A method for constructing an animal model for specifically manipulating somatostatin neurons

Abstract

The invention discloses a method for constructing an animal model of specifically operating somatostatin neurons, and belongs to the field of animal model construction. The invention successfully creates an animal model of specifically operating somatostatin neurons by injecting AAV9-SST-Cre adeno-associated virus and AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus in an animal model according to a volume ratio of 1:1-3, with an injection volume of 0.3-0.5 μL and transfection for 2-3 weeks, and realizes specific labeling of somatostatin neurons and their projection in the rat insular lobe, providing a new tool for the study of brain neuroscience. The method of the invention is not limited by species, can realize specific regulation of the expression of somatostatin SST in rats, and also solves the objective problems such as high cost, low survival rate, slow gene construction process, and difficulty in breeding of Cre tool mice.

Description

Technical Field

[0001] The invention belongs to the field of animal model construction, and in particular relates to a method for constructing an animal model for specifically operating somatostatin neurons. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Rats are often used as model organisms in various neuro-endocrine experimental studies because of their well-developed pituitary-adrenal system and sensitive stress response. In particular, research on neural circuits is very popular. The main reason is that many tool viruses can be used to easily manipulate certain gene expression through stereotaxic injection. Among the many tool viruses, the Cre-DIO system tool is the most commonly used.

[0004] The common Cre-DIO system tools include two types of Cre tool mice and Cre element viruses. There are four main construction strategies for the construction of Cre tool mice: plasmid expression vector Cre mouse model, site-directed knock-in Cre mouse model, BAC transgenic Cre mouse model, and safe site knock-in Cre mouse model.

[0005] However, in the construction of an animal model for specifically manipulating somatostatin SST neurons, there are many problems in the construction of Cre tool mice: first, other phenotypic changes or complications may be caused in the construction tool rats; second, the safer construction model cannot be applied to rat research; and in addition to the above technical problems, there are also common problems that are difficult to solve in a short period of time, such as the low survival rate of Cre tool mice, slow gene construction process, difficulty in breeding, high price, and unstable subculture. Summary of the invention

[0006] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a method for constructing an animal model for specifically manipulating somatostatin neurons. The method for constructing an animal model for specifically manipulating somatostatin neurons provided by the present invention uses a mixed virus of a Cre element adeno-associated virus and a DIO element adeno-associated virus as an inducer, has a short modeling time, can specifically label and manipulate somatostatin neurons, and achieves specific labeling of somatostatin neurons and their projections in the rat insular lobe.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides a method for constructing an animal model for specifically manipulating somatostatin neurons, comprising injecting a mixed virus containing a Cre element adeno-associated virus and a DIO element adeno-associated virus into rats, wherein the volume ratio of the Cre element adeno-associated virus to the DIO element adeno-associated virus is 1:1-3, the injection volume is 0.3-0.5 μL, and the transfection lasts for 2-3 weeks;

[0009] The Cre element adeno-associated virus is AAV9-SST-Cre adeno-associated virus;

[0010] The DIO element adeno-associated virus is AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus.

[0011] Preferably, the titer of the AAV9-SST-Cre adeno-associated virus is 9.42×10 13 vg / ml; AAV9-SST-Cre adeno-associated virus is a somatostatin SST promoter sequence packaged together with the Cre element in the AAV9 vector.

[0012] Preferably, the titer of the AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus is 6.99×10 13 vg / ml; AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus packages the DIO element together with mature neuron hSyn, functional inhibitory element hM4D(Gi), and fluorescent element mcherry on the AAV9 vector.

[0013] AAV9 has a strong ability to transduce multiple tissues and organs. After systemic intravenous or intraperitoneal injection, the virus can be widely expressed in multiple tissues such as bones, muscles, and heart. Molecular biological studies have shown that AAV9 can effectively cross the endothelial cell barrier and can efficiently pass through the blood-brain barrier while inducing effective expression of the target gene.

[0014] The Cre-loxP site-specific recombination system originates from the bacteriophage P1, which contains the Cre recombinase (343 amino acid protein, composed of 4 subunits) and a pair of 34bp DNA sequences (i.e., loxP sites) that it can specifically recognize. Based on the directionality of the two loxP sites, the DNA sequence between the two loxP sites (also called the floxed region) is specifically recombined and cut or reversed. If the Cre recombinase is specifically expressed in the corresponding cells / tissues, and the cells / tissues also contain the floxed target region of the two loxP site sequences, the recombination effect of the Cre-loxP system can be limited to specific cells or tissues, achieving the purpose of specific knockout or expression of the target gene in vivo. T cell-specific Cre transgenic mice were first constructed, providing conditions for the functional study of conditional target genes in T cells. Cre-DIO can achieve the effect of specifically initiating the cre gene sequence.

