PET imaging system construction method for in-vivo visual forward neural circuit connection

Abstract

The invention discloses a PET (positron emission tomography) imaging system construction method for in-vivo visual forward neural circuit connection. The method comprises the following steps: constructing a neuron-specific promoter hSyn to drive a tool virus vector expressed by a mammal codon optimized wheat germ agglutinin (mWGA)-tyrosinase (TYR) reporter gene, injecting the virus vector into a target brain region, and transferring the TYR to a downstream brain region connected with the target brain region by utilizing the characteristic of mWGA cross-single synapse smooth tracing, the TYR can catalyze tyrosine to generate multi-step reaction in cells to synthesize melanin, and the nuclide labeled probe targeting melanin can realize in-vivo visualization of a brain region connected with a neural circuit formed by neurons at an injection site. The construction of the system provides a powerful research tool for loop connection between neurons and brain function research in vivo.

Description

Technical Field

[0001] The present invention belongs to the field of nuclear medicine molecular imaging, and more particularly, relates to a method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections. Background Art

[0002] The efficient execution of brain functions highly depends on the structure and plasticity of neural circuits. As the basic unit for information processing, a neural circuit is a dynamic network formed by neurons in specific brain regions through synaptic connections, and its connection pattern and activity synchronization directly determine the realization of higher functions such as perception, movement, cognition, and emotion. For example, the synaptic plasticity of the prefrontal cortex - amygdala circuit regulates emotional memory encoding, while the pulse synchronization of the thalamus - cortex circuit supports the integration of sensory information, etc. When the circuit structure undergoes abnormal remodeling due to diseases (such as the degeneration of the hippocampal - entorhinal cortex connection in Alzheimer's disease) or the activity rhythm is disordered (such as the oscillation disorder of the basal ganglia circuit in Parkinson's disease), specific brain dysfunctions will occur. Revealing the operation rules of neural circuits is the core breakthrough for analyzing the mechanism of brain functions and diagnosing and treating neuropsychiatric diseases.

[0003] However, the complexity of the brain's structure and function makes traditional tissue biopsy methods difficult to apply to the brain. Protected by the skull, it is difficult to perform a biopsy on the brain. Moreover, many brain functions can only be fully demonstrated in the living state. Coupled with the extremely delicate structure of the brain, any intervention on it may bring irreversible effects, which further increases the challenges of research and diagnosis. Therefore, although the development of neurobiological technologies has greatly promoted the research of brain science, the previous nerve circuit tracing technologies based on neurotropic viruses can only observe the connection state at a certain time point in the ex vivo state and cannot achieve in vivo dynamic detection of the connection state. Therefore, more complex functional research must be supported by in vivo imaging means.

[0004] Positron Emission Tomography (PET) is a molecular imaging technology that can spatiotemporally visualize and quantify in vivo biological processes at the cellular and molecular levels. PET is currently the most mature molecular imaging technology, especially showing incomparable advantages in functional research. Neural activity depends on glucose metabolism, and the most commonly used PET molecular probe 18 fluorine - fluorodeoxyglucose ( 18 F - FDG) indirectly reflects neural activity by detecting glucose metabolism. By using the property that the increase or decrease in neural activity in a specific brain region will synchronously increase or decrease the uptake of 18F - FDG in itself and downstream brain regions, it helps to evaluate the whole - brain metabolic network pattern and potential brain region connections. However, it is difficult for imaging methods to achieve the connection evaluation of specific neural circuits (such as 18The changes in F-FDG PET signals are also affected by various external factors and cannot be used to distinguish the upstream-downstream relationships between different brain regions (e.g., changes in downstream brain regions can also lead to compensatory changes in upstream brain regions). PET reporter gene imaging technology transfects an exogenous reporter gene (encoding a specific enzyme, transporter, or receptor) together with the target gene into target cells, enabling their simultaneous expression in the cells. Subsequently, a radioactive probe is used to specifically recognize and bind to the reporter gene product, thereby achieving the visualization and evaluation of the target gene. However, due to limitations such as the blood-brain barrier and natural expression in the brain, for example, the most widely used HSV1-tk reporter gene currently lacks a PET probe that can penetrate the blood-brain barrier, and the dopamine type 2 receptor reporter gene is highly expressed in the basal ganglia region. There is still a lack of PET reporter gene methods that can enter the nervous system. Tyrosinase (TYR) is a rate-limiting enzyme in the process of melanin synthesis. In previous studies, TYR has been widely used as a reporter gene in multiple fields such as stem cell transplantation tracing after myocardial infarction and tumor treatment monitoring. Moreover, melanin is naturally expressed at a low level in the nervous system, having unique advantages for intracerebral tracing. In vivo visualization of neural circuits is of great significance for studying the functional connections between brain regions and the pathogenic mechanisms of diseases.

