Social disorder animal model based on Calretinin positive interneuron target as well as construction method and application of social disorder animal model

Abstract

The invention discloses a social disorder animal model based on a Calretinin positive internals neuron target as well as a construction method and application of the social disorder animal model. According to the invention, an MSEW normal form is applied to construct a social disorder mouse model in a CR-Cre animal, and an action mechanism of Cllresinin positive interneurons in an anterior cingulation cortex region participating in regulation and control of MSEW-induced social disorder is explored. It is found that the Calretinin positive interneurons participate in regulation of MSEW-induced animal social disorders, and a brand new scientific theoretical basis is provided for exploring a mechanism that the Calretinin positive interneurons participate in regulation of the social disorders in the anterior subtraction cortex region.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to an animal model of social disorder based on Calretinin-positive interneuron targets, its construction method, and its application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Patients with neuropsychiatric disorders whose main symptom is social impairment, such as autism spectrum disorder (ASD), anxiety disorders, and schizophrenia, experience severe difficulties in social interaction and are unable to integrate into normal social life. Animal models and experimental methods are important tools for exploring the mechanisms of these diseases.

[0004] Numerous methods exist for establishing animal models of ASD, primarily categorized into genetic and non-genetic models. Genetic models include single-gene mutations, copy number variations (CNVs), and idiopathic models. These models mostly utilize mice, based on existing research identifying human ASD genetic loci, by knocking out / inserting the corresponding gene in mice. The mechanisms of genetic models are well-defined and can be used to study the effects of specific genes. Non-genetic models involve biochemical induction, maternal autoantibodies, and maternal immune activation models. Biochemical induction involves using valproic acid (VPA), propionic acid (PPA), bisphenol A (BPA), and other chemical drugs on pregnant mice to produce ASD offspring. However, some of these induction methods are complex to perform, have short-lasting effects, or involve long modeling periods and atypical symptoms. Given the abundance of existing ASD modeling methods, the quality of these models varies considerably. Due to the complexity of the disease's etiology, a single modeling method cannot fully replicate the actual etiology and clinical manifestations of ASD patients.

[0005] The anterior cingulate cortex (ACC) plays a crucial regulatory role in cognition, emotion, learning, and pain, and is vital for social behavior. Gamma-aminobutyric acid (GABA) inhibitory interneurons are closely associated with social impairments. GABAergic interneurons are divided into parvalbumin-positive (PV) interneurons. + Interneurons, neuropeptide Y positive (NPY) + Interneurons and calreticulin positive (CR) +Interneurons, and different types of interneurons, possess unique morphological structures and electrophysiological characteristics. In human patients, especially those with ASD and schizophrenia, a reduction in the number of PV interneurons in the ACC region is closely associated with social behavioral impairments. However, the specific role and mechanism of calretinin-positive interneurons in regulating social impairments remain unclear and require further investigation. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention uses Calretinin-positive interneurons as targets in the construction of social disorder models. Therefore, this invention provides an animal model of social disorder based on Calretinin-positive interneurons, its construction method, and its applications. Experiments using this invention demonstrate that Calretinin-positive interneurons play a crucial role in the development of social disorders. This invention provides the following technical solutions: As a first aspect of the present invention, a method for constructing an animal model of social disorder based on Calretinin-positive interneurons is provided. The present invention can induce symptoms of social disorder in animals by intervention with apoptotic viruses and by chemogenetic inhibition of Calretinin-positive interneurons in the anterior cingulate cortex.

[0007] In some embodiments of the present invention, the social disorder model animal is a CR-Cre transgenic animal that specifically expresses Cre recombinase on Calretinin-positive interneurons; the animal is selected from zebrafish, fruit flies, mice, rats, guinea pigs, baboons, cynomolgus monkeys, rhesus monkeys, rabbits, pigs, cattle, sheep, goats, and cats.

[0008] The CR-Cre mice were used to construct a social disorder mouse model through the maternal-infant separation early weaning (MSEW) paradigm.

[0009] In one or more embodiments of the present invention, the apoptotic virus is diphtheria toxin.

[0010] In one or more embodiments of the present invention, chemogenetic inhibition of Calretinin-positive interneurons in the anterior cingulate cortex of CR-Cre animals is achieved by injecting the chemosuppressive virus pAAV-hSyn-DIO-hM4D(Gi)4-mCherry into the anterior cingulate cortex.

[0011] Specifically, in some embodiments of the present invention, apoptosis of Calretinin-positive interneurons can be induced by injecting diphtheria toxin, or by injecting the chemosuppressive virus pAAV-hSyn-DIO-hM4D(Gi)4-mCherry into the anterior cingulate cortex of CR-Cre animals, thereby causing Calretinin-positive interneurons to express hM4D(Gi), which can induce apoptosis of Calretinin-positive interneurons and induce social impairment in animals.

[0012] In one or more embodiments of the present invention, the animal is a mouse.

[0013] In one or more embodiments of the present invention, the social impairment is a social dysfunction; the social dysfunction is autism spectrum disorder (ASD), anxiety disorder, schizophrenia, etc.

[0014] In one or more embodiments of the present invention, the social disorder model animal has specific behavioral phenotypic defects.

[0015] In some embodiments of this invention, a social disorder mouse model was constructed in CR-Cre mice using the Mother-Infant Separation Early Weaning (MSEW) paradigm. The successful construction of the social disorder mouse model was verified through a three-box social behavior experiment. During the experiment, changes in the number, activity, electrophysiology, synaptic function, and presynaptic plasticity of Calretinin-positive interneurons in the anterior cingulate cortex of social disorder mice were observed and recorded. Changes in firing rate and mouse behavior were observed using apoptosis-virus intervention, chemogenetic inhibition, and optogenetic regulation of Calretinin-positive interneurons in the anterior cingulate cortex. The experimental results showed that MSEW induced a decrease in the number, activity, and intrinsic excitability of Calretinin-positive interneurons in the anterior cingulate cortex of social disorder mice. Based on optogenetic regulation of Calretinin-positive interneurons to alleviate social disorder, it was found that monosynaptic inhibitory synaptic connections were formed between Calretinin-positive interneurons and pyramidal neurons, and the inhibitory effect of Calretinin-positive interneurons on the connected pyramidal neurons was enhanced.

[0016] The study of this invention found that the activity of Calretinin-positive interneurons in the anterior cingulate cortex of a social disorder model mouse was reduced.

[0017] In some embodiments of the present invention, synchronous firing of Calretinin-positive interneurons induced by blue light stimulation was observed, thereby confirming that light stimulation can activate Calretinin-positive interneurons. In behavioral tests, mice regained social interest. This study confirms that activation of Calretinin-positive interneurons in the anterior cingulate cortex can improve social impairment.