[0015] The hSyn promoter is derived from the promoter of the human SYN1 gene. The Synapsin I protein expressed by this gene is a protein specifically expressed in neurons. Therefore, the hSyn promoter is generally selected as a neuron-specific promoter.

[0016] hM4D (Gi) is a modified human G protein-coupled receptor. After being activated by clozapine N-oxide (CNO), it activates the Gi signaling pathway to inhibit cells and can control it for 1 to 6 hours.

[0017] mCherry is a red fluorescent dye currently widely used as a tracer in biotechnology, including the labeling of molecules and the localization of cellular components. Its color and monomeric molecules have photostability and low cytotoxicity.

[0018] Preferably, the injection method is nucleus injection. Further preferably, the injection is performed in the INS region of the rat insula.

[0019] Preferably, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus is 1:1, the injection volume is 0.3 μL, and the transfection is carried out for 2 weeks.

[0020] Preferably, the volume ratio of the Cre element adeno-associated virus to the DIO element adeno-associated virus is 1:1, the injection volume is 0.3 μL, and the transfection is for 3 weeks.

[0021] Preferably, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus is 1:1, the injection volume is 0.5 μL, and the transfection lasts for 2 weeks.

[0022] Preferably, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus is 1:1, the injection volume is 0.5 μL, and the transfection is carried out for 3 weeks.

[0023] Preferably, the volume ratio of the Cre element adeno-associated virus to the DIO element adeno-associated virus is 1:2, the injection volume is 0.3 μL, and the transfection is carried out for 2 weeks.

[0024] Preferably, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus is 1:2, the injection volume is 0.3 μL, and the transfection is for 3 weeks.

[0025] Preferably, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus is 1:2, the injection volume is 0.5 μL, and the transfection is carried out for 2 weeks.

[0026] Preferably, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus is 1:2, the injection volume is 0.5 μL, and the transfection is carried out for 3 weeks.

[0027] Preferably, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus is 1:3, the injection volume is 0.3 μL, and the transfection is carried out for 2 weeks.

[0028] Preferably, the volume ratio of the Cre element adeno-associated virus to the DIO element adeno-associated virus is 1:3, the injection volume is 0.3 μL, and the transfection is for 3 weeks.

[0029] Preferably, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus is 1:3, the injection volume is 0.5 μL, and the transfection is carried out for 2 weeks.

[0030] Preferably, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus is 1:3, the injection volume is 0.5 μL, and the transfection is carried out for 3 weeks.

[0031] Preferably, after transfection, rats are intraperitoneally injected with clozapine CNO to activate the virus.

[0032] The second aspect of the present invention provides the use of a mixed virus of the Cre element adeno-associated virus and the DIO element adeno-associated virus in the preparation of a product for constructing an animal model for specifically manipulating somatostatin neurons.

[0033] Preferably, in the animal model for specifically manipulating somatostatin neurons, somatostatin SST neurons are specifically inhibited, and the inhibited SST neurons show fluorescence;

[0034] Alternatively, hybrid viruses can be used to track the projections of somatostatin neurons.

[0035] Further preferably, the somatostatin neuron projections include the nucleus tractus solitarius.

[0036] The beneficial effects of the present invention are:

[0037] The present invention provides a method for constructing an animal model for specifically operating somatostatin neurons. The method comprises the following steps: specifically operating somatostatin neurons by injecting a mixture of AAV9-SST-Cre adeno-associated virus and AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus into an animal model, and specifically labeling somatostatin neurons and their projections in the insular lobe of rats, thereby providing a new tool for the study of brain neuroscience.

