WO2023151701A1

WO2023151701A1 - System for evaluating treatment effect of mental diseases and/or neurodegenerative diseases

Info

Publication number: WO2023151701A1
Application number: PCT/CN2023/083089
Authority: WO
Inventors: 朱心红; 朱旻桢; 李环宇
Original assignee: 人工智能与数字经济广东省实验室(广州)
Priority date: 2022-07-12
Filing date: 2023-03-22
Publication date: 2023-08-17
Also published as: CN117437971A

Abstract

Disclosed in the present invention is a system for evaluating the treatment effect of mental diseases and/or neurodegenerative diseases. According to the present invention, it is found that the early activation of PV positive neurons in the primary auditory cortex can generate an effect of preventing depression in advance; when resisting the external persistent or intermittent but long-term adverse stimulation, the living body resists depression-like behavior which may be caused by long-term stimulation by means of increasing the activity of PV positive neuronal cells of the primary auditory cortex. Therefore, the drug for specifically activating PV positive neurons in the primary auditory cortex can effectively treat depression, and a new school of thought and direction are provided for the development of drugs for depression.

Description

A system for assessing the effectiveness of treatments for psychiatric and/or neurodegenerative diseases

Technical field:

The invention relates to the technical field of psychiatric diseases, in particular to a system for evaluating therapeutic effects of psychiatric diseases and/or neurodegenerative diseases.

Background technique:

At present, a large number of people around the world are plagued by mental illness, causing a serious social burden. Moreover, with the rapid development of society, the social burden caused by mental illness continues to grow rapidly. Among mental illnesses, depression is a common mood disorder, with significant and persistent depression as the main clinical feature, and it is the main type of mood disorder.

Depression (Major depressive disorder) has become a public health problem due to its high incidence, high disability rate and serious suicidal tendency. With the rapid development of society, the increase of stress factors such as fast-paced life and high competitive pressure have led to an increase in the incidence of depression year by year. Among the social burdens caused by all mental illnesses, depression ranks first in both developed and developing countries. Depression has a high disability rate and will become the first cause of disability. Anhedonia is the main symptom of depression, usually accompanied by difficulty concentrating, abnormal appetite, sleep disturbance, sense of worthlessness and even recurrent suicidal tendencies (the vast majority of suicides originate from depression and are minor deaths primary reason), which has brought endless misery to patients and their families, and seriously endangered human life and health.

Stress is the main cause of depression, and stress can seriously disrupt the balance of an individual's physiology and psychology. Stress-related psychopathology, such as major depressive disorder, anxiety, conduct disorder, and post-traumatic stress disorder, disrupts behavior, cognition, and social interaction, and exacerbates people's responses to stressful events. However, traumatic stress does not affect everyone similarly. While susceptible individuals are less adaptive to stress and express inappropriate responses that can become a persistent stressful state, individuals with depressive insensitivity can perceive adversity as the smallest threat and develop adaptive physical and psychological responses . Over the past decade, there has been increasing attention to the phenomenon of depressive non-vulnerability: most people maintain normal mental and physical functioning and avoid serious mental illness even in times of great stress and trauma. Resilience, in this context, refers to an individual's ability to avoid the negative social, psychological, and physical consequences of extreme stress that would otherwise compromise their mental or physical health. Recent reports suggest that resilience in humans represents an active, adaptive process rather than merely the absence of pathological responses in more susceptible individuals.

Regarding the treatment of depression, since the pathogenesis of depression is still unclear, at this stage, the clinical treatment of depression is still based on drug treatment. However, the use of antidepressants faces problems such as slow onset of effect, poor medication compliance, low drug efficacy, serious side effects, and high recurrence rate after drug withdrawal. Therefore, finding a new antidepressant method with rapid onset is a key scientific issue in current depression research.

Past research on mental illness and emotional disorders mainly focused on the prefrontal cortex, hippocampus, amygdala, etc. in the brain, and believed that these are important for regulating memory, fear, excitement, anxiety, etc. Abnormalities in these brain regions are closely related to anxiety, depression and other mental diseases. But no one had found that the sensory cortex, especially the primary sensory cortex, was involved in emotion regulation. It is generally believed that the primary auditory cortex (A1) is mainly involved in the processing of auditory information, and no specific studies have shown that neurons in the auditory cortex may be involved in the processing of emotional information.