[0018] In some embodiments of the present invention, optogenetic excitation of Calretinin-positive interneurons in MSEW mice was performed to record light-induced inhibitory postsynaptic currents (IPSCs) on pyramidal neurons. These currents could be blocked by the antagonist tetrodotoxin (TTX), and the combined action of 4-aminopyridine (4-AP) could partially restore IPSCs. A It was completely blocked again under the action of the blocker picric acid (PTX).

[0019] Therefore, this invention provides the use of Calretinin-positive interneurons as therapeutic targets for social disorder.

[0020] As a second aspect of the invention, it provides the use of Calretinin-positive interneurons as targets in screening drugs or methods for treating social disorders.

[0021] As a third aspect of the invention, it is to provide a model constructed by the method for constructing the social disorder model described in the second aspect, and its application in screening drugs or methods for treating social disorders.

[0022] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides the application of Calretinin-positive interneurons as targets in constructing social disorder models and screening drugs for treating social disorder. This invention demonstrates that Calretinin-positive interneurons in the anterior cingulate cortex play a crucial role in maternal-infant separation-induced social disorder. Using CR-Cre animals, this invention explores the mechanism by which CR-positive interneurons in the anterior cingulate cortex regulate MSEW-induced social disorder. The discovery that Calretinin-positive interneurons regulate MSEW-induced animal social disorder provides a novel scientific basis for exploring the mechanism by which Calretinin-positive interneurons in the anterior cingulate cortex regulate social disorder.

[0023] 2. This invention regulates the activity of Calretinin-positive interneurons through optogenetics and chemogenetics, observing behavioral changes in animal models of social disorder. It was found that both induction of Calretinin-positive interneuron apoptosis and chemogenetic inhibition of Calretinin-positive interneurons lead to social disorder in animals, while optogenetic excitation of Calretinin-positive interneurons improves social disorder in mice. The mechanism of action of Calretinin-positive interneurons in treating social disorder is investigated, providing a new treatment approach for patients with social disorder in clinical practice. It also provides a new model for screening drugs or methods for treating social disorder. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0025] Figure 1 Figure 1 shows the results of the MSEW paradigm-induced social impairment experiment in mice. A represents the MSEW paradigm process. B is an electrophoresis diagram of PCR identification in CR-Cre genotype mice. C is a behavioral heatmap of the three-box social recognition stage, with the upper graph representing the control group and the lower graph representing the MSEW group. D is a bar chart of the time mice spent in each box, with the control group showing S1 = 351.53 ± 5.45 s and NO = 132.83 ± 3.87 s. n =20, P <0.001; MSEW group: n =20, P >0.05. E is a bar chart showing the socialization time of mice in each cage for each group. In the control group: S1 = 195.74 ± 3.70 s, NO = 82.20 ± 3.40 s. n =20, P <0.001; MSEW group: n =20, P >0.05. F is the discrimination index, where S1 = 0.41 ± 0.02 s. n =20; NO=-0.01±0.02 s, n =20, P <0.001. G is a behavioral heatmap of the social novelty preference stage, where the upper graph represents the control group and the lower graph represents the MSEW group. H is a bar chart of the time mice spent in each box, where the control group: S1=142.26±3.56 s, S2=346.14±5.78 s. n =20, P <0.001; MSEW group: S1=297.45±4.30 s, S2=191.34±4.55 s, n =20, P <0.001. I is a bar chart of the social time of mice in each cage in each group, where, control group: S1=83.02±4.11 s, S2=181.87±4.42 s, n =20, P <0.001; MSEW group: S1=163.44±2.60 s, S2=80.92±3.63 s, n =20, P <0.001. J is the preference index, where S1 = 0.37 ± 0.03 s. n=20; S2=-0.34±0.03 s, n =20, P <0.001. Data are expressed as mean ± standard error. n =20). Univariate ANOVA analysis and independent samples t-test: compared with S1, ns >0.05, *** P <0.001, $$$ P <0.001, ^^^ P <0.001; compared with the control group, ### P <0.001.

[0026] Figure 2 The results of the photoactivation combined with behavioral experiments in Example 6 are shown. A is a schematic diagram of the injection of viral AAV-hSyn-DIO-hChR2-EYFP-WPRE or AAV-DIO-EYFP into the ACC of MSEW group mice. B is an image of CR-positive interneurons in the ACC brain region containing YFP fluorescence, recorded using whole-cell patch-clamp. C is a schematic diagram of representative recordings of evoked action potentials in MSEW and control mice (stimulation current 200 pA, duration 300 ms). D is a statistical graph of resting membrane potentials in the two groups of mice. E is a statistical graph of the half-width at half-maximum (WWHM) of evoked action potentials in the two groups of mice. n =24; P >0.05, P >0.05). F is the statistical graph of the threshold potential of the two groups of mice (control group = -34.93±0.52mV, n =24; MSEW group = -31.79±0.52mV, n =24; P<0.001). G is the baseline strength statistics of the two groups of mice (control group = 36.67±5.06pA, n =24; MSEW group =53.33±4.28 pA, n =24; P <0.001). H is a statistical graph of the frequency and injected depolarization current in the two groups of mice (current stimulation was 140 pA). n =24; P <0.05; current stimulation was 160 pA. n =24; P <0.01; current stimulation was 180 / 200 / 220 / 240 / 260 / 280 / 300 / 320 pA. n =24; P<0.001). I is a schematic diagram of representative sIPSCs records from the two groups of mice, with the upper figure representing the control group and the lower figure representing the MSEW group. J is a statistical plot of sIPSCs frequencies from the two groups of mice, with the control group having a frequency of 5.96 ± 0.16 Hz. n =24; MSEW group = 2.99 ± 0.19 Hz, n =24; P <0.001. K represents the amplitude statistics of the two groups of mice ( n =24; P >0.05). Data are expressed as mean ± standard error. n =24). One-way ANOVA, repeated measures ANOVA, and independent samples analysis were used. t Test: Compared with the control group, ns >0.05, ** P <0.01, *** P <0.001.

[0027] Figure 3 This is a graph showing the results of the immunofluorescence staining experiment in Example 3. AD represents the immunofluorescence images of CR-positive interneurons (green) and c-Fos-positive cells (red) in the ACC region during the three-stage social phase in the MSEW group and the control group. E is a bar chart showing the percentage of CR and c-Fos-labeled neurons in the ACC region of mice in the MSEW group and the control group (control group = 87.86 ± 2.16%). n =5; MSEW group = 60.86 ± 3.45%, n =5, P <0.01). F is a bar chart showing the percentage of CR and c-Fos dual-labeled neurons in the ACC region of the two groups of mice among CR-positive interneurons (control group = 90.94 ± 1.99%). n =5; MSEW group = 57.74 ± 3.60%, n =5, P <0.01. Data are expressed as mean ± standard error. n =5). Univariate ANOVA analysis and independent samples t Test: Compared with the control group, *** P <0.01.