[0038] The animal model construction method of the present invention is not limited by species, can achieve specific regulation of the expression of somatostatin SST in Wistar rats, and also solves the objective problems of high cost, low survival rate, slow gene construction process, and great difficulty in breeding of Cre tool mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0040] Figure 1 AAV9-SST-Cre adeno-associated virus and

[0041] Statistical graph of the average fluorescence intensity of rat brain patches obtained by AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus at different volume ratios, different total injection volumes, or different transfection times;

[0042] Figure 2 AAV9-SST-Cre adeno-associated virus and

[0043] Statistical graph of fluorescence area of ​​rat brain patches obtained by AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus at different volume ratios, different total injection volumes, or different transfection times;

[0044] Figure 3 The immunohistochemical images of rat brain slices were obtained 3 weeks after transfection with AAV9-SST-Cre adeno-associated virus and AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus at a ratio of 1:1 and a total injection volume of 0.5 μL;

[0045] Figure 4 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:1, the total injection volume was 0.5 μL, and the 40X brain slice of the nucleus tractus solitarius of rats was transfected 3 weeks later;

[0046] Figure 5 The ratio of AAV9-SST-Cre adeno-associated virus and AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:1, the total injection volume was 0.5 μL, and the 100X brain slice of the nucleus tractus solitarius of the rat was transfected 3 weeks later;

[0047] Figure 6 The gastric motility diagram of rats at 3 weeks after transfection with AAV9-SST-Cre adeno-associated virus and AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus in a ratio of 1:1 and a total injection volume of 0.5 μL;

[0048] Figure 7 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:1, and the total injection volume was 0.3 μL. The rat brain patch was obtained 2 weeks after transfection.

[0049] Figure 8 for Figure 7 A partial enlarged view of the box in the middle;

[0050] Fig. 9 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:1, and the total injection volume was 0.5 μL. The rat brain patch was obtained 2 weeks after transfection.

[0051] Fig.10 for Fig. 9 A partial enlarged view of the box in the middle;

[0052] Fig.11 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:1, and the total injection volume was 0.3 μL. The rat brain patch was obtained 3 weeks after transfection.

[0053] Fig.12 for Fig.11 A partial enlarged view of the box in the middle;

[0054] Fig.13 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:1, and the total injection volume was 0.5 μL. The rat brain patch was obtained 3 weeks after transfection.

[0055] Fig.14 for Fig.13 A partial enlarged view of the box in the middle;

[0056] Fig.15 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:2, and the total injection volume was 0.3 μL. The rat brain patch was obtained 2 weeks after transfection.

[0057] Fig.16 for Fig.15 A partial enlarged view of the box in the middle;

[0058] Fig.17 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:2, and the total injection volume was 0.5 μL. The rat brain patch was obtained 2 weeks after transfection.

[0059] Fig.18 for Fig.17 A partial enlarged view of the box in the middle;

[0060] Fig.19 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:2, and the total injection volume was 0.3 μL. The rat brain patch was obtained 3 weeks after transfection.

[0061] Fig. 20 for Fig.19 A partial enlarged view of the box in the middle;

[0062] Fig.21 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:2, and the total injection volume was 0.5 μL. The rat brain patch was obtained 3 weeks after transfection.

[0063] Fig. 22 for Fig.21 A partial enlarged view of the box in the middle;

[0064] Fig.23 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:3, and the total injection volume was 0.3 μL. The rat brain patch was obtained 2 weeks after transfection.

[0065] Fig.24 for Fig.23 A partial enlarged view of the box in the middle;

[0066] Fig.25The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:3, and the total injection volume was 0.5 μL. The rat brain patch was obtained 2 weeks after transfection.

[0067] Fig.26 for Fig.25 A partial enlarged view of the box in the middle;

[0068] Fig. 27 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:3, and the total injection volume was 0.3 μL. The rat brain patch was obtained 3 weeks after transfection.

[0069] Fig.28 for Fig. 27 A partial enlarged view of the box in the middle;

[0070] Fig.29 The ratio of AAV9-SST-Cre adeno-associated virus to AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:3, and the total injection volume was 0.5 μL. The rat brain patch was obtained 3 weeks after transfection.

[0071] Fig.30 for Fig.29 A partial enlarged view of the box in the middle;

[0072] Fig.31 This is a rat brain patch image of a rat injected with only 0.5 μL AAV9-SST-Cre adeno-associated virus at 3 weeks after transfection;

[0073] Fig.32 This is a rat brain patch image of a rat injected with only 0.5 μL AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus at 3 weeks after transfection;

[0074] Fig.33 The ratio of AAV9-SST-Cre adeno-associated virus and AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:1, and the total injection volume was 0.5 μL. The projection of somatostatin neurons in the amygdala of rats that were transfected for 3 weeks;

[0075] Fig.34 The ratio of AAV9-SST-Cre adeno-associated virus and AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:1, and the total injection volume was 0.5 μL. The projection of somatostatin neurons in the dorsal thalamic nucleus of rats transfected for 3 weeks;

