.4X 2.4mm screw was attached to the left hippocampus and right cerebellum. Then, the electrodes were implanted with virus measuring hippocampal CA3 (coordinate points are the same as those at the virus injection site), and reference wire electrodes were fixed to screws on the frontal lobe cranium. The brain fixing sheet is placed. Finally, the denture fixing sheet, the screw, the skull and the electrode are tightly adhered and fixed together by denture fixing water and denture fixing powder. After the dental cement had dried, the mice were housed for at least 1 week and allowed to recover.

(2) In the procedure of optical fiber implantation, a mouse injected with a 2-week optogenetic virus at the CA3 site was anesthetized in the same manner as described above and the scalp was cut to expose bregma and the central cruciate suture, 1X 1.4X 2.4mm screws were mounted above the left hippocampus and right cerebellum, respectively, and then the cranial plane was leveled with a holder and an optical fiber. The skull of a target nucleus injected with far-end bregma virus (CA 3: AP-2.3mm ML, -2.5mm DV, -2.7 mm) is embedded with a first optical fiber at the window, and the embedding depth of the optical fiber is 0.05mm shallower than the injection depth of the virus, so as to leave an irradiation space for light stimulation. Then fixing the two optical fibers and the screws on the surface of the skull by dental cement layer by layer.

CNO intraperitoneal injection and light stimulation to activate neurons to induce epileptic seizure

A. CNO injection to ignite epileptic seizure

CNO powder was dissolved in 1% DMSO (dimethylsulfoxide) to prepare CNO at a final concentration of 2mg/kg. Mice 3 weeks after the injection of the chemical genetics virus were intraperitoneally injected according to the standard of 0.1ml/10g (CNO concentration of 2 mg/kg), and the behavior was observed for 2 to 3 hours. Mice that could be fired for seizures were marked. The ignited mouse is subjected to field potential detection, after electrodes are embedded and recovered for 1 week, 2mg/kg CNO is injected into the abdominal cavity, and then video ethological observation and in-vivo multichannel local field potential recording are simultaneously carried out.

B. Epileptic seizure ignited by light stimulation

After the mouse recovers for 1 week, the optical fiber on the top of the mouse head is connected to an optical cable, and the other end of the optical fiber rotator is connected with a blue laser with the wavelength of 473 nm. A waveform signal generator is connected with the blue laser to control the emission of the laser. The laser was turned on and the optical power at the end of the optical cable was tested with an optical power meter and adjusted to 10-15 mW. After the optical power is adjusted, the laser is turned off, the optical cable is connected with the optical fiber on the mouse head again, and the control parameters of the waveform signal generator are set, wherein the parameters are set to be the frequency (20 Hz) and the duty ratio (0.2). The stimulation duration was fixed for 20s or persistence, respectively. The remaining output parameters are string parameters Burst Duration (2, 3, 5) s, burst Interval (0.5, 1, 2) s, series parameters Train Delay (0, 0.05, 0.1, 0.5) s, and Train Duration 20s. And (3) performing behavioral observation, video recording and the like after the blue light stimulation through the parameter adjustment, and rejecting experimental groups when the observed mice are subjected to brain tissue fluorescence detection and the virus injection position is inaccurate or the optical fiber embedding is inaccurate. Through experiments of different parameters, the stimulation parameters are finally determined as follows: frequency (20 Hz) and duty cycle (0.2), stimulation duration is 20s. The string parameters Burst Duration 5s, burst Interval 0.5s, and the string parameters Train Delay 0s, train Duration 20s.

4. Behavioural and associated field potentials

4.1 behavioral Observation

4.1.1CNO post-injection behavioural observations, mortality compared with PTZ model

And (3) performing behavioral observation after igniting the epileptic seizure by CNO intraperitoneal injection, observing the epileptic seizure condition of the mice according to the Racine score, and recording the epileptic seizure level and seizure times. After the statistics, corresponding statistics are performed, and the result is shown in fig. 1. The ignition rates and attack levels of the experimental groups did not differ significantly: (CaMKIIa-hM 3D (Gq) control 6/10; test 1.

In FIG. 1, (a) is a schematic view showing the process of virus injection, behavioral observation and field potential experiment. FIG. b is a fluorescent image after virus injection. (c) The figures compare the seizure rate of mice after intraperitoneal injection of CNO (2 mg/kg) with the CaMKIIa-hM3D (Gq) control group (control group) at various ratios, where (CaMKIIa-hM 3D (Gq) control group n =10, experimental group 1. (f) Comparison of mortality for the PTZ model for experiment 1.