[0028] Figure 4The figures show the results of detecting neuronal activity using fiber optic calcium signaling technology in Example 4. A is a schematic diagram of the injection site (green) and fiber optic placement in the GCaMP7s adeno-associated virus (AAV) of MSEW mice (left image); the right image shows immunofluorescence staining confirming that GCaMP7s-infected neurons in CR-Cre mice are CR-positive interneurons. B is a bar chart verifying co-labeling of CR-positive interneurons and GCaMP7s protein. C is a representative fluorescence signal image, heatmap, and ΔF / F event curve of calcium signal changes in CR-positive interneurons during the social recognition phase of the three-box social behavior test in the control group mice. D is a representative fluorescence signal image, heatmap, and ΔF / F event curve of calcium signal changes in CR-positive interneurons during the social recognition phase of the three-box social behavior test in the MSEW group mice. E is the area under the ΔF / F curve for both groups of mice during the social recognition phase of the three-box social behavior test, S1 = 23.08 ± 1.34%. n =8; NO=2.77±0.52%, n =8, P <0.001. F is a bar chart showing the peak values ​​of social behavior in the two groups of mice during the social recognition phase of the three-box social behavior test, S1=5.31±0.59%. n =8; NO=1.58±0.22%, n =8, P <0.001. G represents the representative fluorescence signal map, heatmap, and ΔF / F event curve of calcium signal changes in CR-positive interneurons during social behavior in the social preference phase of the three-box social behavior test in the control mice. Data are expressed as mean ± standard error (SSE). n =8). H represents the representative fluorescence signal map, heatmap, and ΔF / F event curve of calcium signal changes in CR-positive interneurons during social behavior in the MSEW group mice during the social preference phase of the three-box social behavior test. I is a bar chart of the area under the ΔF / F curve and peak value during social behavior in both groups of mice during the social preference phase of the three-box social behavior test, S1=3.15±0.89%. n =8; S2=24.60±2.17%, n =8, P <0.001. J is a bar chart of the area under the ΔF / F curve and the peak value of the two groups of mice during the social preference phase of the three-box social behavior test, with S1=1.31±0.30%. n =8; S2=5.51±0.46%, n =8, P <0.001. Univariate ANOVA analysis and independent samples t Test: Compared with S1, ns >0.05,*** P <0.001.

[0029] Figure 5 The results of the apoptosis virus intervention combined with behavioral experiments in Example 5 are shown. A is a schematic diagram of injecting CR-Cre mice with the virus AAV-mCherry-flex-dtA-WPRE or AAV-DIO-mCherry via ACC. B is an immunofluorescence image of CR-positive interneurons co-labeled with diphtheria toxin. C is an immunofluorescence image of CR-positive interneurons co-labeled with mCherry control virus. D shows the number of CR-positive interneurons bound by the control virus and the apoptosis virus, demonstrating the intervention efficiency of the apoptosis virus (control group = 8.63 ± 0.50%). n =8; MSEW group = 0.05 ± 0.27%, n =8, P <0.001). E is a heatmap of social recognition in the three boxes, where the upper graph represents the mCherry group and the lower graph represents the DTA group. F is a bar chart of the time mice spent in each box (mCherry group: S1=356.77±7.22 s, NO=128.93±4.91 s, n =20, P <0.001; DTA group: P >0.05). G is a bar chart of socialization time per cage for mice in each group (mCherry group: S1=255.70±6.79 s, NO=90.67±4.87 s, n =20, P <0.001; DTA group: P >0.05). H represents the preference index of mice in each group (S1 = 0.47 ± 0.02 s, n =20; NO=-0.01±0.04s, n =20, P <0.001). I is a heatmap of social novelty preferences for each group of mice, with the top graph representing the mCherry group and the bottom graph representing the DTA group. J is a bar chart of the time mice spent in each chamber, with the mCherry group showing S1 = 165.71 ± 5.24 s and S2 = 325.94 ± 6.07 s. n =20, P <0.001; DTA group: S1=296.35±7.64 s, S2=186.04±6.61 s, n =20, P <0.001. K is a bar chart of social time per cage for mice in each group (mCherry group: S1=116.92±6.06 s, S2=242.82±6.98 s,n =20, P <0.001; DTA group: S1=159.49±9.29 s, S2=84.42±4.81 s, n =20, P <0.001). L is the discrimination index of mice in each group (mCherry = 0.35 ± 0.03 s, n =20; DTA=-0.32±0.04 s, n =20, P <0.001. Data are expressed as mean ± standard error. n =20). One-way ANOVA and independent samples t-test were used: compared with S1, ns >0.05, *** P <0.01, $$$ P <0.001, ^^^ P <0.001; compared with the mCherry group, ### P <0.001.

[0030] Figure 6 The results of the chemical genetics combined with behavioral experiments in Example 5 are shown. A is a schematic diagram of injecting CR-Cre mice with the virus AAV-hSyn-DIO-hM4D(Gi)4-mCherry or AAV-DIO-mCherry into the ACC. B shows the injection of the chemically inhibiting virus hM4Di, causing CR-positive interneurons to express hM4D (red); the right figure shows immunofluorescence staining to verify that hM4Di-infected neurons in CR-Cre mice are CR-positive interneurons. C is a bar chart verifying the co-labeling of CR-positive interneurons and hM4Di virus. D is a whole-cell patch-clamp recording of action potential firing before and after CNO perfusion. E shows the change in firing rate before and after CNO perfusion (PreCNO = 21.2 ± 1.05 Hz) in whole-cell patch-clamp recording. n =8; CNO=4.54±1.24Hz, n =8, P <0.001). F and G represent the changes in the firing rate of CR-positive interneurons in mice before and after intraperitoneal injection of CNO in an awake state, as recorded in vivo via multichannel recordings (PreCNO=40.63±0.94Hz). n =8; CNO=14.00±1.27Hz, n =8, P<0.001). H is the heatmap of social recognition in the three boxes, where the upper figure is the mCherry group and the lower figure is the hM4Di group. I is a bar chart of the time mice spent in each box (S1=334.79±6.67 s, NO=156.75±5.27 s). n =20, P <0.001), J is a bar chart of social time per cage for mice in each group (S1=181.54±4.67 s, NO=99.92±4.85 s, n =20, P <0.001). K is the discrimination index of mice in each group (mCherry=0.29±0.03 s, n =20; hM4Di=0.02±0.05 s, n =20, P <0.001). L is a heatmap of social novelty preferences for each group of mice, where the upper graph represents the mCherry group and the lower graph represents the hM4Di group. M is a bar chart of the time mice spent in each chamber (mCherry group: S1=147.60±6.20 s, S2=347.05±6.97 s, n =20, P <0.001; hM4Di group: S1=301.93±8.04 s, S2=158.90±5.91 s, n =20, P <0.001). N is a bar chart of the socialization time of mice in each cage for each group (mCherry group: S1=99.75±3.19 s, S2=188.86±3.57 s, n =20, P <0.01; hM4Di group: S1=169.11±6.88 s, S2=86.80±4.71 s, n =20, P <0.01). O represents the discrimination index of mice in each group (mCherry = 0.31 ± 0.02 s, n =20; hM4Di=-0.32±0.03 s, n =20, P <0.001. Data are expressed as mean ± standard error. n =20). One-way ANOVA and independent samples t-test were used: compared with S1, ns >0.05, *** P <0.01, $$$ P <0.001, ^^^ P <0.001,$$ P <0.01, ^^ P <0.01; compared with the mCherry group, ### P <0.001.

