Method for capturing and controlling addiction memory imprint

Abstract

The invention discloses a method for capturing and controlling an addiction memory print, which comprises the following steps of: opening or closing a Tet-on system by controlling the intake of tetracycline, and marking the memory print by the expression of a reporter gene FLAG; in the catching and control strategy for the addiction memory imprint, an addiction memory model with condition position preference is used, the model pairs scene memory of an experimental animal to a box body and reward stimulation of an addiction drug, and the addiction memory induced by the addiction drug is evaluated through preference of a mouse to a drug-accompanying box body; during the administration period, tetracycline intake is given to the mouse, and the tetracycline is in a closed state in other time, so that the addiction memory imprint of the mouse is captured and marked; after the memory imprint is formed, the activity of the imprint cell can be inhibited by combining an exogenously given chemical genetics drug with the hM4Di receptor, so that the addictive memory imprint is controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of gene editing and addiction research, and in particular to a method for capturing and manipulating addiction memory engrams. Background Art

[0002] According to the 2024 World Drug Report, there are approximately 292 million drug users worldwide. The market and volume of amphetamines are gradually expanding. Methamphetamine (METH) remains the primary amphetamine seized globally, and it is also the most abused drug in my country. Drug addiction has gradually become a serious global public health issue. Addiction memories play a key role in controlling the onset and relapse of drug addiction. The pathological persistence of addictive memories leads to recurrent drug seeking and compulsive drug use. Drug addiction is increasingly being viewed as a memory-related brain disease.

[0003] Memory engrams are the neural substrate for storing and retrieving memories. Memory engrams meet four characteristics: First, memory engrams are persistent changes in the brain triggered by specific experiences or events; second, memory engrams can be triggered by retrieval cues to form behavioral recall, and retrieval cues can be sensory input, ongoing actions, or autonomous retrieval; third, memory engrams reflect the content of events during memory encoding and retrieval; fourth, memory engrams may be dormant during the transition period between encoding and retrieval; addictive memories rely on the encoding of memory engrams, and tools and strategies for capturing and labeling addictive memory engrams are powerful means to study addictive memories.

[0004] Therefore, this paper proposes a method to capture and manipulate addiction memory engrams. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for capturing and manipulating addictive memory engrams for addictive memory events.

[0006] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions: a method for capturing and manipulating addictive memory engrams, characterized by comprising the following steps:

[0007] S1. Construction of c-fos-Tet3G; TRE-hM4Di-FLAG transgenic mice:

[0008] S1.1. Design and synthesis of gRNA: Use the CRISPOR program to design the gRNA sequence targeting the mouse Hipp11 gene locus, select the high-scoring sequence No. 1, and synthesize the gRNA using crRNA and tracRNA;

[0009] S1.2. Prepare RNP complex: Mix equal volumes of Cas9 protein (40 μM) and gRNA (48 μM) and incubate at 37°C for 5 minutes to prepare RNP complex;

[0010] S1.3. Donor vector construction: Following the basic molecular cloning procedures, a donor vector carrying the c-fos promoter-Kozak-Tet3G-BGH pA-anti[TRE3G promoter-Kozak-FLAG tag-hM4DirBG pA] gene cassette was constructed.

[0011] S1.4. Preparation of fertilized eggs: Inject pregnant mare serum and human chorionic gonadotropin into 3-4 week old C57BL / 6N female mice, 48 hours apart. Mate the injected female mice with adult fertile male mice to fertilize them. The next day, collect the fertilized eggs from the female mice's oviducts and place them in a cell culture incubator until ready for use.

[0012] S1.5, Pronuclear microinjection: Inject the prepared exogenous gene injection solution (RNP complex and donor vector solution) into the nucleus of the fertilized egg and culture;

[0013] S1.6 Embryo Transfer: The fertilized egg injected with the exogenous gene is then transplanted into the oviduct of a pseudopregnant female mouse. After the embryo transfer, the surrogate mouse is placed in a clean cage and kept warm until it wakes up, then returned to the cage for breeding. After the oviduct transplant is successful, wait for the female mouse to give birth. One week after birth, the mice are paw-clipped and numbered, and PCR identification is performed. Three weeks after birth, the mice are caged and raised individually.