[0005] Wheat Germ Agglutinin (WGA) is a lectin protein extracted from wheat germ. Its ability to bind to glycans makes it an ideal cell labeling tool. WGA can be absorbed by neurons / synaptic terminals, transported anterogradely / retrogradely along axons, and transmitted to downstream / upstream neurons to achieve anterograde / retrograde tracing. WGA can be combined with neurotropic viruses / horseradish peroxidase / fluorescent proteins, etc. to achieve neuron tracing and the study of neural circuits. In this invention, mammalian codon-optimized wheat germ agglutinin (mWGA) is used, which has the property of trans-synaptic anterograde tracing and can specifically study the neural circuit connections in the downstream brain regions of the injection area. In this invention, the combination of mWGA and the PET reporter gene can achieve in vivo visualization of neuron tracing and neural circuits. Summary of the Invention

[0006] The object of the present invention is to propose a method for constructing a PET imaging system for in vivo visualization of forward neural circuit connections in view of the deficiencies of the prior art.

[0007] The present invention is implemented by the following technical solutions: A method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections, comprising: constructing a tool viral vector expressing a tyrosinase (TYR) reporter gene; using the tool viral vector to transfect neurons in the target brain region, and neurons in the target brain region and the neurons connected thereto express the TYR reporter gene. The TYR reporter gene catalyzes tyrosine to become melanin through multiple steps in cells; thus, the neural circuit in the downstream brain region of the injection site can be visualized in vivo by detecting melanin through PET molecular imaging.

[0008] As an embodiment, the tool viral vector is an adenovirus or an adeno-associated virus.

[0009] As an embodiment, the expression of the tyrosinase reporter gene in cells is driven by a conditional expression promoter of the neural circuit, and the conditional expression promoter is hSyn.

[0010] As an embodiment, the gene sequence of the tyrosinase is as shown in SEQ ID NO.1.

[0011] As an embodiment, the gene sequence of the mammalian codon-optimized wheat germ agglutinin is as shown in SEQ ID NO.2.

[0012] As an embodiment, the amino acid sequence of the tyrosinase is as shown in SEQ ID NO.3.

[0013] As an embodiment, the amino acid sequence of the mammalian codon-optimized wheat germ agglutinin is as shown in SEQ ID NO.4.

[0014] As an embodiment, the tool viral vector expresses a fluorescent protein gene, including but not limited to GFP, RFP, BFP, EGFP, mCherry, mStrawberry, mApple, mRuby or EosFP.

[0015] As an embodiment, it is driven alone by a constitutive promoter, and the constitutive promoter includes but not limited to CMV promoter, EF1a promoter, EFS promoter, CAG promoter, Cbh promoter, SFFV promoter; SV40 promoter, Ubc promoter, Ubi promoter, hPGK promoter, β-actin promoter.

[0016] As an embodiment, the PET molecular imaging probe is 18 F-FPABZA.