[0031] Figure 7 The following are the results of the photoactivation combined with behavioral experiments in Example 6. A shows a schematic diagram of the injection of the virus AAV-hSyn-DIO-hChR2-EYFP-WPRE or AAV-DIO-EYFP into the ACC of MSEW mice. In B, the left image shows the injection of the optogenetic virus ChR2 (green) into the ACC region of MSEW mice via AAV and the placement of the optical fiber; the right image shows immunofluorescence staining confirming that ChR2-infected neurons in CR-Cre mice are CR-positive interneurons. C is a bar chart verifying the co-labeling of CR-positive interneurons and the ChR2 virus. D is a whole-cell patch-clamp recording of blue light-induced action potential firing. E and F are in vivo multi-channel recordings of the changes in the firing rate of CR-positive interneurons carrying EYFP and ChR2 in mice under wakeful conditions (EYFP = 24.25 ± 0.75 Hz). n =8; ChR2=45.88±0.77Hz, n =8, P <0.001). G is the heatmap of social recognition in the three boxes for each group of mice, where the upper figure is for the EYFP group and the lower figure is for the ChR2 group. H is a bar chart of the time mice spent in each box (S1=303.92±7.29 s, NO=153.14±7.63 s, n =20, P <0.001). I is a bar chart of social time per cage for mice in each group (S1=175.39±5.95 s, NO=91.28±3.98 s, n =20, P <0.001). J represents the discrimination index of mice in each group (EYFP = 0.01 ± 0.04 s, n =20; ChR2=0.32±0.02 s, n =20, P <0.001). K is a heatmap of social novelty preferences for each group of mice, with the top graph representing the EYFP group and the bottom graph representing the ChR2 group. L is a bar chart of the time mice spent in each chamber (EYFP group: S1=255.35±6.39 s, S2=189.18±3.16 s, n =20, P <0.001; ChR2 group: S1=167.77±4.84 s, S2=311.27±6.03 s, n=20, P <0.001). M is a bar chart of socialization time per cage for mice in each group (EYFP group: S1=168.45±5.49 s, S2=87.42±4.24 s, n =20, P <0.001; ChR2 group: S1=84.85±3.92 s, S2=178.74±7.83 s, n =20, P <0.001). N is the discrimination index of mice in each group (EYFP = -0.32 ± 0.02 s, n =20; ChR2=0.35±0.03 s, n =20, P <0.001. Data are expressed as mean ± standard error. n =20). One-way ANOVA and independent samples t-test were used: compared with S1, ns >0.05, *** P <0.01, $$$ P <0.001, ^^^ P <0.001; compared with the EYFP group, ### P <0.001.

[0032] Figure 8 The following diagram shows the results of the photoactivation combined with electrophysiological experiment in Example 6. A is a schematic diagram of the effect of CR-positive interneurons in the ACC region on pyramidal neurons recorded by patch clamp; B is a schematic diagram of representative records of photoinduced IPSC firing under artificial cerebrospinal fluid (ACSF) with TTX, TTX+4-AP, and PTX; C is the amplitude ratio of photoinduced oIPSCs in the MSEW group mice under blue light stimulation; D is a schematic diagram of representative records of photoinduced IPSCs in the two groups of mice; E is the amplitude ratio of photoinduced IPSCs in the two groups of mice (control group = 26.86 ± 0.67 pA). n =24; MSEW group =44.96±1.06 pA, n =24; P <0.001). F shows the changes in the paired pulse ratio of the two groups of mice at different time intervals. G shows the changes in the paired pulse ratio of the two groups of mice at different time intervals (PPR). 50 ms =0.82±0.03, P <0.05; PPR 100 ms =0.48±0.19, P <0.001; PPR 150 ms =0.36±0.02, P<0.001; PPR 200 ms =0.26±0.01, P <0.001. Data are expressed as mean ± standard error. n =24). Repeated measures ANOVA and independent samples were used. t Test: Compared with the control group, *** P <0.001, ### P <0.001. Detailed Implementation

[0033] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] The present invention will be further described below with reference to the embodiments.

[0035] In some embodiments of the present invention, a method for constructing an animal model of social disorder based on Calretinin-positive interneurons is provided, which induces symptoms of social disorder in animals by intervention with apoptotic viruses and chemogenetic inhibition of Calretinin-positive interneurons in the anterior cingulate cortex.

[0036] In some embodiments of the present invention, the social disorder model animal is a CR-Cre transgenic animal that specifically expresses Cre recombinase on Calretinin-positive interneurons.

[0037] The animals are selected from zebrafish, fruit flies, rodents, sheep, dogs, or primates. The rodents include mice, rats, guinea pigs, and gerbils; the primates include baboons, cynomolgus monkeys, and rhesus monkeys. Further, the animals may be selected from zebrafish, fruit flies, mice, rats, guinea pigs, baboons, cynomolgus monkeys, rhesus monkeys, rabbits, pigs, cattle, sheep, goats, and cats.

[0038] Preferably, the animal is a rodent, more preferably a mouse, and even more preferably a C57BL / 6J mouse.

[0039] Among the animals mentioned, mice have good reproductive, developmental and environmental adaptability, and produce a large number of offspring, which can ensure the number of experimental individuals and thus obtain reliable results. Moreover, the operation is simple. Therefore, mice were used in the specific embodiments of the present invention, but are not limited thereto.

[0040] Main reagents and experimental materials All mice used were C57BL6 / J mice. The CR-Cre strain mice used in the experiment specifically express Cre recombinase in CR-positive interneurons and were donated by Professor He Miao of the Institute of Brain Science, Fudan University.

[0041] The experimental mice were kept in an environment with 12 hours of light / 12 hours of darkness, a temperature of 22 ± 2℃, and a relative humidity of 55 ± 2%, and were allowed to freely consume food and water.

[0042] Example 1 Pregnant mice housed in individual cages are designated as giving birth on the day of delivery as P0.

[0043] Model mouse treatment: The model mice were treated using the Maternal Separation with Early Weaning (MSEW) behavioral paradigm. Specifically, the offspring mice were separated from their mothers for 4 hours each day (10:00-14:00) from day 2 (P2) to day 5 (P5), and for 8 hours each day (10:00-18:00) from day 6 (P6) to day 16 (P16). During the separation period, the mother mice and the pups did not have any odor or sound contact or communication. A heating pad was placed under the pups' cages. Early weaning was carried out on day 17 (P17).