Neurons in the brain, namely neuron cells, are the most basic structural and functional units of the nervous system. According to the type of neurotransmitter it releases, it is mainly divided into: glutamatergic excitatory neurons that release excitatory neurotransmitters such as glutamate and neurons that release inhibitory neurotransmitters 4-aminobutyric acid (GABA ) of GABAergic inhibitory interneurons (GABAergic interneurons). CaMKII (calcium/calmodulin-dependent protein kinase II, calcium/calmodulin-dependent protein kinase II) is specifically expressed in excitatory neurons, so CaMKII positive neurons are excitatory neurons.

GABAergic inhibitory interneurons can be further subdivided into microalbumin positive neurons (PV-positive neurons), somatostatin-expressing neurons (SST-positive neurons) according to the characteristic proteins expressed by them. neurons) and others.

In the primary auditory cortex, PV-positive neurons and SST-positive neurons accounted for about 65% of inhibitory neurons. Previously, no research has shown that the above neurons in the auditory cortex are involved in neurological diseases such as depression and anxiety. And it is generally believed that the important symptoms of diseases such as depression are significant and persistent depression, loss of interest, etc. Therefore, the commonly used drugs are tricyclic imipramine and norepinephrine that increase the excitability of neurons and increase the activity of excitatory neurons. stetin reuptake inhibitors, such as sitines.

The Cre/loxP recombinase system is a widely used in vivo gene targeting technology, which can achieve conditional, inducible and spatiotemporal specific gene targeting. Inducible Cre recombinase (cyclization recombinase estrogen receptor; Tamoxifen, Cre-ERT) is a kind of fusion protein containing the ligand-binding region mutant (ERT) of estrogen receptor (ER) and Cre recombinase. After intraperitoneal injection of Tamoxifen, the metabolite 4-OHT (estrogen analogue) of Tamoxifen binds to ERT, allowing Cre-ERT2 to enter the nucleus to exert Cre recombinase activity. The FLP/FRT recombinase system is similar to the Cre/loxP recombinase system, in which FLP is a recombinase (flippase, Flp), and its recognition site is FRT (short flippase recognition target, FRT).

Invention content:

The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art, and to provide the application of the activator of the PV-positive neuron cells of the primary auditory cortex in the preparation of antidepressant drugs.

The first object of the present invention is to provide a product for detecting the activation of PV-positive neuron cells in the primary auditory cortex in the preparation and diagnosis of mental diseases and/or neurodegenerative diseases, assessment of the risk of mental diseases and/or neurodegenerative diseases and and/or applications in products that assess the efficacy of treatments for psychiatric and/or neurodegenerative diseases.

A second object of the present invention is to provide a system for evaluating the effects of treatments for psychiatric and/or neurodegenerative diseases.

The third object of the present invention is to provide the application of the product for activating or enhancing the activation of PV-positive neuron cells in the primary auditory cortex in the preparation of antipsychotic and/or neurodegenerative disease products.

The fourth object of the present invention is to provide a product that activates or enhances the activation of neurons projecting to the medial geniculate nucleus of the primary auditory cortex in the preparation of products for antipsychotic and/or neurodegenerative diseases.

The fifth object of the present invention is to provide a product that activates or enhances the projection of the medial geniculate nucleus to the primary auditory cortex in the preparation of products for antipsychotic and/or neurodegenerative diseases.

A sixth object of the present invention is to provide a tangible computer-readable medium encoded with a program.

The seventh object of the present invention is to provide a method for antipsychotic and/or neurodegenerative diseases.

In order to achieve the above object, the present invention is achieved through the following schemes:

Products that detect the activation of PV-positive neurons in the primary auditory cortex are used in the preparation of diagnosis of psychiatric diseases and/or neurodegenerative diseases, assessment of the risk of psychiatric diseases and/or neurodegenerative diseases and/or evaluation of mental diseases and/or neurodegenerative diseases Application of products for the treatment of degenerative diseases.

Preferably, the PV-positive neuron cells in the primary auditory cortex specifically activate only the PV-positive neuron cells in the primary auditory cortex that receive MG projections.

