WO2021114255A1

WO2021114255A1 - Method for studying brain-intestine neural circuit and use thereof

Info

Publication number: WO2021114255A1
Application number: PCT/CN2019/125268
Authority: WO
Inventors: 湛琴琴; 李志玲; 詹阳
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2021-06-17

Abstract

A method for studying a brain-intestine neural circuit. The method comprises: injecting a transsynaptic virus carrying a target gene into an intestinal wall of a mammal, and allowing the virus to simultaneously express the target gene in a central nervous system and a peripheral nervous system, so as to study the brain-intestine neural circuit. In the involved method, the transsynaptic virus is selected as a viral vector, and an intestinal wall injection method is specifically implemented on an animal, so as to study the formation of the brain-intestine neural circuit for regulating social behavior; an intestinal neuron is successfully transfected for the first time, and a neuron can be rapidly transfected reversely in a transsynaptic manner, so as to achieve long-distance projection tracing of the neuron.

Description

A method for studying the brain-enteric nerve circuit and its application

Technical field

This application belongs to the technical field of neurology, and specifically relates to a method for studying the brain-enteric nerve circuit and its application.

Background technique

Social communication is an important and complex cognitive behavior. The regulation of social communication ability requires specific brain functional areas to achieve. The synchronicity between different brain regions reflects the way the brain works in coordination under different behaviors and tasks and the participation state of neural circuits. The study of synchronization between different brain areas of the brain has been a hot topic in brain science research in recent years. In normal subjects, brain connection strength and synchronization relationship can reflect the network state between different brain regions under related tasks; in patients with mental illness, the changes in synchronization between brain regions can be used as the biological characteristics of brain diseases. Many mental illnesses related to abnormal social interactions have synchronization changes between brain regions. Studies have shown that there is synchronization between the hippocampus and the prefrontal lobe, and there are functional abnormalities in the circuit connection between the hippocampus and the prefrontal lobe in patients with autism and schizophrenia. Autism is a neurodevelopmental disorder. Its diagnosis is based on the existence of social interaction and lack of communication, as well as restrictive and repetitive behaviors. These patients bring a huge financial burden to the family, but the neuroanatomy and neural circuit regulation mechanisms behind these abnormal behaviors are still unclear.

In addition, in addition to some diseases of the central nervous system, patients with autism are often accompanied by other system complications. Gastrointestinal dysfunction is the most common. Compared with the general population, the prevalence of gastrointestinal diseases (such as acute constipation, diarrhea, and abdominal pain) in autistic patients is significantly increased. Usually autistic children need two times the normal dose of drugs for the treatment of their gastrointestinal diseases. In a more serious case, compared with pure neurogenic autism patients, up to four times the number of patients with intestinal diseases Of autistic people need to be hospitalized. A meta-analysis of the gastrointestinal dysfunction study of 84 patients with autism found that the prevalence of constipation was 22.2%, the prevalence of diarrhea was 13%, and the prevalence of other gastrointestinal symptoms was 46.8%. Other studies have shown that 60-74% of children with autism have experienced enteritis. Studies have shown that gastrointestinal dysfunction is related to the degree of social damage in autism, and there is also a correlation between the degree of social disorder and the degree of constipation. However, the mechanism leading to abnormal intestinal function in autism is not clear.

As the global incidence continues to increase, exploring the pathogenesis of autism has become a major issue that needs to be resolved urgently. The brain-gut axis serves as a pathway that connects the brain and the intestine, making it possible to communicate bidirectional information between the brain and the intestine. With increasing attention to the brain-gut axis, it has also attracted widespread attention as a new target for the treatment of gastrointestinal and mental diseases (such as inflammatory bowel disease, depression and post-traumatic stress disorder). The brain-gut axis includes the brain, spinal cord, autonomic nervous system (sympathetic, parasympathetic, and enteric nervous system), and adrenal axis. There are many signal pathways and a large number of the same neurotransmitters between the "central nervous system" and the "enteric nervous system" of the second brain. The brain-gut interaction not only plays an important role in regulating gastrointestinal functions, such as the digestion and absorption of nutrients, but also affects certain central cognitive functions, such as decision-making or social behavior. A large number of studies have shown that abnormal brain-gut connections and/or regulatory factors that seriously affect the development of the enteric nerve and central nervous system may be important factors leading to gastrointestinal function and behavioral disorders in patients with autism. Research on the brain-enteric nerve circuit Will play a very important role for us to explore the causes of autism.