[0044] Control mice were weaned normally, and no treatment was given to mice from different litters.

[0045] Identification of CR-Cre homozygous mice: Reagents used: Tail buffer: containing 2 ml of 1 M Tris; 10 ml of 10% SDS; 10 ml of EDTA-2Na; and 1.17 g of NaCl. Adjust the pH to 8, bring the volume to 100 ml, and store at room temperature.

[0046] When the pups born to the pregnant female mice are 1-2 weeks old, cut off a 2-5 mm tail. Place the tail into an enzyme-free centrifuge tube. Then, add 250 μL of Tail buffer and 6 μL of proteinase K (Beijing Solarbio Science & Technology Co., Ltd.) to each tube, and place in a 55℃ water bath for lysis for 8-12 h. Cool the lysed liquid to room temperature and centrifuge at 12000 rpm for 15 min at 4℃. After centrifugation, discard the precipitate and retain the supernatant. Add 150 µL of isopropanol to each tube of supernatant and mix thoroughly. Centrifuge the mixed liquid again at 12000 rpm for 15 min at 4℃. Discard the supernatant and retain the precipitate. Add 1 mL of 75% ethanol to the precipitate, invert to mix the solution thoroughly, and then centrifuge at 12000 rpm for 15 min at 4℃. Finally, discard the supernatant and retain the precipitate. Let it stand for 20-30 minutes until the ethanol has completely evaporated. Add 100 μL of enzyme-free water, vortex, and store at -20°C. The primer sequences used for PCR amplification are as follows:

[0047] Add the reaction solution (1 μL each of primers, 10 μL of Premix Taq, 6 μL of ultrapure water, and 2 μL of DNA) to PCR tubes, with each tube containing 20 µL. The PCR amplification program is as follows: 34 cycles of 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 51 / 56℃ annealing for 30 s, and 72℃ extension for 30 s, followed by a final extension at 72℃ for 5 min, and storage at 12℃.

[0048] Preparation of agarose gel: Prepare a 1.5% agarose gel by heating at high temperature until it is completely dissolved and the solution is colorless and transparent. Pour this solution into a gel plate and quickly insert a comb. Allow the gel to solidify completely for subsequent electrophoresis.

[0049] Electrophoresis: After the amplification process is completed, the prepared agarose gel is placed in an electrophoresis tank containing TBE buffer for electrophoresis at 120 V for 30 min. After amplification, it is analyzed in a chemical imaging system.

[0050] Example 2: Three-box social behavior experiment This invention uses the Three-Box Social Interaction Test (TBSIT) to assess whether it can induce social impairment in mice. The Three-Box Social Interaction Test consists of two phases: Phase 1 is a social recognition test, and Phase 2 is a social novelty preference test.

[0051] The experimental method is as follows: (1) The experimental setup consisted of three interconnected transparent boxes (each measuring 60 cm × 38 cm × 20 cm), namely the middle box and the two side boxes. Openings were provided between the boxes to allow the test animals to move freely between them. The laboratory environment was kept quiet and well-lit to avoid interfering with the animals' behavior. Mice were allowed to acclimatize to the environment 1 hour before the behavioral tests.

[0052] (2) Place the experimental mouse in the middle box of the three-box setup and allow it to move freely between the three boxes for 10 minutes to adapt to the experimental environment. Then, place a strange adult mouse (S1) in a different cage into a small circular cage on one side of the three boxes, and place a novel object (NO) in a similar small circular cage on the other side. The test animal can move freely between the three boxes.

[0053] (3) During the behavioral test, the fiber optic recording system was turned on and a 10-minute video recording was performed to observe the mouse’s activities in each box, including the number of times it entered each box and the time it stayed in each box, as basic data. These indicators can reflect the test animal’s social interest, social ability and behavioral preference towards unfamiliar animals.

[0054] (4) After the recording is finished, another unfamiliar adult mouse (from a different nest and cage than the previous tool mouse, S2) is placed in an empty round cage, and the timing is started and a 10-minute video is recorded. (5) After the experiment, the behavioral videos were imported into the ANY-maze software to analyze the time and trajectory of the test mice in each cage.

[0055] (6) Based on the time the mouse spends in a specific area and the time it interacts with others, the preference index is calculated as follows: (time spent interacting with S1 - time spent interacting with NO) / (time spent interacting with S1 + time spent interacting with SE); (time spent interacting with S2 - time spent interacting with S1) / (time spent interacting with S1 + time spent interacting with SE).

[0056] Figure 1 A in the diagram is a schematic of the MSEW operating procedure. The result is as follows: Figure 1 As shown in Figure B, a DNA band was detected at 175 bp, indicating that the mouse is a CR-Cre homozygous genotype mouse. Figure 1As shown in the CF, there were significant differences in the social recognition test results between the MSEW group mice and the control group mice. Specifically, the control group mice spent significantly more time in the Stranger 1 (S1) box than in the Novelty Object (NO) box, and their social time with S1 was also significantly longer than their social time with NO. In contrast, the MSEW group mice showed no significant difference in the time spent in the S1 and NO boxes, and their social time with both S1 and NO did not show significant differences. The discrimination index of the two groups of mice showed significant differences.

[0057] like Figure 1 As shown in the GJ, the social preference test results revealed different social behaviors in the two groups of mice after being introduced into the stranger mouse 2 (S2) enclosure. The control group mice spent more time in the S2 enclosure than in the S1 enclosure, indicating a greater preference for social interaction with S2 mice than S1 mice. Conversely, the MSEW group mice significantly preferred to stay in the S1 enclosure rather than the S2 enclosure, and were also more inclined to socialize with S1 mice. A significant difference in preference indices was observed between the two groups (S1 = 0.37 ± 0.03 s, n = 20; S2 = -0.34 ± 0.03 s, n = 20, P < 0.001).

[0058] The above results indicate that in the two-stage tests of the three-box social behavior model—the social recognition test and the social novelty preference test—the control group mice performed normally, while the MSEW group mice exhibited social impairment, suggesting that the MSEW paradigm leads to a decline in the social ability of mice, and a social impairment mouse model was successfully constructed.

[0059] Example 3, Immunofluorescence staining experiment Ninety minutes after the behavioral experiment, mice were anesthetized and then perfused via the heart. The brains were first harvested and fixed. The following day, the mouse brain tissue was immersed in a 30% sucrose solution for dehydration for approximately 36 hours, maintained at 4°C, until the brain tissue settled to the bottom of the solution due to dehydration. Mouse brain sections were prepared, stained with immunofluorescence, and mounted. Images were acquired and exported under a laser confocal microscope at different magnifications. Using a mouse brain atlas, c-Fos-positive brain regions were identified and located. ImageJ's cell counting function was used to count CR-positive interneurons, c-Fos-positive cells, and positive neurons co-labeled by CR and c-Fos.