[0014] S1.7. Identification of first-generation F0 mice: Collect tissues (tail or toe tissue) from 1-2 week-old pups, lyse the tissues, and extract the genome. Perform PCR amplification and electrophoresis using primers specific for the target gene to screen for offspring F0 mice that have integrated the exogenous gene.

[0015] S1.8. Passaging and establishing F1 mouse lines: F0 mice were mated with wild-type mice to verify germline transmission and to screen for positive F1 mice.

[0016] S1.9. Perform sibling mating of positive F1 strains and screen for homozygous F2 mice. Establish stable lineages of homozygous F2 mice and record the generation status and pedigree.

[0017] S2. Prepare experimental mice: Custom cannulas are implanted into the hippocampus and nucleus accumbens of transgenic mice and fixed with dental cement. The transgenic mice are then allowed to recover after surgery.

[0018] S3. Environmental adaptation: 3 days before the experiment, transgenic mice were placed in the CPP apparatus for acclimatization for 30 minutes per mouse every day;

[0019] S4. Pre-experimental testing to determine the natural preference box and companion box of transgenic mice;

[0020] S5. Prepare different groups of transgenic mice according to different experimental conditions:

[0021] S5.1. All mice were injected intraperitoneally with saline for 2 days in a natural preference box. Mice were confined to the natural preference box for 30 minutes after each injection.

[0022] S5.2. After the injection, mice that were to be injected with methamphetamine were given Dox (4 mg / ml) in drinking water, while the control group was given normal drinking water. The methamphetamine concentration was 2 mg / kg.

[0023] S5.3. Mice were then injected intraperitoneally with methamphetamine for 4 days in a companion box, while the control group was injected with an equal volume of saline;

[0024] S5.4. After each injection, confine the mice to the drug box for 30 minutes. After the methamphetamine injection, replace the mice's drinking water with normal drinking water.

[0025] S5.5. All mice were then injected intraperitoneally with saline for 2 days in a natural preference box. Mice were confined to the natural preference box for 30 minutes after each injection.

[0026] S6. Second test: Place the mice after drug administration into the CPP device, start the Visu Track v3.0 system, and record the time the mice spend in the black box and the white box;

[0027] S7. Chemogenetic inhibition:

[0028] S7.1. Secure the injection needle to the microsyringe pump, fill the polyurethane tubing with mineral oil, connect the injection needle and the inner syringe with a locking nut at each end of the PU tubing, respectively. Start the microsyringe pump and aspirate the chemogenetic drug DCZ at a concentration of 20 ng / 1 μl.

[0029] S7.2. Anesthetize the mouse with isoflurane inhalation using an animal anesthesia machine. After disinfecting the cannula with 75% alcohol, remove the catheter cap, insert the injection tube, and install the locking nut. Start the microinjection pump and infuse DCZ.

[0030] S7.3. After the drug injection is complete, unscrew the locking nut, remove the injection tube, replace the catheter cap, and transfer the mouse to a warm place to wake up.

[0031] S8. Third test: The DCZ-infused mice were placed in the CPP apparatus, and the Visu Track v3.0 system was activated to record the time the mice spent in the black and white boxes.

[0032] S9. Select mice for sampling:

[0033] S9.1. Use an animal anesthesia machine to anesthetize mice with isoflurane inhalation;

[0034] S9.2. Rapidly open the chest cavity of anesthetized mice and perfuse them transcardially with normal saline. After the liver, lungs, and other organs turn pale, perform transcardial perfusion with 4% paraformaldehyde fixative. Rapidly decapitate and remove the brain. Immerse the entire brain in 4% paraformaldehyde fixative and store at 4°C until needed.

[0035] S10. Detection: Prepare mouse brain slices using a slice ice machine, and co-label memory engram cells using c-fos antibody and FLAG antibody.

[0036] Furthermore, the exogenous gene injection solution is a mixture of RNP complex and Donor vector.