[0076] Fig.35 The ratio of AAV9-SST-Cre adeno-associated virus and AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was 1:1, with a total injection volume of 0.5 μL, and the fiber connections between the amygdala and dorsal thalamic nucleus of rats transfected for 3 weeks. DETAILED DESCRIPTION

[0077] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0078] Example 1

[0079] A method for constructing an animal model for specifically manipulating somatostatin neurons

[0080] 1. Materials

[0081] AAV9-SST-Cre adeno-associated virus (titer 9.42X10 13 vg / ml) and

[0082] AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus (titer 6.99X10 13 vg / ml) were purchased from Shandong Weizhen Biotechnology Co., Ltd.

[0083] 2. Steps

[0084] 1. Virus mixing ratio and storage method

[0085] AAV9-SST-Cre adeno-associated virus was mixed with AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus in a volume ratio of 1:1, 1:2, and 1:3 and stored at -80 degrees Celsius.

[0086] 2. Grouping and processing

[0087] The experimental animals were randomly divided into two groups: experimental group and control group.

[0088] Set up the experimental group:

[0089]

[0090] In addition, a control group was set up:

[0091]

[0092] 3. Experimental operation

[0093] First, the rats were anesthetized with mixed anesthetics; second, the rat heads were fixed, and after routine disinfection, the rat scalp was cut open to expose the skull. The anterior fontanel was used as a bony landmark, and the injection site was determined with reference to the rat brain stereotaxic brain atlas (1.2mm in front of the anterior fontanel; 5.5mm lateral; 7.0mm below the dura mater). A hole was made with a skull drill, and then a microsyringe was used for injection. After transfection at different times, 1ml of clozapine CNO was injected intraperitoneally for 40 minutes to activate the virus, and the brain tissue was fixed by cardiac perfusion, sliced, and patched to observe the results.

[0094] 3. Beneficial Effects

[0095] First, the experimental results show that Fig.31 As shown, only 0.5 μL of AAV9-SST-Cre adeno-associated virus was injected and no red fluorescence was observed in the brain patch 3 weeks after transfection. Fig.32 As shown in the figure, only 0.5 μL AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus was injected and transfected for 3 weeks, and no red fluorescence was displayed in the brain patch. Therefore, when only AAV9-SST-Cre or AAV9-hSyn-DIO-hM4D(Gi)-mcherry was injected, no red fluorescence was displayed in the brain patch.

[0096] Figure 7-Figure 30 AAV9-SST-Cre adeno-associated virus and

[0097] The rat brain patch images of different ratios of AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus, different total injection volumes, or different transfection times. It can be clearly seen from the figure that when the two viruses are mixed and injected at different ratios and doses, red fluorescence is displayed in the brain patch, but the transfection effect is different. Through software analysis and data processing, it was found that when the two viruses were mixed at a ratio of 1:1, the injection volume was 0.5 microliters, and the transfection was 3 weeks, the average fluorescence intensity of the red fluorescence in the rat brain patch was the strongest, and the transfection area could best cover the INS brain area, such as Figure 1 , 2 shown.

[0098] Secondly, the brain patches with a mixing ratio of 1:1 and an injection volume of 0.5 μl were transfected for 3 weeks to perform immunohistochemistry to verify SST expression. Figure 3 As shown, red is the virus-labeled somatostatin neurons, green is the somatostatin protein shown by immunohistochemistry, and the co-localized yellow can show the overlap of the two. Figure 3 The red neurons in the image are basically covered by green protein and appear yellow, indicating that mixed injection of the two viruses can specifically mark somatostatin neurons.

[0099] Importantly, by using AAV9-SST-Cre and AAV9-hSyn-DIO-hM4D(Gi)-mcherry virus mixed injection, it was found that the projection of somatostatin neurons can be tracked, providing a new tracking tool for neurobiological research. Figure 4 , Figure 5 The nucleus tractus solitarius is shown. Fig.33 , which shows the projection of somatostatin neurons in the amygdala when the mixed virus was injected into the insula of rats; Fig.34 The projection of somatostatin neurons in the dorsal nucleus of the thalamus when the mixed virus was injected into the insula of rats. Fig.35 It is the fiber connection between the amygdala and the dorsal nucleus of the thalamus. Literature indicates that the insula can have fiber connections with the cingulate gyrus, amygdala, hippocampus, somatosensory cortex, basal ganglia, thalamus, hypothalamus, aqueductal gray matter, pons and solitary nucleus, and has extensive intercortical and subcortical input and output projection connections. Fig.33 , Fig.34 , Fig.35 All of them had red fluorescence, which verified the projection brain area of ​​the AAV9-SST-Cre and AAV9-hSyn-DIO-hM4D(Gi)-mcherry mixed viruses, and proved that the projection of somatostatin neurons can be tracked by mixed injection of AAV9-SST-Cre and AAV9-hSyn-DIO-hM4D(Gi)-mcherry viruses.