[0060] c-Fos, as a marker of neuronal activation, shows a positive correlation between its expression level and neuronal activity. This study uses immunofluorescence staining to detect c-Fos expression levels and assesses the effect of the MSEW paradigm on the activity of CR-positive interneurons in the ACC region of mice.

[0061] The results are as follows Figure 3As shown, in the social recognition test and the social novelty preference test, compared with the control group, the percentage of c-Fos positive neurons in the ACC region of MSEW mice was significantly reduced. These results indicate that the activity of CR positive interneurons in the ACC region is reduced in mice with social impairment.

[0062] Example 4: Detection of neuronal activity using fiber optic calcium signaling technology Fiber optic calcium signaling technology can accurately reveal the corresponding changes in calcium concentration during neuronal action potential generation by capturing fluctuations in fluorescence signals. This embodiment combines fiber optic calcium signal recording with behavioral experiments to visually demonstrate the activity state of a specific type of neuron during a specific behavior.

[0063] This embodiment uses fiber optic calcium signaling technology combined with a three-box social behavior test to observe intracellular calcium in CR-positive interneurons. 2+ Concentration changes. The effectiveness and specificity of GCaMP7s expression were verified by injecting adeno-associated virus pAAV-hSyn-FLEX-jGCaMP7s-WPRE virus into the ACC region, which labeled CR-positive interneurons with GCaMP7s.

[0064] The effectiveness of GCaMP7s expression (CR) + / GCaMP7s + = 85.15 ± 1.53%, n = 8) and specificity (GCaMP7s) + / CR + = 86.79 ± 1.46%, n = 8; Figure 4 The results of A and B are as follows: Figure 4 As shown in Figures A and B. Three weeks after viral expression, calcium signaling in CR-positive interneurons of mice engaging in social behavior was recorded. Results are as follows... Figure 4 As shown in C and D, in the social recognition test and the social novelty preference test, compared with the novel object (NO), the control mice showed increased mean area under the curve (ΔF / F) and peak value of the fluorescence signal of CR-positive interneurons in the ACC region when interacting with stranger 1 (S1) and when socializing with the newly appeared stranger 2 (S2). This indicates that CR-positive interneurons are activated during social interaction. In contrast, the MSEW group mice showed no difference in the social recognition and social novelty preference stages (P>0.05). Figure 4The changes in calcium indicator fluorescence intensity, the area under the curve (AUC) and peak value of ΔF / F were significantly lower in the MSEW group mice than in the control group mice during social behavior. These results indicate that during social interaction, the calcium ion fluorescence intensity and activity of CR-positive interneurons in the ACC region of the MSEW group mice were weakened. Consistent with the c-Fos results, this suggests that MSWE induces social impairment, and fiber optic calcium signaling records reduced activity of CR-positive interneurons in the ACC region of socially impaired mice in a conscious state.

[0065] Example 5: Apoptosis Virus Intervention and Chemogenetics Combined with Behavioral Science 1. Apoptosis virus intervention combined with chemogenetics and behavioral science Solution: (1) Diphtheria toxin virus was injected into the bilateral ACC brain regions of CR-Cre mice to induce apoptosis of CR-positive interneurons. Another group of mice was injected with mCherry as a control group. The apoptotic virus was pAAV-mCherry-flex-dtA-WPRE, and the viral titer was 5.54 × 10⁻⁶. 12 vg / ml.

[0066] Behavioral experiments were conducted 3 weeks after the virus began expressing its behavior. A three-box social behavioral experiment was performed and video was recorded simultaneously. The experimental procedure for the three boxes was the same as that described in Example 2, the three-box social behavioral experiment. After the experiment, the behavioral videos were imported into the ANY-maze analysis software for analysis.

[0067] (2) A chemosuppressive virus was injected into the bilateral ACC brain regions of CR-Cre mice to inhibit CR-positive interneurons. The chemosuppressive virus was pAAV-hSyn-DIO-hM4D(Gi)-mCherry, 300 nl, viral titer: 2.21 × 10⁻⁶. 13 The dose was vg / ml. After the virus injection, the needle remained at the injection site for 10 minutes, and then the mouse scalp was carefully sutured. After the procedure, the mouse was placed on a heating pad to maintain its body temperature, and returned to its original cage after waking up. Three weeks after the virus began to express, the mice were intraperitoneally injected with clozapine-N-oxide (CNO, 1 mg / ml, CNO containing 0.001 g CNO and 1 ml DMSO) 30 minutes before the behavioral experiment. The CNO needed to be dissolved in physiological saline containing 5% DMSO beforehand. The injection dose was 1 mg / kg. The behavioral experiment was conducted after the drug took effect. The behavioral experiment procedure was the same as described in Example 2, the three-box social behavior experiment. After the experimental mice adapted to the environment before the test, the behavioral recording began.

[0068] The results are as follows Figure 5As shown in Figure A, the DTA group involved injecting the apoptotic virus pAAV-mCherry-flex-dtA-WPRE into the ACC brain region of control CR-Cre mice to induce apoptosis of CR-positive interneurons. Mice in the mCherry group were selected as the control group after being injected with the mCherry virus. Immunofluorescence results are shown below. Figure 5 As shown in Figures B and C, compared with the mCherry group mice, the number of CR-positive interneurons in the ACC region of the diphtheria toxin group was significantly reduced, confirming the intervention efficiency of the apoptotic virus.

[0069] In behavioral experiments, the mCherry group mice exhibited normal social behavior: during the social recognition phase, mice spent more time in the S1 box and interacted with S1; during the social novelty preference phase, mCherry group mice spent more time in the S2 box and were more inclined to interact with S2. In contrast, the DTA group mice showed no preference for either S1 or NO in the social recognition test, with a significant difference in preference indices between the two groups; during the social novelty preference phase, mice spent more time in the S1 box than in the S2 box, and compared to S2, the time spent interacting with S1 was relatively increased, with a significant difference in preference indices between the two groups. These results suggest that CR-positive interneuron-induced apoptosis leads to social impairment in mice.

[0070] 2. Chemical inhibition combined with electrophysiology Based on the principles of apoptosis-viral intervention and chemogenetics combined with behavioral studies, the scalp was sutured after viral injection, and mice were returned to their original cages after recovery. Patch-clamp experiments were performed 3 weeks after viral expression was established. Fluorescent CR-positive interneurons were located under a microscope. Electrophysiological data were recorded after 5 min of CNO perfusion at a concentration of 5 μmol / L. The electrophysiological data were then processed using Mini Analysis and Origin 2024.