[0037] Furthermore, the basic steps of the molecular cloning include fragment amplification → ligation of the vector backbone and the fragment → bacterial testing → plasmid extraction from positive clones → enzyme digestion identification → sequencing → preparation of plasmid for injection; the object of the molecular cloning is the c-fos promoter-Kozak-Tet3G-BGH pA-anti[TRE3G promoter-Kozak-FLAG tag-hM4DirBG pA] gene cassette.

[0038] Furthermore, the pronuclear microinjection specifically comprises the following steps:

[0039] ① Add the prepared exogenous gene injection solution into the microinjection needle;

[0040] ②Select fertilized eggs with normal morphology and place them in the injection dish;

[0041] ③ Under an inverted microscope with a magnification of 200-400 times, inject the exogenous gene injection solution into the nucleus of the fertilized egg by microinjection;

[0042] ④ Transfer the injected fertilized eggs into M16 culture medium and culture them in a cell culture incubator for 1 hour before transplantation; this completes the pronuclear microinjection.

[0043] Furthermore, the preparation of surrogate mother mice includes the following steps: selecting fertile female mice and mating with male mice that have been sterilized after vasectomy, stimulating the female mice to undergo a series of pregnancy changes to obtain pseudo-pregnant female mice, which serve as surrogate mice after the fertilized eggs are genetically modified.

[0044] Furthermore, the preparation of experimental mice specifically includes the following steps:

[0045] ① Use an animal anesthesia machine to perform isoflurane inhalation anesthesia on mice;

[0046] ②Use a stereotaxic device to locate the hippocampus and nucleus accumbens of anesthetized mice;

[0047] ③After positioning, the customized cannula is loaded into the adapter and slowly implanted into the hippocampus and nucleus accumbens;

[0048] ④ Prepare dental cement and drip it around the cannula to completely cover the cannula root and skull pin;

[0049] ⑤ After the dental cement is fully dried, the mice are transferred to a warm place to wake up. After 7 days of recovery, the experimental mice are obtained.

[0050] Furthermore, in S4, selecting the mouse's natural preference box and drug-companion box specifically includes the following steps: placing the mouse in the CPP apparatus, activating the Visu Track v3.0 system, recording the time the mouse spends in the black box and the white box, and calculating the percentages. The box with the higher percentage is defined as the mouse's natural preference box, and the box with the lower percentage is defined as the mouse's drug-companion box.

[0051] The beneficial effects of the present invention are:

[0052] The present invention controls the intake of tetracycline (Tet), turns on or off the Tet-on system, and marks the memory engram with the expression of the reporter gene FLAG. By combining the conditioned place preference addiction memory model, the methamphetamine addiction memory engram is captured and marked in the hippocampus and nucleus accumbens of mice. After the memory engram is formed, exogenously administered chemogenetic drugs bind to the hM4Di receptor to inhibit the activity of the engram cells, thereby manipulating the addiction memory engram. Furthermore, by inhibiting the addiction memory engram activity in the hippocampus and nucleus accumbens, the retrieval of the conditioned place preference memory of mice is hindered.

[0053] The present invention provides a powerful tool for studying addiction memory and has an important promoting effect on addiction research. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0055] Figure 1Schematic diagram of targeted knock-in of transgenic mice of the present invention;

[0056] Figure 2 Schematic diagram of the principle of capturing and manipulating methamphetamine addiction memory engrams in transgenic mice of the present invention;

[0057] Figure 3 This is a schematic diagram of the gene knock-in identification results of the transgenic mice of the present invention;

[0058] Figure 4 This is a schematic diagram of the genotype identification results of the F2 mice of the present invention;

[0059] Figure 5 The transgenic mice of the present invention capture and manipulate methamphetamine addiction memory engrams. DETAILED DESCRIPTION

[0060] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0061] Example 1

[0062] A method for capturing and manipulating addictive memory engrams, comprising the following steps:

[0063] 1) Construction of c-fos-Tet3G; TRE-hM4Di-FLAG transgenic mice, including the following steps:

[0064] ① gRNA Design and Synthesis: gRNA sequences were designed using the CRISPOR program, and high-scoring sequences were selected. crRNA was synthesized by Nanjing GenScript Biotech, and tracRNA was purchased from Integrated DNA Technologies. The crRNA and tracRNA were combined to form gRNA. The final synthesized gRNA sequence was: GAACACTAGTGCACTTATCCTGG.