[0100] In addition, in terms of functional expression, because there are inhibitory functional elements on AAV9-hSyn-DIO-hM4D(Gi)-mcherry, its inhibitory effect was verified in the brain-gastrointestinal axis and it was found that after clozapine cno activated the virus, somatostatin neurons were inhibited, and then gastric motility occurred. After the inhibitory somatostatin neurons were changed, gastric motility was significantly weakened, such as Figure 6 shown.

[0101] In summary, the mixed virus injection method can fully achieve specific labeling and manipulation of somatostatin neurons, and solve many problems existing in Cre tool mice.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing an animal model for specifically manipulating somatostatin neurons, characterized in that: The method comprises injecting a mixed virus containing a Cre-element adeno-associated virus and a DIO-element adeno-associated virus into rats, wherein the volume ratio of the Cre-element adeno-associated virus to the DIO-element adeno-associated virus is 1:1-3, the injection volume is 0.3-0.5 μL, and the transfection lasts for 2-3 weeks; The Cre element adeno-associated virus is AAV9-SST-Cre adeno-associated virus; The DIO element adeno-associated virus is AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus; AAV9-SST-Cre adeno-associated virus is a somatostatin SST promoter sequence packaged together with the Cre element in the AAV9 vector; AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus is a vector that packages the DIO element together with mature neuron hSyn, functional inhibitory element hM4D(Gi), and fluorescent element mcherry on the AAV9 vector; Injection into the INS region of the rat insula; The injection method is nuclear injection.

2. The method for constructing an animal model of specifically operating somatostatin neurons according to claim 1, characterized in that: The titer of AAV9-SST-Cre adeno-associated virus is 9.42X10 13 vg / ml.

3. The method for constructing an animal model of specifically manipulating somatostatin neurons according to claim 1, characterized in that: The titer of the AAV9-hSyn-DIO-hM4D(Gi)-mcherry adeno-associated virus is 6.99X10 13 vg / ml.

4. The method for constructing an animal model of specifically manipulating somatostatin neurons according to claim 1, characterized in that: The volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:1, the injection volume was 0.3 μL, and the transfection lasted for 2 weeks; Alternatively, the volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:1, the injection volume was 0.3 μL, and the transfection lasted for 3 weeks; Alternatively, the volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:1, the injection volume was 0.5 μL, and the transfection lasted for 2 weeks; Alternatively, the volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:1, the injection volume was 0.5 μL, and the transfection was performed for 3 weeks.

5. The method for constructing an animal model of specifically operating somatostatin neurons according to claim 1, characterized in that: The volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:2, the injection volume was 0.3 μL, and the transfection lasted for 2 weeks; Alternatively, the volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:2, the injection volume was 0.3 μL, and the transfection lasted for 3 weeks; Alternatively, the volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:2, the injection volume was 0.5 μL, and the transfection lasted for 2 weeks; Alternatively, the volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:2, the injection volume was 0.5 μL, and the transfection was performed for 3 weeks.

6. The method for constructing an animal model of specifically manipulating somatostatin neurons according to claim 1, characterized in that: The volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:3, the injection volume was 0.3 μL, and the transfection lasted for 2 weeks; Or, the volume ratio of Cre element adeno-associated virus to DIO element adeno-associated virus was 1:3, the injection volume was 0.3 μL, and the transfection lasted for 3 weeks; Alternatively, the volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:3, the injection volume was 0.5 μL, and the transfection lasted for 2 weeks; Alternatively, the volume ratio of Cre-element adeno-associated virus to DIO-element adeno-associated virus was 1:3, the injection volume was 0.5 μL, and the transfection was performed for 3 weeks.

7. The method for constructing an animal model of specifically manipulating somatostatin neurons according to claim 1, characterized in that: After transfection, rats were intraperitoneally injected with clozapine (CNO) to activate the virus.

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