[0071] The results are as follows Figure 6 As shown in Figure A, injecting the chemosuppressive virus pAAV-hSyn-DIO-hM4D(Gi)4-mCherry into the ACC region of control CR-Cre mice induced hM4D(Gi) expression in CR-positive interneurons. The viral efficacy and specificity were as follows: Figure 6 As shown in B and C, the validity is: CR + / hM4Di = 67.91 ± 3.48%, n = 8; Specificity: hM4Di / CR + = 84.55 ± 3.05%, n = 8. Electrophysiological and behavioral tests were performed 3 weeks after viral expression. Brain slices of CNO-perfused hM4Di CR-positive interneurons were analyzed, such as... Figure 6As shown in D and E, the firing frequency of action potentials in CR-positive interneurons in the ACC region decreased after whole-cell recording perfusion. Figure 6 As shown in F and G, in vivo multichannel recordings showed a decrease in the firing rate of CR-positive interneurons after CNO injection, confirming the effectiveness of chemical inhibition.

[0072] Thirty minutes before the behavioral test, CR-Cre mice expressing hM4Di were activated by intraperitoneal injection of 1 mg / kg CNO, and their social behavior was observed using a three-box social test. Results are as follows: Figure 6 As shown in the HK diagram, compared to the mCherry group, the hM4Di group mice exhibited social impairment in both phases of the three-box social test. In the social recognition phase, Figure 6 As shown in Figures I and J, the mCherry group mice tended to remain in the S1 box and socially interact with S1, while the hM4Di group mice showed no preference for social interaction with S1 and NO (P>0.05). Figure 6 As shown in K, the two groups of mice showed differences in discrimination index performance. During the social novelty preference stage, such as... Figure 6 As shown in M ​​and N, the mCherry group mice tended to stay in the S2 box and socially interact with S2, while the hM4Di group mice spent relatively more time in the S1 box, with reduced social interaction time in both S1 and S2, and relatively more social interaction time in S1. Figure 6 As shown in Figure O, there was a significant difference in the discrimination index between the two groups of mice. In this embodiment, by using chemogenetic inhibition of CR-positive interneurons in the ACC region, the mice exhibited social impairment.

[0073] Example 6: Photoactivation combined with behavioral science 1. Photoactivation experiment The photoactivation experiment was conducted using the following method: Virus injection: 300 nl of optogenetic virus pAAV-hSyn-DIO-hChR2(H134R)-EYFP-WPRE was injected into the bilateral ACC brain regions of CR-Cre mice in the MSEW group. The viral titer used was 6.63 × 10⁻⁶. 12 Vg / ml. Fiber optic cables were implanted, and after 3 weeks of viral expression, optogenetic stimulation was performed simultaneously with a three-box social behavioral experiment. A fiber optic jumper was connected to a ceramic insert on the mouse's head, and the blue light stimulation parameters were set as follows: light intensity 5 mW, stimulation frequency 20 Hz, with continuous light stimulation throughout the behavioral experiment. Optogenetic stimulation was initiated simultaneously with the start of recording the three-box social behavioral experiment. After the experiment, the behavioral videos were imported into ANY-maze analysis software for analysis.

[0074] The results are as follows Figure 2As shown in Figures A-E, compared with the control group mice, the MSEW group mice showed no significant differences in resting membrane potential and evoked action potential half-width. Figure 2 As shown in Figure F, the threshold potential of mice in the MSEW group increased, as... Figure 2 As shown in G, the base strength increases, as... Figure 2 As shown in Figure H, the evoked action potential frequency decreased. These results indicate that the intrinsic excitability of CR-positive interneurons in the ACC region of socially impaired mice is reduced.

[0075] The frequency and amplitude of spontaneous inhibitory post-synaptic currents (sIPSCs) in fluorescently labeled CR-positive interneurons in the ACC region of two groups of mice were observed. Figure 2 The diagram in I represents a representative record of sIPSCs, as shown below. Figure 2 As shown in Figure J, compared to the control group, the frequency of sIPSCs (CR-positive interneurons) in the ACC region of mice in the MSEW group was reduced, such as... Figure 2 As shown in K, the amplitude remains unchanged. This indicates that the inhibitory synaptic transmission efficiency of CR-positive interneurons in socially impaired mice is weakened, and the synaptic transmission function of CR-positive interneurons in socially impaired mice is impaired.

[0076] 2. Optogenetics combined with in vivo multichannel electrophysiological recording The photoelectrode was implanted into the mouse brain tissue and pushed into the ACC position. After the signal stabilized, the electrode and cranial screw were fixed with dental cement. After the dental cement hardened, the mouse was placed on a heating pad to await recovery. After the mouse awoke, it was housed individually and electrophysiological recordings were performed one week later. The electrode connector on the mouse's head was connected to the amplifier, and the outer end of the photoelectrode was connected to a blue laser generator. Laser stimulation was applied through an optical fiber for 10 seconds. Data was sampled at a frequency of 40 kHz, the fluorescence signal was filtered, and the output current signal was converted. The observed characteristic local field potential signals and neuronal firing characteristics were imported into an offline strainer for processing and analysis.

[0077] The results are as follows Figure 7 As shown in Figure A, firstly, in the MSEW group: CR-Cre mice were injected with pAAV-hSyn-DIO-hChR2-EYFP-WPRE into the ACC region, selecting CR-positive interneurons in the ACC region to express ChR2; in the control group: CR-Cre mice in the MSEW group were injected with EYFP virus (EYFP group). Figure 7 As shown in Figure C, the effectiveness results of ChR2 virus expression are: CR + / ChR2=84.71±2.07%, n =8; Specificity result: ChR2 / CR+ = 82.44 ± 3.00%, n = 8; Three weeks after viral expression, electrophysiological and behavioral experiments were conducted. For example... Figure 7 As shown in Figure D, in in vitro electrophysiological experiments, ChR2-expressing CR-positive interneurons were irradiated with 20 Hz, 473 nm blue light, and whole-cell recordings were used to induce action potential firing. Synchronous firing of CR-positive interneurons was induced by blue light stimulation using both in vitro and in vivo electrophysiological recordings. Figure 7 As shown in E and F, in vivo and in vitro electrophysiological experiments confirmed that optogenetics can selectively activate CR-positive interneurons in the ACC region, inducing an increase in neuronal firing rate.

[0078] In behavioral tests, socially impaired mice were subjected to continuous stimulation of CR-positive interneurons with 20 Hz, 473 nm blue light, and their social behavior was observed in both groups. In the EYFP group, CR-positive interneurons were irradiated with blue light, and in social recognition tests, the mice exhibited the following behaviors: Figure 7 As shown in H and I, the time the EYFP group spends in chambers S1 and NO is ( P >0.05, n = 20) and no preference for time spent socially interacting with S1 and NO ( P >0.05, n = 20), mice exhibited social recognition impairment; such as Figure 7 As shown in H and I, blue light irradiation of the ChR2 group mice resulted in increased time spent in the S1 chamber and increased social interaction time with S1; as Figure 7 As shown in Figure J, there was a significant difference in the discrimination index between the two groups of mice.