[0065] ② Preparation of RNP complex: Mix equal volumes of Cas9 protein (40 μM) and gRNA (48 μM) and incubate at 37°C for 5 minutes to prepare the RNP complex.

[0066] ③ Donor vector construction: According to the basic steps of molecular cloning, the fragment amplification → vector backbone and fragment connection → bacterial testing → positive clone plasmid extraction → enzyme digestion identification → sequencing → preparation of injection plasmid to construct the Donor vector.

[0067] ④ Preparation of fertilized eggs: Inject pregnant mare serum and human chorionic gonadotropin into 3-4 week old C57BL / 6N female mice, with an injection interval of 48 hours; mate the injected female mice with adult fertile male mice to fertilize them; the next day, collect the fertilized eggs from the oviduct of the female mice and place them in a cell culture incubator for later use.

[0068] ⑤ Pronuclear microinjection: Prepare a microinjection needle and a fixing needle, and add the prepared exogenous gene injection solution into the microinjection needle; select fertilized eggs with normal morphology and place them in an injection dish. Under an inverted microscope with 200-400 times magnification, inject the exogenous gene injection solution into the nucleus of the fertilized egg by microinjection; transfer the injected fertilized eggs to M16 culture medium and place them in a cell culture incubator for 1 hour before transplantation.

[0069] ⑥ Preparation of surrogate mice and embryo transplantation: Select fertile female mice of appropriate age and mate them with male mice that have been sterilized after vasectomy, stimulate the female mice to undergo a series of pregnancy changes and obtain pseudo-pregnant female mice, which serve as surrogate mice after the genetically modified fertilized eggs; transplant the fertilized eggs that have been injected with exogenous genes into the oviducts of surrogate female mice on the day of thrombosis; after transplantation, place the surrogate female mice in a clean cage and keep them warm until they wake up and then return them to the cage for breeding; after successful oviduct transplantation, wait for the female mice to give birth; one week after the mice are born, clip their claws and number them, and perform PCR identification at the same time; three weeks after the mice are born, raise them independently in separate cages.

[0070] ⑦ Identification of F0 mice at birth: Collect tissues (tail or toe tissues) from 1-2 week old mice, lyse the tissues and extract the genome, perform PCR amplification and electrophoresis detection using specific primers for the target gene, and screen out offspring with exogenous gene integration.

[0071] ⑧Passaging and establishment of F1 mouse lines: F0 generation mice are mated with wild-type mice to verify germline transmission and screen positive F1 mice.

[0072] ⑨Purification and propagation of F2 mice: The positive F1 mice were sired and homozygous F2 mice were screened. The homozygous F2 mice were stably propagated and the propagation status and pedigree were recorded.

[0073] 2) Capturing and manipulating addiction memory engrams in c-fos-Tet3G;TRE-hM4Di-FLAG mice, including the following steps:

[0074] ① Microdose Cannula Implantation: Mice were anesthetized with isoflurane inhalation using a small animal anesthesia machine. The hippocampus and nucleus accumbens were located using a stereotaxic apparatus. Custom cannulas were inserted into adapters and slowly implanted into the hippocampus and nucleus accumbens. Dental cement was prepared and dripped around the cannula, completely covering the base of the cannula and the cranial pin. After the cement was fully dried, the mice were transferred to a warm place to recover and allowed to recover for 7 days.

[0075] ② Acclimation: 3 days before the experiment, place the mice in the CPP apparatus for acclimation for 30 minutes per mouse.

[0076] ③ Pre-test: Place the mouse in the CPP apparatus and activate the Visu Track v3.0 system. Record the time the mouse spends in the black and white boxes and calculate the percentages. The box with the higher percentage is defined as the mouse's natural preferred box, and the box with the lower percentage is defined as the mouse's drug-companion box.