FIG. 1 shows the simultaneous stimulation of glutamatergic and GABAergic neurons by a mixture of viruses of different proportions. The results in table 1 show that: glutamic acid and GABAergic neurons can induce epileptic seizure under a specific state; in particular, when a small amount of GABAergic neurons are excited and glutamatergic neurons are also in an excited state simultaneously, seizures are more likely to be induced. The model has a lower mortality rate of about 18% compared to the conventional PTZ model, which has a mortality rate of more than 70% after the cavity injection of PTZ (24 mg/kg).

4.1.2 post-light stimulation behavioural observations

Based on the above research results, the present inventors found that there is a difference between the excitation of only glutamatergic neurons and the simultaneous excitation of glutamatergic neurons and gabaergic neurons, and the number of epileptic seizures, and assumed that the activity of the above neurons can be more accurately regulated and controlled by adjusting different stimulation parameters by applying the optogenetic method, whether the duration of epileptic seizures can be prolonged, or even whether epileptic status can be induced.

For this purpose, rAAV-Ef1 α -DIO-hCR 2 (H134R) -EYFP-WPRE-pA virus and rAAV-CaMKII-CRE-WPRE-pA virus were mixed and injected into DG and CA3 regions (200 nl per site) to serve as a control group. rAAV-Ef1 alpha-DIO-hCHR 2 (H134R) -EYFP-WPRE-pA (short for short)

After "DIO-hCHR2 (H134R) -EYFP") was mixed with rAAV-CaMKII-CRE-WPRE-pA (abbreviated as "CaMKII-CRE") and AAV-VGAT1-CRE-WPRE-pA (abbreviated as "VGAT 1-CRE") 1, respectively, the mixture was again mixed as 1: (DIO-hCHR 2 (H134R) -EYFP + CaMKII-CRE)) to obtain a mixed virus, and 200nl of the mixed virus was injected into the CA3 region to prepare an experimental group. After 2 weeks of virus infection, the fiber was embedded at the site where the virus was injected. Blue light stimulation was performed after week 3. The frequency (20 Hz) and duty cycle (0.2) were fixed, and the stimulation duration was fixed to be persistent. The firing rate and duration of the seizures were then observed by adjusting the corresponding stimulation parameters, and the results are shown in fig. 2.

FIG. 2 shows the characteristics of the duration of the light-genetic stimulation. Wherein, a) is a schematic diagram of the DG + CA3 group injection of the optogenetic virus for exciting glutamatergic neuron and a corresponding fluorescence diagram. (b) FIG. is a schematic representation and corresponding fluorescence map of CA3 injection of optogenetics viruses that excite GABAergic neurons and glutamatergic neurons (1. (c) The firing rate of optogenetically ignited seizures (1. (d) The graph shows the duration of seizures when the group DG + CA3 photostimulates glutamatergic neurons and two different stimulation parameters ignite the seizures (n.s.p >0.05, paired t-test). (e) Figure is a graph of 1.

The results of fig. 2 show that: under the same stimulation conditions, the epilepsy ignition rate of the control group and the experimental group is about 70 percent (figure 2 c), and no obvious difference exists. When the series parameters Burst Duration 5s, burst Interval 0.5s, series parameters Train Delay 0s, train Duration 20s; neither group of mice had twitch times exceeding 2 minutes (Pulse 1). When the series parameters Burst Duration 2s, burst Interval 2, train Delay 0s, train Duration 20s; no significant difference in seizure duration was found in the GD + CA3 group (fig. 2 d). Whereas the seizure duration was significantly prolonged in group 1. Indicating that the two models do differ. It was also further confirmed by optogenetic methods that the 1.

4.2 Observation of 7 week behavioural Observation and field potential experiment of chemical genetics Virus infection

The groups were re-ignited at week 7. The results are shown in FIG. 3. FIG. 3 shows the 7 th week behavioural characteristics of the two viruses after mixing in different proportions. Wherein (a) is a plot of the firing rates of each group of mice at week 7 (1 group n =9,1, 2 groups n =11, 1. (b) Figure each proportion group compares the number of episodes in week 3 with week 7 (p >0.05,. P <0.01,. P <0.001,. P <0.0001, two-wayaanova); (c) The corresponding seizure levels (Ranice score) (p >0.05, two-wayANOVA) are shown.