[0079] During the novelty-seeking phase of social interaction, such as Figure 7 As shown in Figure L, the EYFP group spent more time in the S1 box and interacted with S1 mice for a longer period than with S2 mice, indicating that the mice exhibited social novelty preference disorder. In contrast, the ChR2 group mice spent more time in the S2 box than in the S1 box and showed a greater inclination to interact with S2 mice. Figure 7 As shown in Figure N, there was a significant difference in the discrimination index between the two groups of mice. These results indicate that optogenetic activation of CR-positive interneurons in the ACC region can alleviate social impairment in mice.

[0080] 3. Photoactivation combined with electrophysiological assay Based on the photoactivation experiment in step 1, the scalp was sutured after viral injection, and the mice were returned to their original cages after recovery. Patch-clamp experiments were performed 3 weeks after viral expression. Fluorescent CR-positive interneurons were located under a microscope, and electrophysiological data were recorded by applying blue light stimulation. The specific procedures for the patch-clamp experiment were as follows: Mice were anesthetized with isoflurane and perfused with cold cutting solution. The brain was then quickly removed and stabilized in cutting solution containing a mixed gas (95% O2 + 5% CO2) for 1-2 minutes. Coronal sections (300 µm) containing the ACC brain region were then prepared using a vibratory microtome. After incubation, glass electrodes were drawn, and well-formed, plump neurons with fluorescent expression were located under a microscope under a fluorescent light source. These neurons were then sealed and their membranes ruptured. In current-clamp mode, no blue light stimulation was applied to the model group and the control group. The resting membrane potential, action potential half-width, frequency, threshold potential, and current base intensity were recorded, as well as the action potential frequency induced by blue light stimulation (20 Hz) in CR-positive interneurons. The optogenetic activation procedure was as follows: In whole-cell mode, with a clamp voltage of 0 mV, 1 μM of sodium channel blocker tetrodotoxin (TTX) and 1 mM of potassium channel blocker 4-aminopyridine (4-AP) were perfused for 5 min to block the original oIPSCs and record the newly induced oIPSCs; after rinsing for 5 min, GABA was perfused again. A PTX 50 μM was administered for 5 min as an inhibitor. Inhibitory postsynaptic currents (oIPSCs) induced by pyramidal neurons forming synaptic connections with CR-positive interneurons under blue light stimulation (473 nm, 5-10 mW, pulse width 2 ms) were recorded, as well as the photo-evoked paired-pulse ratio (PPR) in both groups of mice. Action potential frequencies after CNO perfusion were recorded. Images and signals were acquired. Electrophysiological signals were amplified, filtered to remove noise, and then acquired using Patchmaster software. Electrophysiological data were imported into Mini Analysis and Origin 2024 for analysis and processing.

[0081] The results are as follows Figure 8 As shown in Figure A, after injecting ChR2 virus and expressing it for 3 weeks, an in vitro electrophysiological experiment was performed. The photoinduced inhibitory postsynaptic currents of pyramidal neurons surrounding fluorescently labeled neurons were recorded under a microscope. Figure 8 As shown in Figure B, under 0 mV clamping, blue light stimulation of CR-positive interneurons in the MSEW group mice induced IPSCs, and the IPSC current could be completely blocked by TTX (ACSF = 292.07 ± 6.92 pA). n= 24; TTX = 2.88 ± 0.30 pA, n = 24; P <0.001), under the combined action of 4-AP, it can partially recover IPSCs (TTX = 2.88 ± 0.30 pA, n = 24; TTX +4-AP = 277.20 ± 6.01 pA, n = 24; P <0.001), and was completely blocked again under the action of PTX (TTX + 4-AP = 277.20 ± 6.01 pA, n = 24; PTX = 1.81 ± 0.20 pA, n = 24; P <0.001), confirming that the current is GABA. A Receptor-mediated, indicating the existence of monosynaptic inhibitory connections between CR-positive interneurons and pyramidal neurons.

[0082] Analyze the amplitude of light-induced IPSCs in the two groups of mice, such as Figure 8 Results from D and E showed that, compared to the control group, the MSEW group mice had increased oIPSCs amplitude, such as Figure 8 As shown in Figures F and G, the light-induced PPR in the MSEW group mice was lower than that in the control group mice at different time intervals. This result suggests that blue light stimulation of CR-positive interneurons in the MSEW group mice can enhance inhibitory synaptic transmission to pyramidal neurons.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of social disorder based on Calretinin-positive interneurons, characterized in that the animal develops symptoms of social disorder by intervening with apoptotic viruses or by inhibiting Calretinin-positive interneurons in the anterior cingulate cortex through chemogenetics.

2. The method for constructing an animal model of social disorder based on Calretinin-positive interneurons according to claim 1, characterized in that the social disorder model animal is a CR-Cre transgenic animal that specifically expresses Cre recombinase on Calretinin-positive interneurons; the animal is selected from zebrafish, fruit flies, mice, rats, guinea pigs, baboons, cynomolgus monkeys, rhesus monkeys, rabbits, pigs, cattle, sheep, goats, and cats.

3. The method for constructing an animal model of social disorder based on Calretinin-positive interneurons according to claim 1, characterized in that the apoptotic virus is diphtheria toxin, and apoptosis of Calretinin-positive interneurons is induced by injection of diphtheria toxin.

4. The method for constructing an animal model of social disorder based on Calretinin-positive interneuron targets according to claim 1, characterized in that, by injecting the chemosuppressive virus pAAV-hSyn-DIO-hM4D(Gi)4-mCherry into the anterior cingulate cortex of CR-Cre animals, chemogenetic inhibition of Calretinin-positive interneurons in the anterior cingulate cortex is achieved.

5. The method for constructing an animal model of social disorder based on Calretinin-positive interneurons according to claim 4, characterized in that a chemosuppressive virus pAAV-hSyn-DIO-hM4D(Gi)4-mCherry is injected into the anterior cingulate cortex of CR-Cre animals to induce Calretinin-positive interneurons to express hM4D(Gi), leading to apoptosis of Calretinin-positive interneurons and inducing social disorder in the animals.

6. The method for constructing an animal model of social disorder based on Calretinin-positive interneuron targets according to claim 1, characterized in that the social disorder is a social dysfunction.

7. The method for constructing an animal model of social disorder based on Calretinin-positive interneuron targets according to claim 1, characterized in that the animal model of social disorder has specific behavioral phenotypic defects.

8. The model constructed by the method for constructing a social impairment model based on Calretinin-positive interneuron targets as described in claim 1.

9. The application of the social disorder model constructed by the method of claim 8 based on Calretinin-positive interneuron targets in screening drugs or methods for treating social disorders.

10. The application of Calretinin-positive interneurons as targets in screening drugs or methods for treating social disorders.

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