Preferably, the psychiatric diseases and/or neurodegenerative diseases include but are not limited to depression, major depressive disorder, schizophrenia, bipolar disorder, post-traumatic stress disorder, anorexia, anxiety disorder, dementia (including Alzheimer's disease) Alzheimer's disease), drug addiction, deficit and hyperactivity disorder Or one or more of autism, etc.

As a specific embodiment, the mental disease and/or neurodegenerative disease is depression.

A system for evaluating the therapeutic effect of mental illness and/or neurodegenerative disease, including an information collection module, a data processing module and a result output module;

The information collection module is used to obtain the activation of PV positive neuron cells in the primary auditory cortex of the patient before and after treatment, or to obtain the activation of PV positive neuron cells in the primary auditory cortex of the treatment group and the control group;

The data processing module is used to compare the activation degree of PV positive neuron cells in the primary auditory cortex before and after the treatment, or to compare the activation degree of PV positive neuron cells in the primary auditory cortex of the treatment group and the control group;

The result output module is used to output the treatment effect:

The activation degree of PV-positive neurons in the primary auditory cortex after treatment is higher than that of PV-positive neurons in the primary auditory cortex before treatment, and the treatment is effective; otherwise, the treatment is ineffective;

Alternatively, the activation degree of PV-positive neuron cells in the primary auditory cortex of the treatment group is higher than that of the primary auditory cortex in the control group, and the treatment is effective; otherwise, the treatment is ineffective.

Use of the product for activating or enhancing the activation of PV-positive neuron cells in the primary auditory cortex in the preparation of products for antipsychotic and/or neurodegenerative diseases.

Preferably, the activator specifically activates PV-positive neuronal cells of the primary auditory cortex.

Use of a product that activates or enhances activation of neurons projecting to the medial geniculate nucleus (MG) of the primary auditory cortex in the preparation of a product for antipsychotic and/or neurodegenerative diseases.

Preferably, said activation or enhanced activation is a sustained, or intermittent but long-term activation of the product of medial geniculate nucleus (MG) neurons projecting to primary auditory cortex, or intermittent but long-term activation of MG neurons projecting to primary auditory cortex.

Preferably, the medial geniculate nucleus neurons projecting to the primary auditory cortex specifically activate only PV-positive neurons in the primary auditory cortex.

Use of a product that activates or enhances the projection of the medial geniculate nucleus to the primary auditory cortex in the preparation of a product for antipsychotic and/or neurodegenerative diseases.

Preferably, the psychiatric diseases and/or neurodegenerative diseases include but are not limited to depression, major depressive disorder, schizophrenia, bipolar disorder, post-traumatic stress disorder, anorexia, anxiety disorder, dementia (including Alzheimer's disease) Alzheimer's disease), drug addiction, deficit and hyperactivity disorder, or autism.

Preferably, the projection of the medial geniculate nucleus to the primary auditory cortex is the projection of the medial geniculate nucleus to the PV-positive neurons of the primary auditory cortex.

More preferably, the antipsychotic and/or neurodegenerative disease is treatment and/or prevention of mental disease and/or neurodegenerative disease.

More preferably, the device is one or more of transcranial magnetic stimulators, brainwave recorders, vibrators, sound frequency generators, and current stimulators.

A tangible computer readable medium encoded with a program which, when executed, causes a device to perform a treatment that The method includes at least one of the following methods:

Activation or enhanced activation of PV-positive neuronal cells in the primary auditory cortex;

Activation or enhanced activation of medial geniculate nucleus neurons projecting to the primary auditory cortex;

Activation or enhanced activation of MG projections to primary auditory cortex;

The treatment of suppressing the activity of medial geniculate nucleus neurons for less than 14 days is a course of treatment;

To treat the subject's psychiatric disease and/or neurodegenerative disease or reduce the possibility of the subject developing a psychiatric disease and/or neurodegenerative disease.

Preferably, the treatment comprises one or more courses of treatment.

As a specific embodiment, the inhibition of the neuron activity of the medial geniculate nucleus for less than 14 days is: 8 times of light for 10 seconds per day for three days inhibits the activity of the neurons of the medial geniculate nucleus.

Preferably, the device is an instrument that stimulates neurons through nuclear magnetic resonance, sound waves, vibration or electric current.