[0077] ④ Administration: All mice were injected intraperitoneally with normal saline for 2 days in the natural preference box, and the mice were confined to the natural preference box for 30 minutes after each injection; after the injection, the mice that were about to receive methamphetamine (2 mg / kg) injection were given Dox (4 mg / ml) in drinking water, and then the mice were injected intraperitoneally with methamphetamine for 4 days in the companion drug box, and the control group was injected with an equal amount of normal saline, and the mice were confined to the companion drug box for 30 minutes after each injection; after the injection, the mice were replaced with normal drinking water, and then all mice were injected intraperitoneally with normal saline for 2 days in the natural preference box, and the mice were confined to the natural preference box for 30 minutes after each injection.

[0078] ⑤ Post-test: Place the mice after drug administration into the CPP device, start the Visu Track v3.0 system, and record the time the mice stay in the black box and the white box.

[0079] ⑥ Chemogenetic Inhibition: Before the second post-test, secure the injection needle to the microsyringe pump, fill the polyurethane tubing with mineral oil, connect the injection needle and the inner syringe with a locking nut at each end of the tubing, respectively. Start the microsyringe pump and aspirate the chemogenetic drug DCZ. Anesthetize the mouse with isoflurane inhalation using a small animal anesthesia machine. After disinfecting the cannula with 75% alcohol, remove the catheter cap, insert the inner syringe, and install the locking nut. Start the microsyringe pump and infuse DCZ at a concentration of 20 ng / 1 μl. After the drug injection is completed, remove the locking nut, remove the inner syringe, replace the catheter cap, and transfer the mouse to a warm place to revive.

[0080] ⑦ Second post-test: Place the DCZ-infused mice into the CPP device, start the Visu Track v3.0 system, and record the time the mice spend in the black box and the white box.

[0081] ⑧ Sampling: Anesthetize mice with isoflurane inhalation using a small animal anesthesia machine. Quickly open the chest cavity and perfuse the mice transcardially with normal saline. After the liver, lungs and other organs turn white, perfuse the mice transcardially with 4% paraformaldehyde fixative. Quickly decapitate and remove the brain. Immerse the whole brain in 4% paraformaldehyde fixative and store at 4°C until needed.

[0082] ⑨ Visualization of memory engrams: Mouse brain slices were prepared using a slice ice machine, and memory engram cells were co-labeled using c-fos antibodies and FLAG antibodies.

[0083] 3) Result analysis

[0084] (1) Figure 3 F1 mice were identified by PCR as shown, and the genotype identification strategy was as follows:

[0085] ① PCR identification of F1 mice using identification primers 1 and 2 ( Figure 3 A), screened out positive mice No. 17, 19 and 21, and designed primers as follows:

[0086] Primer 1 (annealing temperature: 60.0°C):

[0087] 5'arm forward primer(F2):5'-GTGGGCATTGGTTATTGGTCGTAG-3'

[0088] 3'KI reverse primer(R2):5'-CGCGTGTAGGATTTCGGAGATG-3'

[0089] Primer 2 (annealing temperature: 60.0°C):

[0090] 5'KI forward primer(F1):5'-CCACCGTACACGCCTAAAGC-3'

[0091] 3'arm reverse primer(R1):5'-GCCTTGACCTAAGAGATGATG CGAC-3'

[0092] ② Southern blot analysis was performed on DNA samples from positive mice No. 17, 19 and 21 ( Figure 3 B) Confirm the correctness of the exogenous gene targeting, Southern blot analysis strategy such as:

[0093] Expected fragment size:

[0094] 5'Probe-MfeI:10.99kb-WT,3.33kb-MT

[0095] 3'Probe-SspI:4.12kb-WT,8.00kb-MT

[0096] Primer for 5' probe:

[0097] 5'Probe forward primer:5'-GGCACAATGTTAATCCAGCCTGACTC-3'

[0098] 5'Probe reverse primer:5'-GTGACCAGTTTGTCCTCCTCCAGTA GA-3'

[0099] Primer for 3' probe:

[0100] 3'Probe forward primer:5'-GATGTGAACAAAGCACCCTATGGCTC-3'

[0101] 3'Probe reverse primer:5'-GTGTCGATCATCCATTAGCCTAGCC-3'

[0102] ③ Sequencing analysis was performed on the No. 17 positive F1 mice ( Figure 3 C) It is clear that the exogenous gene has been knocked in. The sequencing primers are as follows:

[0103] Sequencing primers for PCR product 1:

[0104] 5'Sequence primer(F3):5'-CTCTACTGGAGGAGGACAAACTG-3'

[0105] Sequencing primers for PCR product 2:

[0106] 3'Sequence primer(R3):5'-GTCTTCCACCTTTCTTCAGTTAGC-3'

[0107] (2) Figure 4 The F2 mice were genotyped by PCR and homozygous mice were screened. The genotype identification strategy is as follows:

[0108] Primer 1 (annealing temperature: 60.0°C):

[0109] F1:5'-TCTTGTCGTCATCGTCTTTGTAGT-3'

[0110] R1:5'-ACACATTTAATATCCCCTTGTTCCC-3'

[0111] Primer 1 product fragment size: 594 bp

[0112] Primer 2 (annealing temperature: 60.0°C):

[0113] F2:5'-CTCTACTGGAGGAGGACAAACTG-3'

[0114] R2:5'-GTCTTCCACCTTTCTTCAGTTAGC-3'

[0115] Primer 2 product fragment size: 519 bp

[0116] Internal reference primer:

[0117] F:5'-CATGCCAATGGTTCACTCTAAGGT-3'

[0118] R:5'-TCTCTATGTCCCAAAGTGCAGACAC-3'

[0119] Product fragment size of internal reference primer: 335bp

[0120] Homozygotes: two bands, 594 bp and 335 bp respectively

[0121] Heterozygote: 3 bands, 594 bp, 335 bp, and 519 bp

[0122] Wild type: 2 bands, 519 bp and 335 bp respectively

[0123] ③ Such as Figure 5 As shown, transgenic mice and CPP modeling were used together to capture and manipulate methamphetamine addiction memory engrams ( Figure 5 A). The results of the pre-test showed that there was no significant difference in CPP scores between the methamphetamine group and the control group; the first post-test showed that there was a significant difference in CPP scores between the methamphetamine group and the control group (P < 0.001), indicating that the addiction memory of mice had been formed; the second post-test showed that chemical genetic inhibition of hippocampus ( Figure 5 B) or nucleus accumbens ( Figure 5C) can weaken the CPP scores of mice (P < 0.001 or P < 0.01), indicating that inactivation of engram cells can hinder the retrieval of addiction memory in mice; the use of cfos and FLAG antibodies can achieve visual labeling of methamphetamine addiction memory engram ( Figure 5 F).

[0124] Example 2

[0125] The crRNA described in the present invention was synthesized by Nanjing GenScript Biotech Co., Ltd., and the tracRNA was purchased from Integrated DNA Technologies.

[0126] Figure 2 Middle A indicates that the Tet-on system is turned on or off by controlling the intake of doxycycline (Dox; a tetracycline), and the memory engram is marked by the expression of the reporter gene FLAG; Figure 2 Figure B shows mice ingesting Dox during methamphetamine administration, while remaining in a Dox-blocked state. This captures and labels the methamphetamine addiction memory engram. After the engram is formed, exogenously administered chemogenetic drug (deschloroclozapine, DCZ) specifically binds to hM4Di, inhibiting engram cell activity and thus inactivating the methamphetamine addiction memory engram.

[0127] Figure 3 A in the middle represents the PCR identification results of mice No. 17, 19, and 21 in the F1 mice; Figure 3 Middle B shows the Southern blot analysis results of F1 mice No. 17, 19 and 21. Figure 3 Middle C represents the gene sequencing results of mouse No. 17 among F1 mice.

[0128] Figure 5 A in the middle represents the experimental flow chart; Figure 5 Middle B shows the CPP scores of mice in the experiment of chemical genetic inhibition of hippocampal engram cells; Figure 5 Middle C represents the CPP score of mice in the experiment of chemical genetic inhibition of nucleus accumbens engram cells; Figure 5 Middle D shows the trajectory of hippocampal cannula implantation. Figure 5 Middle E indicates the trajectory of nucleus accumbens cannula implantation; Figure 5 Middle F represents the visualization markers of methamphetamine addiction memory engrams in the hippocampus and nucleus accumbens.