As the most effective neural circuit research tool at present, viral vectors have been widely used in the central nervous system. However, in the peripheral nervous system, especially the enteric nervous system, because the intestinal mucosa is the body’s largest natural barrier, it can resist Invasion of germs is immunogenic to most viral vectors successfully transfected into the central nervous system, thus limiting the choice of viral vectors. At present, because of its low immunogenicity and low toxicity, recombinant adeno-associated viruses (rAAVs) have been reported to be able to successfully transfect the enteric nervous system to achieve high expression of target genes in the enteric nervous system. However, in view of its inability to infect neurons across synapses, and the time to transfect neurons is generally more than three weeks, it is not conducive to the rapid collection of experimental data, which greatly limits its application in the study of brain-enteric nerve circuits. Secondly, there are many ways to inject the virus. Common injection methods to infect intestinal tissues include oral, enema, intraperitoneal injection, tail vein injection, mesenteric artery injection, etc. The use of different virus injection methods and injection positions will also affect its transfection efficiency and expression time.

Summary of the invention

In view of the shortcomings of the prior art, the purpose of this application is to provide a method for studying the brain-enteric nerve circuit and its application.

In order to achieve the purpose of this application, this application adopts the following technical solutions:

On the one hand, the present application provides a method for studying the brain-enteric nerve circuit, the method comprising: injecting a transsynaptic virus carrying a gene of interest into the intestinal wall of mammals so that it can be in the central nervous system and peripheral The nervous system simultaneously expresses the target gene and uses it to study the brain-enteric nerve circuit.

Because the brain-gut axis involves a large span of neural circuits and needs to span multiple levels of synapses, although viral vectors are used in the study of enteric neural circuits, they cannot meet the research needs of long-range neural projections. The method involved in this application creatively uses the transsynaptic virus as the carrier of the target gene, and by injection into the intestinal wall of mammals, it can effectively transfect neurons and specifically express the target gene in the central nervous system at the same time. In neurons and peripheral neurons, neural projections can be tracked through identifiable target genes to conduct research on the brain-enteric nerve circuit that regulates social behavior. Moreover, the time required to achieve the expression of the target gene in this method is very short, which makes the experimental process relatively short, thereby expanding its scope of application.

Preferably, the target gene includes a gene for marking neurons.

Preferably, the gene for marking neurons includes an enhanced green fluorescent protein gene (eGFP).

Preferably, the transsynaptic virus includes pseudorabies virus (PRV).

The PRV virus used in the method involved in this application can not only reversely infect neurons, but also spread across synapses. At the same time, its expression time is very short compared to rAAVs. After the intestinal wall is injected with PRV, it usually only takes no more than 5 days The time can be projected to the cerebral cortex. Therefore, in addition to realizing the neural projection tracking of the brain-gut axis, the experiment period is greatly shortened, thereby improving the efficiency of the experiment.

Preferably, the pseudorabies virus is PRV-152.

Preferably, the mammals include mice, rats, guinea pigs, pigs, dogs or rabbits.

Preferably, the method for injecting a transsynaptic virus carrying a gene of interest into the intestinal wall of a mammal includes the following steps: anesthetize the mammal and perform an abdominal laparotomy to expose the intestinal tissue; and inject the virus solution into the intestinal wall tissue ; Return the intestines and suture them.

Those skilled in the art know that the use of different virus injection methods, different injection positions, injection doses, etc. will also affect the efficiency of virus transfection, expression time and expression effects. Determining the appropriate injection method, injection site, injection dose, injection rate, virus concentration, and expression time is a very important part of the brain-gut axis research, which is also a problem to be solved in this application.

The method of anesthesia is as follows: anesthetize the animal by injecting sodium pentobarbital into the abdominal cavity of the animal, fix it on the dissection table, shave the hair of the operation site and disinfect with iodophor. The skin on the abdomen needs to be tightened, but not completely tight.