A method for evaluating the effect of antidepressant treatment. Experimental animals are modeled with a social failure model. After successful modeling, they are randomly divided into two groups, the control group and the treatment group, and the experimental animals in the treatment group are treated with antidepressants. After treatment, the control group is compared. The PV positive neuron cell activation degree of the primary auditory cortex of experimental animals in the treatment group and the treatment group, the PV positive neuron cell activation degree of the primary auditory cortex of the treatment group, is higher than the PV positive neuron cell activation degree of the primary auditory cortex of the control group, and the treatment is effective. , otherwise the treatment is ineffective.

The present invention also claims a method for treating and/or preventing depression, using one or more of the following methods for treatment:

Activation or enhanced activation of projections of the medial geniculate nucleus to the primary auditory cortex;

To inhibit the activity of neurons in the medial geniculate nucleus, one course of treatment is less than 14 days, but not limited to one course of treatment.

Preferably, the activation or enhanced activation is performed using one or more of transcranial magnetic stimulators, brain wave recorders, vibrators, sound frequency generators, and current stimulators.

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

The present invention finds that early activation of PV-positive neurons in the primary auditory cortex can prevent depression in advance; when resisting external continuous or intermittent but long-term adverse stimuli, the organism can increase the activity of PV-positive neurons in the primary auditory cortex. Depression-like behaviors that may result from resistance to long-term stimuli. Therefore, drugs that specifically activate PV-positive neurons in the primary auditory cortex can effectively treat depression, and provide new ideas and directions for the development of depression drugs.

Description of drawings

Figure 1 shows that PV positive neurons in the primary auditory cortex of depression non-susceptible mice are activated in the social failure model: a is the co-staining result of CamkII positive neurons in the primary auditory cortex and c-fos protein in the social failure model; b is the co-staining result of SST-positive neurons in the primary auditory cortex and c-fos protein in the social failure model; c is the co-staining result of PV-positive neurons in the primary auditory cortex and c-fos protein in the social failure model; d is the three states of the mice in the social failure model; e is the calcium signal result of the PV positive neurons in the primary auditory cortex of the RES mice (depression non-susceptible mice); f is the calcium signal result of the SS mice (depression-susceptible mice) Calcium signal results of PV-positive neurons in the primary auditory cortex of ).

Figure 2 shows that compound-targeted activation of PV-positive neurons in the primary auditory cortex can produce antidepressant-like effects: a is the compound-targeted activation of PV-positive neurons in the primary auditory cortex, and its antidepressant effect is verified by FST experiments; Cortical PV-positive nerves The neuron does not affect the motor ability of the mice; c is the targeted activation of the compound's PV-positive neurons in the primary auditory cortex can reverse the depression-like behavior.

Figure 3 shows that the chemical genetic method specifically activates the PV positive neurons of the primary auditory cortex to produce an antidepressant-like effect: a shows that the chemical genetic method specifically activates the PV positive neurons of the primary auditory cortex to verify its antidepressant effect through FST experiments; b The specific activation of PV-positive neurons in the primary auditory cortex by chemical genetic method does not affect the exercise ability of mice; c is the specific activation of PV-positive neurons in the primary auditory cortex by chemical genetic method can significantly reduce the possibility of stress-induced depression-like behavior Sex; d is chemical genetic method to specifically activate PV-positive neurons in the primary auditory cortex can reverse depression-like behavior.

Figure 4 shows that PV-positive neurons in the primary auditory cortex are mainly regulated by neurons in the MG brain area: a is the main brain area of PV-positive neurons projecting to the primary auditory cortex; b is the PV-positive neurons projecting to the primary auditory cortex MG neuron types.

Figure 5 shows that the specific activation of neurons in the MG brain area projecting to the primary auditory cortex can produce antidepressant-like effects: a is the specific activation of neurons in the MG brain area projecting to the primary auditory cortex to verify its antidepressant effect through FST experiments; b The specific activation of neurons in the MG brain area projecting to the primary auditory cortex does not affect the motor ability of mice; c is the specific activation of neurons in the MG brain area projecting to the primary auditory cortex can significantly reduce the possibility of stress-induced depression-like behavior Sex; d, specific activation of neurons in the MG brain area projecting to the primary auditory cortex can reverse depression-like behavior.