Claims

1. A method for capturing and manipulating addictive memory engrams, characterized in that: The following steps are involved: S1. Construction of c-fos-Tet3G; TRE-hM4Di-FLAG transgenic mice: S1.

1. Design and synthesis of gRNA: Use the CRISPOR program to design gRNA sequences targeting the mouse Hipp11 gene locus, select high-scoring sequences, and synthesize gRNA using crRNA and tracRNA; S1.

2. Prepare RNP complex: Mix equal volumes of Cas9 protein (40 μM) and gRNA (48 μM) and incubate at 37°C for 5 minutes to prepare RNP complex; S1.

3. Construct a donor vector carrying the c-fos promoter-Kozak-Tet3G-BGH pA-anti[TRE3G promoter-Kozak-FLAG tag-hM4DirBG pA] gene cassette according to the basic molecular cloning procedures. S1.

4. Preparation of fertilized eggs: Inject pregnant mare serum and human chorionic gonadotropin into 3-4 week old C57BL / 6N female mice, 48 hours apart. Mate the injected female mice with adult fertile male mice to fertilize them. The next day, collect the fertilized eggs from the female mice's oviducts and place them in a cell culture incubator until ready for use. S1.5, Pronuclear microinjection: Inject the prepared exogenous gene injection solution into the nucleus of the fertilized egg and culture it; S1.6 Embryo Transfer: The fertilized egg injected with the exogenous gene is then transplanted into the oviduct of a pseudopregnant female mouse. After the embryo transfer, the surrogate mouse is placed in a clean cage and kept warm until it wakes up, then returned to the cage for breeding. After the oviduct transplant is successful, wait for the female mouse to give birth. One week after birth, the mice are paw-clipped and numbered, and PCR identification is performed. Three weeks after birth, the mice are caged and raised individually. S1.

7. Identification of first-generation F0 mice: Collect tissues (tail or toe tissue) from 1-2 week-old pups, lyse the tissues, and extract the genome. Perform PCR amplification and electrophoresis using primers specific for the target gene to screen for offspring F0 mice that have integrated the exogenous gene. S1.

8. Passaging and establishing F1 mouse lines: F0 mice were mated with wild-type mice to verify germline transmission and to screen for positive F1 mice. S1.

9. Perform sibling mating of positive F1 strains and screen for homozygous F2 mice. Establish stable lineages of homozygous F2 mice and record the generation status and pedigree. S2. Prepare experimental mice: Custom cannulas are implanted into the hippocampus and nucleus accumbens of transgenic mice and fixed with dental cement. The transgenic mice are then allowed to recover after surgery. S3. Environmental adaptation: 3 days before the experiment, transgenic mice were placed in the CPP apparatus for acclimatization for 30 minutes per mouse every day; S4. Pre-experimental testing to determine the natural preference box and companion box of transgenic mice; S5. Prepare different groups of transgenic mice according to different experimental conditions: S5.

1. All mice were injected intraperitoneally with saline for 2 days in a natural preference box. Mice were confined to the natural preference box for 30 minutes after each injection. S5.

2. After the injection, mice receiving methamphetamine were given Dox (4 mg / ml) in drinking water, while the control group received normal drinking water. The methamphetamine concentration was 2 mg / kg. S5.

3. Mice were then injected intraperitoneally with methamphetamine for 4 days in a companion box, while the control group was injected with an equal amount of saline; S5.

4. After each injection, confine the mice to the drug box for 30 minutes. After the methamphetamine injection, replace the mice's drinking water with normal drinking water. S5.

5. All mice were then injected intraperitoneally with saline for 2 days in a natural preference box. Mice were confined to the natural preference box for 30 minutes after each injection. S6. Second test: Place the mice after drug administration into the CPP device, start the Visu Track v3.0 system, and record the time the mice spend in the black box and the white box; S7. Chemogenetic inhibition: S7.

1. Secure the injection needle to the microsyringe pump, fill the polyurethane tubing with mineral oil, connect the injection needle and the inner syringe with a locking nut at each end of the PU tubing, respectively. Start the microsyringe pump and aspirate the chemogenetic drug DCZ at a concentration of 20 ng / 1 μl. S7.