Preferably, the mammal is a mouse, and the laparotomy refers to a distance of 0.8-1.2cm (for example, 0.8cm, 0.9cm, 1.0cm, 1.1cm or 1.2cm, etc.) from the longitudinal line of the genitals on the left side of the mouse's abdomen. ) Is the starting point. Make a vertical incision of 3-4cm (for example, 3cm, 3.2cm, 3.5cm, 3.8cm or 4cm, etc.) from the pelvis to the breastbone.

The injection location is specifically chosen to be the place on the left side of the abdomen of the mouse about one finger away from the longitudinal line of the genitals as the starting point for laparotomy. A vertical incision (about 3-4 cm) is made from the pelvis to the sternum to expose the cecum, following the cecum to the tail. The extension is the colon. The colon of adult mice is about 7-8cm long. Due to structural differences, the middle part of the posterior colon is usually selected as the site of virus injection, and the ink is used as a horizontal mark. The reason is that the muscular layer of the posterior segment of the colon is thicker than the anterior segment, which facilitates injection.

In addition, it should be noted that if there is a formed stool mass in the colon, the injection is usually in the middle of the two stool masses; if the colon is full of stool, a part of the stool mass can be manually separated gently to expose the colon segment for injection.

Preferably, the mammal is a mouse, and the injection site is the middle part of the posterior colon of the mouse.

Preferably, the injection volume of the virus solution is 6-10 μL, such as 6 μL, 6.5 μL, 7 μL, 7.5 μL, 8 μL, 8.5 μL, 9 μL, 9.5 μL, or 10 μL.

Preferably, the concentration of the virus solution is 10 ^5-10 ⁷ pfu/mL, such as 10 ⁵ pfu/mL, 2x10 ⁵ pfu/mL, 5x10 ⁵ pfu/mL, 10 ⁶ pfu/mL, 5x10 ⁶ pfu/mL or 10 ⁷ pfu/mL and so on.

Preferably, the injection rate is 0.5-1.5 μL/min, such as 0.5 μL/min, 0.7 μL/min, 0.8 μL/min, 1.0 μL/min, 1.2 μL/min, 1.4 μL/min, or 1.5 μL/min. min etc.

During the specific injection operation, it is specifically: wipe the microinjection needle with alcohol, then suck the virus solution, gently control the colon injection site with the left index finger and thumb to keep it level; The parallel direction of the muscle is slowly injected at a slight angle, and the injection is held for about 1 minute to prevent backflow.

Note here: Insert the microinjection needle into the intestinal wall, that is, the needle can move freely in the intestinal wall and is clearly visible. Do not insert into the intestinal cavity, and then slowly push the syringe. After the injection, you can see that the surface of the intestine is raised. Form, stay for about 1 min, and then pull out the needle.

Preferably, the suture includes muscle layer suture and epidermal layer suture.

Preferably, the suture of the muscle layer adopts an intermittent suture method.

Preferably, the suture of the epidermal layer adopts a continuous suture method.

The suture operation is divided into two layers, in which the muscle layer adopts the intermittent suture method and the epidermal layer adopts the continuous suture method. Only on the basis of layering can the protection of internal organs and the accuracy of suture be ensured.

Preferably, the expression time is 110-120h (for example, 110h, 115h, 120h, etc.) after the transsynaptic virus carrying the gene of interest is injected into the intestinal wall of the mammal. Because the shorter time the target gene cannot infect the entire neural circuit, and the longer time will cause related lesions and affect the survival of mice, so the best expression effect can be achieved within the above time range.

In order to explore the effect of simultaneous expression of the target gene in the intestine and brain tissue, this application collects the intestine and brain tissues separately, fixes them with paraformaldehyde, strips off the intestinal tissues and/or slices the brain tissues, and then performs a fluorescence microscope Observe the expression intensity of the target gene to detect the transfection of the virus to the tissue. Among them, according to the routine operation (0.01M PBS-4%PFA), the brain tissue was taken out by cardiac perfusion, and the tissue was completely soaked in 4% PFA solution overnight. After 24h, it was soaked in 30% sucrose solution until completely dehydrated (usually It takes at least 3 days), then you can use a cryostat to slice, and then observe the expression of the target gene under a fluorescence microscope after coverslipping.

On the other hand, this application provides an application of the method for studying the brain-enteric nerve circuit as described above in studying the brain-enteric nerve circuit.