Figure 6 shows that the specific activation of PV-positive neurons in the primary auditory cortex that accepts MG brain area projections can produce antidepressant-like effects: a is the specific activation of PV-positive neurons in the primary auditory cortex that accepts MG brain area projections through FST Experiments verify its antidepressant effect; b is the specific activation of PV positive neurons in the primary auditory cortex that accepts the projection of the MG brain area does not affect the motor ability of the mice; c is the specific activation of the primary auditory cortex that accepts the projection of the MG brain area of PV-positive neurons can reverse depression-like behavior.

Figure 7 shows that antidepressant individuals show the characteristics of decreased neuron activity in the MG brain area projected to the primary auditory cortex in the early stage of stress stimulation: a is SS mice (depression susceptible mice) in the early stage of stress stimulation, Calcium activity of neurons in the MG brain area projecting to the primary auditory cortex; b is the calcium activity of neurons in the MG brain area projecting to the primary auditory cortex in RES mice (depression non-susceptible mice) in the early stage of stress stimulation; c is The calcium signal activity when the mice were challenged (Attack) in the early stage (2nd day) was negatively correlated with the Interaciotn Ratio value after 10 days of modeling.

Figure 8 shows that short-term inhibition of neurons in the MG brain area projecting to the primary auditory cortex can activate PV-positive neurons in the primary auditory cortex, thereby having an antidepressant effect: a shows that short-term inhibition of neurons in the MG brain area projecting to the primary auditory cortex can activate PV-positive neurons in the primary auditory cortex; b, short-term inhibition of MG neurons projecting to the primary auditory cortex can significantly reverse the depression-like phenotype.

Detailed ways:

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

1. Immunofluorescence labeled antibody

Primary antibody: mouse-derived anti-PV (Swant Company, product number: 235), mouse-derived anti-CamkIIα (Invitrogen Company, product number: MA1-048), rabbit-derived anti-c-fos (CST Company, product number: 2250s)

2. Experimental animals

Male C57BL/6J mice (8-12 weeks old) used in the experiment were from the Experimental Animal Center of Southern Medical University (permit number: SCXK-2011-0015, Guangzhou, China).

The sources of transgenic mouse strains used are as follows:

PV-Cre (No. 008069), SST-Cre (No. 013044), Camk2-Cre ^ERT (No. 012362) and ROSA26-tdTomato Reporter mice (No. 007909) were purchased from The Jackson Laboratory, USA.

PV; tdTomato mice are obtained by mating PV-Cre and ROSA26-tdTomato, SST; tdTomato mice are obtained by mating SST-Cre and ROSA26-tdTomato, CamkⅡ-Cre ^ERT ; tdTomato mice are obtained by mating CamkⅡ-Cre ^ERT and ROSA26 -tdTomato mating obtained.

All transgenic mouse lines were of C57BL/6J background.

3. Viruses and article numbers used: The following viruses are from Wuhan Privy Technology Co., Ltd.: rAAV-Ef1a-DIO-mCherry-WPRE-pA (article number PT-0013), rAAV-Ef1a-DIO-hM3D(Gq)-mCherry-WPRE- pA (Cat. No. PT-0042),

rAAV-Ef1a-DIO-GCaMp6f-WPRE-hGH pA (Cat. No. PT-0106-9),

rAAV-hSyn-CRE-WPRE-hGH polyA (Cat. No. PT-0136),

RV-ENVA-ΔG-dsRed (Product No. R01002),

rAAV-Ef1a-DIO-His-EGFP-2a-TVA-WPRE-pA (Cat. No. 9-21-K180918),

rAAV-CMV-DIO-RVG-WPRE-pA (Cat. No. 9-991-K180726),

rAAV-nEF1a-fDIO-eNpHR3.0-mCherry-WPRE-hGH polyA (Cat. No. PT-1261),

rAAV2/R-hSyn-NLS-FLP-bGH pA (Cat. No. PT-0133).