[0017] As an embodiment, the method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections can construct and obtain a tracer.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention provides a method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections. A neural circuit tracing viral vector with a neuron-specific promoter hSyn driving the mWGA-tyrosinase TYR reporter gene is constructed; the constructed viral vector is injected into a local brain region to make the infected neurons express the mWGA-TYR reporter gene; then, by utilizing the characteristic of mWGA trans-synaptic transmission, TYR is transmitted to the downstream brain regions connected to the neurons in the injection region; the TYR reporter gene catalyzes tyrosine to become melanin through multiple steps in cells; a radionuclide-labeled probe targeting melanin can achieve in vivo visualization of the brain regions forming neural circuit connections with the neurons at the injection site; the present invention detects melanin through PET molecular imaging, visualizes neural circuits in vivo, can evaluate the brain regions expressing the TYR reporter gene in the brain, thereby realizing in vivo visualization of the brain regions forming anterograde neural circuit connections with the injection brain region, and constructs a powerful research tool for in vivo research on the circuit connections between neurons and brain function research.

[0020] 2. The tracing method provided by the present invention, by virtue of the continuous expression and continuous transmission characteristics of mWGA, is a potential method for realizing the dynamic changes of neural circuit remodeling (such as the recovery of ischemic stroke).

[0021] 3. The tracer provided by the present invention can directly achieve in vivo visualization of neural circuits in the brain by binding to the PET molecular imaging probe 18 F-FPABZA. The probe has good blood-brain barrier penetration ability and does not require additional physical or chemical operations (such as ultrasound microbubbles, small molecule drugs) to open the blood-brain barrier.

[0022] 4. The tracing method provided by the present invention is not limited by the imaging depth and does not require invasive imaging by inserting optical fibers or lenses into the brain additionally. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is a schematic diagram of the imaging result of the present invention; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention and should not be construed as a limitation to the present invention. Those of ordinary skill in the art can understand that: various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. For the experimental methods without specific conditions noted in the following embodiments, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0026] In the present invention, a tool virus vector transfection is used to enable neurons in the target brain region to express TYR (catalyzing the synthesis of melanin), and by utilizing the characteristics of the cross-synaptic transmission of mWGA, TYR is transmitted to the neurons that establish neural circuit connections with the target brain region and catalyzes the production of melanin. By 18 binding F-FPABZAPET to melanin, the in vivo visualization of the neural circuit connected to the target brain region can be achieved. The specific operation examples are as follows:

[0027] 1. Production and packaging of adeno-associated virus particles

[0028] In the present invention, first, the hSyn-mWGA-TYR-P2A-EGFP reporter gene is cloned into the tool virus vector plasmid, and then the recombinant plasmid is co-transfected into HEK-293T cells (providing the trans-acting factors required for AAV replication and packaging) together with pHelper (carrying genes from adenovirus) and pAAV-RC (carrying AAV replication and capsid genes). The infected HEK-293T cells are collected and lysed to obtain AAV virus particles. Finally, high-titer AAV virus particles are obtained through concentration and purification steps.

[0029] 2. In vivo transfection of neurons with adeno-associated virus

[0030] In the present invention, adeno-associated virus is injected into the mouse sensorimotor cortex in situ to transfect neurons, and the in vivo visualization effect of the neural circuit is evaluated 3 weeks after transfection.

[0031] 3. 18 F-FPABZAPET imaging

[0032] In the present invention, 18 F-FPABZA is injected into the mouse tail vein. After injection, the mouse is placed back in the cage to move freely. After 60 minutes, the mouse is anesthetized and 18 a static F-FPABZA PET scan is performed for 10 minutes.

[0033] 4. Ex vivo immunofluorescence staining of mouse brain slices

[0034] In the present invention, the brain is taken by perfusing the heart of a mouse, and after cryosectioning, immunofluorescence staining is performed to co-localize and evaluate wheat germ agglutinin WGA, tyrosinase TYR, and green fluorescent protein GFP.

[0035] As Figure 1 shown in the implementation results of the present invention, 3 weeks after rAAV-hSyn-mWGA-TYR-P2A-EGFP infects the sensorimotor cortex of C57 mice, 18 F-FPABZA PET signals can be seen in the in situ brain region (upper figure) and remote brain region (lower figure) of the virus injection site. Figure 1 The upper figure in Figure 1 shows the in situ brain region of the injection. GFP is a punctate signal in the cell body. The triple-positive cells of GFP, WGA, and TYR are virus-infected neurons. The GFP nerve fiber-like signal in the subcortex indicates the neurites of virus-infected neurons. The double-positive cells of WGA and TYR and negative for GFP are adjacent neurons that establish connections with virus-infected neurons. 18 F-FPABZA PET targets the TYR product melanin, thereby achieving in vivo visualization of the brain regions that establish neural circuit connections with the in situ brain region of the injection.