In another aspect, the present application provides an application of the method for studying the brain-enteric nerve circuit as described above in the study of social behavior.

Compared with the prior art, this application has the following beneficial effects:

The method involved in this application selects the transsynaptic virus as the viral vector, and specifically uses the intestinal wall injection method to study the formation of the brain-enteric nerve circuit that regulates social behavior, and successfully transfects enteric neurons for the first time. It can quickly reversely transfect neurons (including enteric neurons) across synapses to achieve long-range neuron projection tracking.

Description of the drawings

Figure 1 shows the expression of eGFP in the myenteric nerve plexus after injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 ⁶ pfu/ml) into the posterior segment of the colon of C57BL mice for 120 hours; the arrow indicates the positive expression of eGFP Neuron; scale bar: 50μm.

Figure 2 shows that after 120 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 ⁶ pfu/ml) into the posterior segment of the colon of C57BL mice, eGFP was found in the paraventricular nucleus (PVN) and central amygdala (CeA) of the hypothalamus. ); the box is a partial enlarged view of PVN and CeA; scale bar: left 100μm and right 50μm.

Figure 3 shows the C57BL mice injected with colon segment carrying the green fluorescent protein of pseudorabies virus (PRV152-eGFP, 8μl, 1x10 7 pfu / ml) after 120h, eGFP expression in the myenteric plexus; scale: 100μm.

Figure 4 shows the expression of eGFP in the paraventricular nucleus (PVN) of the hypothalamus after injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 ^{7 pfu/ml) into the posterior segment of the colon of C57BL mice for 120 hours; the box is} Partial enlarged view of PVN; scale bar: 100μm.

Figure 5 shows the expression of eGFP in the myenteric nerve plexus after 96 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 ^{6 pfu/ml) into the posterior segment of the colon of C57BL mice; the arrow indicates the positive expression of eGFP} Neuron; scale bar: 100μm.

Figure 6 shows the expression of eGFP in the paraventricular nucleus (PVN) of the hypothalamus after 96 hours after injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 ^{6 pfu/ml) into the posterior segment of the colon of C57BL mice; the box is} Partial enlarged view of PVN; scale bar: 100μm.

Detailed ways

The technical solutions of the present application will be further explained through specific implementations below. It should be understood by those skilled in the art that the described embodiments are merely to help understand the application and should not be regarded as specific limitations to the application.

Example 1

This embodiment provides a method for studying the brain-enteric nerve circuit, and the method includes the following steps:

(1) Inject the mouse (model C57BL-6J) intraperitoneally with 1% sodium pentobarbital (dose 35mg/kg) for anesthesia, fix it on the dissection table, shave the hair of the surgical site and disinfect with iodophor;

(2) The left side of the mouse's abdomen is 1cm away from the longitudinal line of the genitals as the starting point for laparotomy. A 4cm vertical incision is made from the pelvis to the sternum to expose the cecum, extending along the cecum to the tail end is the colon. Choose the middle position of the posterior part of the colon. As the site of virus injection, and mark horizontally with ink;

(3) Wipe the microinjection needle with alcohol, then suck the pseudorabies virus solution (PRV152-eGFP) carrying the eGFP gene, and gently control the colon injection site with the left index finger and thumb to keep it level; the right hand holds the needle at the far end of the mark ^{Inject 8μL of virus solution (concentration 1x10 6} pfu/mL, rate of 1μL/min) at a slight angle in a direction parallel to the longitudinal muscle, and stay for 1 minute after injection to prevent backflow;

(4) Return the intestines, close the abdominal cavity, suture the abdominal wall and skin, and divide the suture into two layers, the muscle layer is sutured intermittently, and the epidermis layer is sutured continuously; wait for the eGFP gene to be expressed.