The following viruses are from Shanghai Taiertu Biotechnology Co., Ltd.:

AAV2/1-CAG-FLEX-Flpo-WPRE-pA (Cat. No. S0273-9),

AAV2/9-hEF1a-fDIO-mCherry-WPRE-pA (Cat. No. S0553-9),

AAV2/9-hEF1a-fDIO-hM4D(Gi)-mCherry-ER2-WPRE-pA (Cat. No. S0336-9),

AAV2/9-hEF1a-fDIO-hM3D(Gq)-mCherry-ER2-WPRE-pA (Cat. No. S0335-9),

AAV2/2Retro-CAG-FLEX-Flpo-WPRE-pA (Cat. No. S0273-2/R).

4. Animal feeding conditions

The experimental mice were all raised in exhaust-ventilated closed cages (EVC), 4 to 5 per cage, and the feeding environment followed the standard laboratory environment, with the temperature controlled at 24°C and a 12-hour day and night cycle (7:00A.M to 7:00AM). 00P.M), with free access to food and water. Before the animal behavior test was carried out, all the mice were touched for 3-4 days (2 times/day, 5min/time). Animal behavior testing was carried out between 1:00P.M and 5:00P.M, and all animal experiments strictly followed the relevant regulations of the China Animal Ethics Committee.

5. Gene identification

After the offspring of the transgenic mice were born, a small amount of tissue (ears, toes, and tail) was cut, and the DNA was extracted by boiling the DNA extraction solution for 1 hour, and then the genetic identification of the transgenic mice was carried out by PCR and agarose gel electrophoresis.

6. Immunofluorescence staining

Brain slice tissue processing and immunohistochemical methods were carried out according to the methods previously used in our laboratory. After deep anesthesia, the mice were perfused with 0.1M PBS and 4% formaldehyde solution. After the perfusion, the brains were dissected out, fixed in 4% formaldehyde solution for 4-6 hours, washed with tap water for 1 hour, and finally soaked in 30% saturated sucrose solution. After the sugar precipitation of the mouse brain was completed, the Leica cryostat (CM1850, Lecia, Nussloch, Germany) was used to slice the brain, and the coronal brain slices (thickness: 40 μm) containing A1 were cut. The brain slices were first blocked with goat serum blocking solution containing 0.3% triton X-100 for 2 hours, then incubated with the primary antibody overnight (4°C), then rinsed with 0.1M PBS (5min×3) and then incubated with the secondary antibody at room temperature in the dark 2 hours. Finally, brain slices were mounted with DAPI-containing mounting medium (Vectro Laboratories Inc.). Eight slices containing A1 from a single mouse brain were selected for colocalization analysis of c-fos with CamkⅡ, PV and SST, respectively.

Immunofluorescence imaging was performed using a Nikon A1R confocal microscope (Nikon Instruments Inc, Japan), and positive cell counts were performed using Image J analysis software (US National Institutes of Health).

7. Stereotaxic technology (microinjection, buried tube)

After the mice were anesthetized, they were fixed on a brain stereotaxic instrument, the hair on the top of the head was removed, the skin was disinfected, the median incision was made, and the bregma was exposed, and the positioning was performed according to the mouse brain atlas. For in vivo adenovirus injection, mice were injected with 0.1 μl of virus bilaterally in MG and 0.15 μl bilaterally in A1, using a 33GA microsyringe needle (Hamilton) and a microsyringe pump (Stoelting, Wood Dale, IL) to inject 0.03 μl The virus was injected at a rate of min ^-1 . Position the needle of the microsyringe to MG (AP=-2.92mm; ML=±1.95mm; DV=3.5mm), A1 (AP=-2.45mm; ML=±4.25mm; DV=0.8mm), double A cranial drill was used to drill a hole above the lateral MG/Au1, and the needle was inserted as deep as needed, and the virus was injected at a rate of 0.03 μl min ^-1 . After the injection, the needle was left for 5 minutes, slowly withdrawn, and finally sutured. All experiments were performed 3 weeks after virus administration. After the experiment, the virus injection site was confirmed by intraperitoneal injection of CNO (2 mg/kg), and the expression of mcherry was detected.

When cannulation was performed using stereotaxic techniques, the cannulas were implanted bilaterally in A1 (AP = -2.45 mm; ML = ±4.25 mm; DV = 0.7 mm). Relevant pharmacological experiments were performed after the mice recovered for 1 week.

8. Social Failure Model

The social failure model is carried out with reference to the existing literature. The social defeat model was performed using CD1 retired breed mice with aggressive behavior. The experimental mice were challenged by different CD1 retired breed mice for 10 minutes every day for 10 consecutive days.