2. Anesthetize the mouse with isoflurane inhalation using an animal anesthesia machine. After disinfecting the cannula with 75% alcohol, remove the catheter cap, insert the injection tube, and install the locking nut. Start the microinjection pump and infuse DCZ. S7.

3. After the drug injection is complete, unscrew the locking nut, remove the injection tube, replace the catheter cap, and transfer the mouse to a warm place to wake up. S8. Third test: The DCZ-infused mice were placed in the CPP apparatus, and the Visu Track v3.0 system was activated to record the time the mice spent in the black and white boxes. S9. Select mice for sampling: S9.

1. Use an animal anesthesia machine to anesthetize mice with isoflurane inhalation; S9.

2. Rapidly open the chest cavity of anesthetized mice and perfuse them transcardially with normal saline. After the liver, lungs, and other organs turn pale, perform transcardial perfusion with 4% paraformaldehyde fixative. Rapidly decapitate and remove the brain. Immerse the entire brain in 4% paraformaldehyde fixative and store at 4°C until needed. S10. Detection: Prepare mouse brain slices using a slice ice machine, and co-label memory engram cells using c-fos antibody and FLAG antibody.

2. The method for capturing and manipulating addictive memory engrams according to claim 1, characterized in that: The exogenous gene injection solution is a Donor vector having a Cas9-gDNA complex targeting the mouse Hipp11 gene site and carrying a c-fos promoter-Kozak-Tet3G-BGH pA-anti[TRE3G promoter-Kozak-FLAG tag-hM4DirBG pA] gene box.

3. The method for capturing and manipulating addictive memory engrams according to claim 1, characterized in that: The basic steps of the molecular cloning include fragment amplification → ligation of the vector backbone and the fragment → bacterial testing → plasmid extraction from positive clones → enzyme digestion identification → sequencing → preparation of plasmid for injection; the target of the molecular cloning is the c-fos promoter-Kozak-Tet3G-BGH pA-anti[TRE3G promoter-Kozak-FLAG tag-hM4DirBG pA] gene cassette.

4. The method for capturing and manipulating addictive memory engrams according to claim 1, wherein: The pronuclear microinjection specifically comprises the following steps: ① Add the prepared exogenous gene injection solution into the microinjection needle; ②Select fertilized eggs with normal morphology and place them in the injection dish; ③ Under an inverted microscope with a magnification of 200-400 times, inject the exogenous gene injection solution into the nucleus of the fertilized egg by microinjection; ④ Transfer the injected fertilized eggs into M16 culture medium and culture them in a cell culture incubator for 1 hour before transplantation; this completes the pronuclear microinjection.

5. The method for capturing and manipulating addictive memory engrams according to claim 1, wherein: The preparation of the surrogate mother mouse comprises the following steps: Fertile female mice are selected to mate with male mice that have been sterilized after vasectomy, stimulating the female mice to undergo a series of pregnancy changes to obtain pseudo-pregnant female mice, which serve as surrogate mice for the genetically modified fertilized eggs.

6. The method for capturing and manipulating addictive memory engrams according to claim 1, wherein: The preparation of experimental mice specifically comprises the following steps: ① Use an animal anesthesia machine to perform isoflurane inhalation anesthesia on mice; ②Use a stereotaxic device to locate the hippocampus and nucleus accumbens of anesthetized mice; ③After positioning, the customized cannula is loaded into the adapter and slowly implanted into the hippocampus and nucleus accumbens; ④ Prepare dental cement and drip it around the cannula to completely cover the cannula root and skull pin; ⑤ After the dental cement is fully dried, the mice are transferred to a warm place to wake up. After 7 days of recovery, the experimental mice are obtained.

7. The method for capturing and manipulating addictive memory engrams according to claim 1, wherein: In S4, selecting the mouse's natural preference box and drug-companion box specifically includes the following steps: placing the mouse in the CPP apparatus, activating the Visu Track v3.0 system, recording the time the mouse spends in the black box and the white box, and calculating the percentages. The box with the higher percentage is defined as the mouse's natural preference box, and the box with the lower percentage is defined as the mouse's drug-companion box.

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