Example 2

(2) The left side of the mouse's abdomen is 1cm away from the longitudinal line of the genitals as the starting point for laparotomy. A 3cm vertical incision is made from the pelvis to the sternum to expose the cecum, extending along the cecum to the tail end is the colon. Choose the middle of the posterior part of the colon. As the site of virus injection, and mark horizontally with ink;

(3) Wipe the microinjection needle with alcohol, then suck the pseudorabies virus solution (PRV152-eGFP) carrying the eGFP gene, and gently control the colon injection site with the left index finger and thumb to keep it level; the right hand holds the needle at the far end of the mark It was imposed and parallel to the longitudinal muscle slight angle injectable solution 8μL virus (concentration of 1x10 ⁷ pfu / mL, the rate of 0.5μL / min), after completion of injection dwell 1min, to prevent reflux;

Evaluation test:

At 120h after the completion of the injection, the intestines and brain tissues of the mice in Examples 1 and 2 were collected at the same time, and the cover slips were processed to observe the expression of eGFP under a fluorescence microscope. The specific operation method is: open the mouse Abdominal cavity, exposing the intestines, quickly cut off the colon and small intestine, soak in the Krebs solution containing oxygen, and process the intestines in time. Place the intestine in a silicone rubber dish filled with 1×PBS solution, and continue to infuse oxygen. After opening the intestinal wall along the middle seam under the microscope, use a steel needle to fix it smoothly along its edge. Use a disposable pipette to suck the PBS solution to wash the intestinal contents, and peel off the mucosa and submucosal nerve layer of the small intestine with fine forceps under the microscope (for the colon, the striated muscle needs to be stripped again). After the stripping is over, rinse with PBS solution twice to wash away excess tissue fragments. Then it was immersed in 4% PFA solution and fixed for 40 minutes. After the fixation, rinse with PBS solution repeatedly to wash off excess PFA solution. Cut the small intestine section by section with a scalpel, take a small piece of tissue, spread it evenly on a glass slide under a microscope, drop an appropriate amount of mounting tablets, and cover it.

In the same way, after removing the intestinal tissue, the mouse was quickly perfused with the heart, the blood in the mouse was washed with 1×PBS solution, and the brain tissue was fixed with 4% PFA solution. After the perfusion is completed, carefully remove the intact brain tissue and preserve the brainstem, and soak in 4% PFA solution for 24 hours. Then, it was completely dehydrated with 30% sucrose solution and then embedded, and the whole brain was frozen sectioned on the embedded brain. After patching the sliced brain slices in order, add an appropriate amount of mounting tablets to cover the slices.

Observe the processed intestinal tissues and brain slices under a fluorescence microscope, select the excitation light that can excite EGFP, and the green fluorescent part observed under the microscope is the neuron marked by the virus infection in the brain-gut loop cell.

The results of Example 1 are shown in Figures 1 and 2 (Figure 1 shows that after ^{120 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 6} pfu/ml) into the posterior segment of the colon of C57BL mice, eGFP In the myenteric plexus (MP) expression, the arrow in the figure points to the neurons expressing positive eGFP; Figure 2 shows the injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 ⁶ pfu/ml) 120h later, the expression of eGFP in the paraventricular nucleus (PVN) and central amygdala (CeA) of the hypothalamus).

The results of Example 2 are shown in Figures 3 and 4 (Figure 3 shows that after ^{120 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 7} pfu/ml) into the posterior segment of the colon of C57BL mice, eGFP Expression in the myenteric plexus (MP); Figure 4 shows that after 120 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 ⁷ pfu/ml) into the posterior segment of the colon of C57BL mice, eGFP is in the hypothalamic ventricle Paranuclear (PVN) expression).

As can be seen from the above figure, the method involved in the present application can successfully transfect enteric neurons, and it can quickly reverse transfect neurons across synapses to achieve long-range neuron projection tracking.

Example 3

In order to explore the influence of waiting time for material collection on the transfection effect, the mice in Example 1 were collected 96h, 120h, and 148h after the injection was completed, and the intestines and brain tissues of the mice were collected according to the above operation in the fluorescence microscope. Observe the expression of eGFP.

The results are shown in Figure 1, Figure 2, Figure 5 and Figure 6 (Figure 5 shows the injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8μl, 1x10 ⁶ pfu/ml) into the posterior segment of the colon of C57BL mice After 96h, the expression of eGFP in the myenteric nerve plexus (MP). The arrow in the figure indicates the neuron with positive expression of eGFP; Figure 6 shows the injection of pseudorabies virus carrying green fluorescent protein (PRV152-) into the posterior part of the colon of C57BL mice. eGFP, 8μl, 1x10 ⁶ pfu/ml) 96h later, the expression of eGFP in the paraventricular nucleus (PVN) of the hypothalamus). After 148h, the mice could not survive and could not collect tissues.