After each challenge, the experimental mice and the CD1 retired breed mice were separated by a transparent plexiglass partition with air holes, and they were rotated before being challenged the next day. The control mice were also raised in two cages, separated by a transparent plexiglass partition with air holes, and rotated once a day.

Social Interaction Detection: Social interaction was detected 24 hours after the last aggressive behavior (Day 11). The detection process consists of two 2.5min. In the first 2.5 minutes, the experimental mice were put into a new test box and allowed to move freely, with a small plexiglass frame with air holes on one side of the test box. Then, after it was put back into the original cage for 1min, the mouse was put back into the detection box again, and the activity of the second 2.5min was recorded. At this time, a strange CD1 retired species ( Target). The activity time of mice in the interaction area was tracked using Ethovision 7.0 software (Noldus, Wageningen, Netherlands). (interaction area time, target)/(interaction area time, no target)×100 is defined as Interaction Ratio.

Depression susceptible group (SS) and depression non-susceptible group (RES) were distinguished by Interaction Ratio. Interaction Ratio less than 100 was the depression-susceptible group, and Interaction Ratio greater than or equal to 100 was the depression-non-susceptible group. In the experiments after modeling, the mice were housed in single cages.

All attacks take place between 16:00 and 17:00. The social interaction behavior test was carried out between 18:00 and 24:00, and the mice to be tested were put into the test room for dark adaptation 1 hour in advance.

9. Forced swimming experiment

The forced swimming experiment was carried out according to the existing reports. The mice whose behavior was to be tested were put into the testing room 1 hour in advance to adapt. A cylindrical transparent glass cylinder (45cm high, 19cm inner diameter) is used for detection, and water is added (water depth 23cm, water temperature 22-25°C). The mice were gently put into the cylinder, allowed to swim freely in the cylinder for 6 minutes, and the immobility time (Immobility) of the mice after 4 minutes was recorded. A higher mouse immobility time indicates a higher degree of depression in the mouse, and conversely, a lower immobility time indicates that the mouse is more resistant to depression.

10. Open field experiment

The mice whose behavior was to be tested were put into the testing room 1 hour in advance to adapt. The detection device is an open detection box (Accuscan Instruments, Columbus, OH), surrounded by transparent plexiglass. Put the mouse into the detection box and allow it to freely explore for 5min. The total amount of mouse movement The distance (Total distance) was analyzed using Versmax analysis software.

11. Data Analysis

All data were statistically analyzed using GraphPad Prism statistical software. Statistical analysis methods include Teo-tailed Student’s t-tests, One-way ANOVA, and Two-way ANOVA. A significant difference was considered when the P value was less than 0.05. The experimental results were drawn using GraphPad Prism software.

Example 1 The number of PVs in the primary auditory cortex brain area in the brain of depressive non-sensitive mice increased and the activity was significantly enhanced

1. Experimental method

In order to explore the reason why mice in the depression non-susceptible group in the social failure model have the ability to resist depression, we used fluorescent reporter mice labeled with different types of neurons (that is, the corresponding neurons will express red fluorescence), social failure model After modeling, observe whether various neurons in the primary auditory cortex of the mouse brain are activated or inhibited, that is, analyze whether a certain type of neuron in the primary auditory cortex of the brain is involved in antidepressant.

The specific method is as follows:

Use the fluorescent reporter mice for each neuron obtained by hybridization:

PV;tdTomato mice (PV positive neuronal reporter mice), SST;tdTomato mice (SST positive neuronal reporter mice) and CamkII-Cre ^ERT ;tdTomato mice (red fluorescent reporter mice for excitatory neurons) .

Three kinds of fluorescent reporter mice were modeled in the classic depression model - social failure model (CSDS), and were divided into: (1) Normal control group (CTRL): no social failure model (CSDS) Failed; (2) Depression-susceptible group (SS): the Interaction Ratio of those who have experienced social failure is less than 100; (3) Depression-non-susceptible group (RES): the Interaction Ratio of those who have experienced social failure is greater than 100.