[0036] Details are as follows in each of the following examples:

[0037] Example 1: Construction of an adeno-associated virus vector plasmid overexpressing a tyrosinase reporter gene

[0038] 1. Synthesized genes: The mWGA-TYR reporter gene is synthesized by a DNA synthesizer. Among them, the gene sequence of tyrosinase is shown in SEQ ID NO.1, the gene sequence of mammalian codon-optimized wheat germ agglutinin is shown in SEQ ID NO.2, the amino acid sequence of tyrosinase is shown in SEQ ID NO.3, and the amino acid sequence of mammalian codon-optimized wheat germ agglutinin is shown in SEQ ID NO.4.

[0039] 2. Amplification of the reporter gene: The reporter gene is amplified in large quantities by polymerase chain reaction (PCR). The primers at the 5' and 3' ends contain KpnI and BamHI restriction endonuclease sequences respectively, and the product is purified using a gel recovery kit.

[0040] 3. The PCR reaction system is as follows

[0041]

[0042] 4. The PCR reaction program is as follows:

[0043]

[0044] 5. Insert the gene fragments digested with BamHI and KpnI into the multiple cloning site (MCS) of the pre-prepared vector, and ligate them using DNA ligase. The vector sequence is hSyn-mWGA-TYR-P2A-EGFP.

[0045] 6. Screen the correct recombinant plasmids by PCR and restriction digestion methods. After sequencing and identifying that the vector construction is correct, start to amplify the recombinant plasmids (hSyn-mWGA-TYR-P2A-EGFP) in large quantities.

[0046] 7. Preparation of HEK-293T cells: Inoculate 4 million HEK-293T cells in a 10-cm culture dish, culture overnight to restore the cell state, and change to fresh complete medium before transfection.

[0047] 8. Transfection of HEK-293T: Add three plasmids (7.5 μg of PxpaX2 plasmid, 2.5 μg of PMD2g plasmid, and 10 μg of TYR recombinant plasmid) to DMEM serum-free and antibiotic-free medium with a total volume of 500 μL according to the ratio, denoted as solution A, and pipette and mix well; Take 50 - 60 μg (1 μg / mL) of transfection reagent PEI and add it to 500 μL of DMEM serum-free and antibiotic-free medium, denoted as solution B, and pipette and mix well; Drop solution B into solution A drop by drop, gently mix with a pipette gun, and let it stand at room temperature for about 15 minutes; Slowly drop the standing transfection reagent (A + B) into the HEK 293T cell culture dish; At 18 h after transfection, replace the supernatant of HEK 293T containing the transfection reagent with 10 mL of DMEM complete medium.

[0048] 9. Collection and concentration of virus particles: Adeno-associated virus samples exist in both cells and culture supernatants. To obtain a better yield, both cells and culture supernatants can be collected. Collect the cells together with the medium, use an AvantiJ-15R centrifuge, centrifuge at 200×g for 3 minutes, collect the culture supernatant and cell pellet respectively, store the supernatant separately, and resuspend the cells with PBS; Lyse the cells by repeated freezing and thawing or sonication and then centrifuge, use an Avanti JXN-26 high-speed centrifuge, centrifuge at 10,000×g to remove cell debris, and collect the cell lysate supernatant. Precipitate the virus in the supernatant with PEG 8000 and filter it with a 0.45-μm filter to obtain a filtrate containing virus particles for subsequent purification.