The results of fig. 3 show that: the ignition rates of the experimental 1. The attack level of each group is not obviously different, although the attack frequency is reduced within 3 hours of each group, the attack frequency is about 5, and the attack frequency of 1. This suggests that gabaergic neurons and glutamatergic neurons are activated simultaneously in specific states, with an effect on seizure status.

The mice were ignited for in vivo field potential recording, and the brain electrical discharge form was found to be continuous after CNO injection, and the results are shown in FIG. 4. Fig. 4 shows the field potential and frequency spectra for the control and experimental groups, caMKIIa-hM3D (Gq) control (n = 5), experimental group 1 (n = 3), 1 (n = 4), 1.

The results of fig. 4 show that: it is further confirmed from field potentials and spectrograms that gabaergic neuronal excitation does play a promoting role during seizures and may also play an important role in seizures in clustered seizures.

The optogenetic group has no obvious difference in restimulation at the 7 th week of fiber embedment, and therefore, the details are not repeated herein.

5. Confirmation of two neuronal infections

To further understand whether there were transfections of two neurons when only a small number of gabaergic neurons were excited, and that small doses of gabaergic neurons were excited, also could lead to seizures, the inventors selected VGAT1-hM3D (Gq) virus to be mixed with a control virus (rAAV-CAMKII-EGFP-WPRE-pA) in 1.3 weeks after the virus infection, CNO (2 mg/kg) was intraperitoneally injected, and the behavior was observed for 3 hours, and the results are shown in FIG. 5.

FIG. 5 infection of VGAT1-hM3D (Gq) virus mixed with CAMKII-EGFP virus. Wherein, the picture (a) is a schematic view of virus injection. (b) The figure is a fluorescence plot of infection of group 1 and group 1. (c) The figures show 1 (n =5, seizures 0) and 1 (6) (n =5, seizures 1) seizure firing rates (n.s.p >0.05, fisher's exact test) for 1.

The results in FIG. 5 show that: control 1, group 1 had no seizures (n = 5), control 1, group 6 had one mouse seizures (1/5) (fig. 5 c), seizure class 4, number of seizures 2; subsequent multiple ignitions all reoccur seizures. The presence of infection by both viruses was further confirmed by fluorescence detection and there was a co-tagging phenomenon. As shown in fig. 5. Demonstrating simultaneous infection of both neurons.

6. Repeated ignition and long-term follow-up experiment

In order to further verify the repeatability and long-term stability of the animal model of epilepsy of the invention. The same batch of mice was subjected to repeated experiments every other week for 5 experiments for each of the chemical genetics experiments 1. The results are shown in FIG. 6. Figure 6 shows the long-time follow-up of chemogenetic activation experiments 1. Wherein (a) is the firing rate at week 12 (1

The results of fig. 6 show that: the animal model of epilepsy of the invention has high success rate of reburning rate at week 12, and the times of epileptic seizure and the seizure level have no obvious difference. The above results indicate that the seizure model which activates both gabaergic neurons and glutamatergic neurons has better stability, especially 1.

Claims (2)

1. A method for constructing an endogenous epileptic seizure animal model comprises the steps of injecting a chemogenetic or optogenetic activating virus into a hippocampal CA3 region of a mouse, and constructing after expressing for 3 weeks, wherein the activating virus is selected from the following:

a) rAAV-CaMKIIa-hM3D (Gq) -mCherry-WPREs-pA and

a mixture of rAAV-VGAT1-hM3D (Gq) -mCherry-WPRE-pA, and b) a mixture of rAAV-Ef1 alpha-DIO-hCHR 2 (H134R) -EYFP-WPRE-pA, rAAV-CaMKII-CRE-WPRE-pA and AAV-VGAT1-CRE-WPRE-pA,

wherein the volume ratio of the rAAV-CaMKIIa-hM3D (Gq) -mCherry-WPRE-pA to the rAAV-VGAT1-hM3D (Gq) -mCherry-WPRE-pA is 6:1, the volume ratio of rAAV-Ef1 alpha-DIO-hCHR 2 (H134R) -EYFP-WPRE-pA), rAAV-CaMKII-CRE-WPRE-pA and AAV-VGAT1-CRE-WPRE-pA is 7:6:1.

2. the method of constructing an animal model of seizures according to claim 1 further comprising igniting the seizures using CNO or light stimulation.

Images