It can be seen from the comparison of Figure 2 and Figure 6 that the pseudorabies virus (PRV152-eGFP) carrying green fluorescent protein (PRV152-eGFP) can be expressed in the intestinal tissue and brain tissue at the same time after 96h or 120h injected into the posterior segment of the mouse colon. The post-colon injection of pseudorabies virus (PRV152-eGFP) carrying green fluorescent protein (PRV152-eGFP) 120h after eGFP expression in the hypothalamic paraventricular nucleus (PVN) is stronger than 96h, which can obtain a good effect of observing and distinguishing brain regions.

The applicant declares that this application uses the above-mentioned examples to illustrate a method of this application for studying the brain-enteric nerve circuit and its application, but this application is not limited to the above-mentioned examples, which does not mean that this application must It can be implemented only by relying on the above-mentioned embodiments. Those skilled in the art should understand that any improvement to this application, the equivalent replacement of each raw material of the product of this application, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

The preferred embodiments of the present application are described in detail above. However, the present application is not limited to the specific details in the foregoing embodiments. Within the scope of the technical concept of the present application, a variety of simple modifications can be made to the technical solution of the present application. These simple modifications All belong to the protection scope of this application.

In addition, it should be noted that the various specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application provides various possible combinations. The combination method will not be explained separately.

Claims

A method for studying the brain-enteric nerve circuit, which includes: injecting a transsynaptic virus carrying a target gene into the intestinal wall of a mammal so that it can express the target gene in the central nervous system and the peripheral nervous system at the same time, and To study the brain-enteric nerve circuit.
The method for studying the brain-enteric nerve circuit according to claim 1, wherein the target gene includes a gene for marking neurons.
The method for studying the brain-enteric nerve circuit of claim 2, wherein the gene for labeling neurons includes an enhanced green fluorescent protein gene.
The method for studying the brain-enteric nerve circuit according to any one of claims 1 to 3, wherein the transsynaptic virus includes pseudorabies virus.
The method for studying the brain-enteric nerve circuit of claim 4, wherein the pseudorabies virus is PRV152.
The method for studying the brain-enteric nerve circuit according to any one of claims 1 to 5, wherein the mammals include mice, rats, guinea pigs, pigs, dogs, or rabbits.
The method for studying the brain-enteric nerve circuit according to any one of claims 1-6, wherein the method of injecting a transsynaptic virus carrying a gene of interest into the intestinal wall of a mammal comprises the following steps : Open the abdomen after anesthetizing the mammal to expose the intestinal tissue; inject the virus solution into the intestinal wall tissue; return the intestine to its position and suture it.
The method for studying the brain-enteric nerve circuit according to claim 7, wherein the mammal is a mouse, and the laparotomy refers to a distance of 0.8-1.2 to the longitudinal line of the genitals from the left side of the mouse’s abdomen. The starting point is at cm, a vertical incision of 3-4 cm is made from the pelvis to the sternum.
The method for studying the brain-enteric nerve circuit according to claim 7 or 8, wherein the mammal is a mouse, and the injection site is the middle of the posterior colon of the mouse.
The method for studying the brain-enteric nerve circuit according to claim 9, wherein the injection volume of the virus solution is 6-10 μL;

Preferably, the concentration of the virus solution is 10 5 -10 7 pfu/mL;

Preferably, the injection rate is 0.5-1.5 μL/min.
The method for studying the brain-enteric nerve circuit according to any one of claims 7-10, wherein the suture includes muscle layer suture and epidermal layer suture.
The method for studying the brain-enteric nerve circuit according to claim 11, wherein the suture of the muscle layer adopts an intermittent suture method;

Preferably, the suture of the epidermal layer adopts a continuous suture method.
The method for studying the brain-enteric nerve circuit according to any one of claims 1-12, wherein the time of expression is a distance from injecting a transsynaptic virus carrying a gene of interest into the intestinal wall of a mammal After 110-120h.
The application of the method for studying the brain-enteric nerve circuit of any one of claims 1-13 in the study of the brain-enteric nerve circuit.
The application of the method for studying the brain-enteric nerve circuit according to any one of claims 1-13 in the study of social behavior.