[0049] 10. Purification of adeno-associated virus: Prepare iodixanol solutions with mass-volume ratios of 15%, 25%, 40%, and 60% respectively, and then spread the freshly prepared solutions of different gradients into ultracentrifuge tubes; (fresh preparation is required each time, and two gradient solutions are spread simultaneously) Add the collected supernatant containing adeno-associated virus particles into the ultracentrifuge tubes respectively, seal them after filling; Centrifuge at 69,000 rpm for 1 h; After centrifugation, the full particle adeno-associated virus sample is between 40% - 60%. Puncture and sample carefully to take out the sample. Note that when taking out, the white band above the 40% gradient solution is empty shell samples, proteins and other impurities. To ensure the separation effect, do not aspirate this part of the solution. Ultrafilter or dialyze the purified virus sample, and the adeno-associated virus particles can be obtained after desalting treatment. To obtain a high-purity sample, a second iodixanol density gradient centrifugation can be performed to further remove adeno-associated virus empty particles and cell debris. During the second centrifugation, 30%, 40%, and 60% iodixanol solutions can be used for separation.

[0050] Example 2: Intracortical injection of adeno-associated virus into the sensorimotor cortex of normal C57 mice

[0051] C57 mice about 2 months old were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital with an injection dose of 50 mg / kg. The mice were fixed on a stereotaxic apparatus, leveled the anterior and posterior fontanelles, drilled a small hole in the skull with a cranial drill, and injected 200 nL of adeno-associated virus using a microinjector pump. The coordinates were 3 mm to the right of the midline, 0 mm in front of the anterior fontanelle, and the depth was 2.5 mm, with a speed of 50 nL / min. After injection, let it stand for 10 minutes and then slowly pull out the injection needle. Suture the skin of the mice, disinfect, and intraperitoneally inject 200,000 units of penicillin for 3 days to prevent infection.

[0052] Example 3: Tracer injection 18 Synthesis of F-FPABZA

[0053] 1. After the fluorine source of fluorinated heavy water is adsorbed on the anion adsorption column (QMA column), elute the QMA column with 0.8 mL of eluent (15 mg K222, 3 mg potassium carbonate, 0.64 mL acetonitrile, 0.16 mL sterile water) and enter the reaction flask. Evaporate the water in the reaction flask liquid at 110 °C, and add 1 mL of anhydrous acetonitrile to azeotropically evaporate and repeat three times during this period.

[0054] 2. After complete drying, add 1 mL of DMF solution dissolved with 7.5 mg of the precursor BrPABZA, and react in a sealed manner at 140 °C for 30 min. Dilute the reaction solution and then enter the HPLC chromatographic column for purification and separation. Use a mobile phase mixture of acetonitrile and 0.5 / 1000 trifluoroacetic acid aqueous solution with a volume ratio of 3:7 to wash the chromatographic column. The product peak appears at about 16 min and is collected.

[0055] 3. Dilute the product peak solution with 30 mL of pure water and then pass it through a C18 separation column. Adsorb the product on the C18 column, and then wash the C18 column with 10 mL of pure water. Elute the product on the C18 column with 1 mL of ethanol, dilute it with 10 mL of physiological saline containing 10 mg / mL ascorbic acid, and finally filter it through a 0.22-μm sterile filter membrane to obtain the final 18 F-FPABZA injection solution.

[0056] Example 4: 18 F-FPABZA PET scan

[0057] 1. 18 F-FPABZA PET scan: Four weeks after the injection of adeno-associated virus, the mice were induced to anesthesia with 4% isoflurane by volume. Approximately 18.5 MBq (500 μCi) 18 of F-FPABZA was injected into the tail vein, and then the mice were put back into the cage to move freely. After 60 minutes, the mice were induced to anesthesia again with 4% isoflurane by volume, fixed in the prone position on the small animal PET scan window for static scanning. The scanning time was 10 minutes, and 2% isoflurane by volume was given to maintain anesthesia during the scanning.

[0058] 2. 18 F-FPABZA PET reconstruction: The initial images obtained by scanning were reconstructed using the ordered subsets expectation maximization method (OSEM), and the reconstructed images were processed using Pmod software.

[0059] Example 5: In vitro immunofluorescence staining of mouse brain slices

[0060] 1. The mice were deeply anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital in mass-to-volume ratio. The heart field was exposed, and a perfusion needle was inserted into the apex of the mouse heart. The needle was placed along the left ventricular outflow tract to the starting point of the aortic arch, and 30 mL of normal saline was rapidly perfused, and then approximately 20 mL of 4% paraformaldehyde in mass-to-volume ratio was slowly perfused until the whole body of the mouse became stiff. The complete brain tissue was carefully removed. The removed mouse brain was placed in a 4% paraformaldehyde solution and externally fixed overnight in a 4°C refrigerator, and then replaced with a 30% sucrose solution in mass-to-volume ratio for dehydration. After the mouse brain was dehydrated, tissue embedding and frozen sectioning were performed.

[0061] 2. Select brain slices and place them in a 24-well plate containing 0.5 mL of PBS. Shake rapidly on a shaker for 5 min and repeat three times. Add 0.2 mL of rapid blocking solution to the 24-well plate and slowly shake on a shaker at room temperature for 1 h for blocking. Wash three times with 0.5 mL of PBS. Prepare the primary antibody at an appropriate ratio according to the antibody instruction manual and incubate overnight with slow shaking on a shaker in a 4°C refrigerator. Recover the primary antibody and add 0.5 mL of PBS to each well and wash repeatedly 3 times. Prepare the secondary antibody at an appropriate ratio according to the primary antibody species and the secondary antibody instruction manual and add it. Incubate with slow shaking at room temperature for 1 h. Recover the secondary antibody and add 0.5 mL of PBS to each well and wash 3 times. Add DAPI staining solution and incubate at room temperature for 10 min. Add 0.5 mL of PBS to each well and wash 3 times. Flatten and mount the brain slices in a Petri dish containing PBS. Drop anti-quenching mounting medium on the brain slices, mount the slices and observe.

[0062] The above embodiments are used to explain and illustrate the present invention, rather than limiting the present invention. Any modifications and changes made within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections, characterized in that: The method comprises the following steps: constructing a tool virus vector expressing a mammalian codon-optimized wheat lectin mWGA-tyrosinase TYR reporter gene, wherein the tool virus vector carries a neuron-specific promoter; injecting the virus vector into a target brain region, utilizing the property of mWGA to propagate forward across a single synapse to deliver TYR to a brain region downstream of the target brain region, wherein TYR catalyzes melanin synthesis, and realizing in vivo visualization of a brain region connected to a neural circuit formed with the injected brain region through a PET probe targeting melanin.

2. The method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections according to claim 1, characterized in that: The tool virus vector is adenovirus or adeno-associated virus.

3. The method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections according to claim 2, characterized in that: The expression of the tyrosinase reporter gene in cells is driven by a conditional expression promoter of a neural circuit, and the conditional expression promoter is a neuron-specific promoter hSyn.

4. The method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections according to claim 1, characterized in that: The gene sequence of the tool virus vector expressing the TYR protein includes the sequence shown in SEQ ID NO.1, and the gene sequence of the tool virus vector expressing the mWGA protein includes the sequence shown in SEQ ID NO.

2.

5. The method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections according to claim 1, characterized in that: The tool virus vector also expresses a fluorescent protein gene, and the fluorescent protein gene is selected from GFP, RFP, BFP, EGFP, mCherry, mStrawberry, mApple, mRuby or EosFP.

6. The method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections according to claim 1, characterized in that: The radionuclide labeled probe targeting melanin is a PET probe 18 Fluoro-pyridineamide-benzamide 18 F-FPABZA.

7. The method for constructing a PET imaging system for in vivo visualization of anterograde neural circuit connections according to claim 1, characterized in that: It is driven solely by a constitutive promoter, which includes but is not limited to CMV promoter, EF1a promoter, EFS promoter, CAG promoter, Cbh promoter, SFFV promoter; SV40 promoter, Ubc promoter, Ubi promoter, hPGK promoter, β-actin promoter.

8. A tracer obtained by constructing the method for constructing a PET imaging system for visualizing anterograde neural circuit connections in vivo as described in any one of claims 1 to 7.

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