Immediately early genes are a group of genes that are rapidly and transiently activated after a series of external stimuli. The earliest immediate genes found in cells include c-fos, c-myc and c-jun. The expression of c-fos is usually observed by immunofluorescence staining to judge whether the neuron is activated or in a silent state: neurons expressing c-fos protein indicate that the neuron is activated rapidly after being stimulated by the outside world, otherwise, there is no c-fos Protein neurons are silent.

Immunofluorescent staining of c-fos was performed on the primary auditory cortex of the three neuron fluorescent reporter mice grouped according to the results of the interactive behavior detection after the above modeling, and the three types of neurons labeled by the above three fluorescent reporter mice were analyzed The social failure model models whether neurons are activated or silenced following this external stimulus.

2. Experimental results

As shown in Fig. 1a, in excitatory neurons, CamkⅡ positive neurons were also detected by c- The number of fos-labeled cells and the proportion of total CamkⅡ-positive neurons were significantly reduced, indicating that the excitatory neurons in the primary auditory cortex of the depression non-susceptible group (RES) were significantly inhibited.

As shown in Figure 1b and Figure 1c, in inhibitory neurons, PV positive neurons in depression non-susceptible mice (RES) compared with normal control group (CTRL) were also marked by c-fos The number of cells accounted for a significant increase in the proportion of the total PV-positive neurons; while the depression-susceptible group (SS) compared with the normal control group (CTRL), the number of PV-positive neurons was also marked by c-fos, The proportion of total PV-positive neuronal cells did not change significantly. However, in SST-positive neurons, another type of inhibitory neurons, the proportion of co-labeled cells of SST-positive neurons and c-fos was lower in the primary depression-sensitive group (RES) than in the normal control group (CTRL). There were no changes in the auditory cortex. The above indicated that the activity of CamkII positive neurons (excitatory neurons) in Figure 1a was inhibited by the activation of PV positive neurons.

To sum up, in the mice of the depression non-susceptible group produced by the classical depression model, the activity of PV-positive neurons in the primary auditory cortex was significantly enhanced, and the activation of PV-positive neurons in this brain area played a dominant role (combined with CamkⅡ and compared to SST-positive neurons), can suppress the activity of excitatory neurons, showing the key role of activating PV-positive neurons in the primary auditory cortex in antidepressant applications.

In addition, it was found that before the social failure model was established, the mice in the depression non-susceptible group had an antidepressant-like phenotype without any treatment, and the activation of PV-positive neurons was characteristic of depression non-susceptible mice. That is, the activation of PV-positive neurons is an instinctive response to anti-depression in antidepressant individuals. From this, it can be judged that early activation of PV-positive neurons in the primary auditory cortex can prevent depression in advance.

Example 2 Real-time detection of PV positive neuron changes in the primary auditory cortex of depression non-susceptible mice

Calcium ion imaging technology (Calcium imaging) is the use of calcium ion indicators to detect the concentration of calcium ions in tissues. Calcium ion imaging is primarily used in the study of the nervous system, where changes in calcium ions indicate neuronal activity. Calcium ion optical fiber recording (Fiber Photometry) is a commonly used method for recording calcium signals in vivo (in vivo), which can be used to examine and record changes in cell activity in real time.

The activity of PV-positive neurons in the primary auditory cortex of mice was detected in real time by calcium ion imaging.

1. Experimental method

In the primary auditory cortex of PV-Cre mice, stereotaxic microinjection of Cre enzyme-dependent virus rAAV-Ef1a-DIO-GCaMp6f-WPRE-hGH pA (product number PT-0106-9) expressing calcium ion indicator and optical fiber recording Fiber optic plug, the virus is cleaved by Cre enzyme after injection to express the GCaMP6f calcium indicator protein with green fluorescence, which is excited by excitation light with a wavelength of 470nm, and the fluorescence signal is collected and recorded by the corresponding program in the computer.

After 14 days, a 10-day social defeat model was performed while the activity of PV-positive neurons in the primary auditory cortex was recorded.

After 10 days of modeling, the depression-susceptible group (SS) and depression-non-susceptible group (RES) of the mice were also separated by social interaction detection.

To analyze the cell activity changes of PV-positive neurons in the primary auditory cortex of the depression-susceptible (SS) and depression-non-susceptible (RES) mice in various states of the model, and to analyze the neurons that experienced social failure. Which process changes in a stressful environment.

2. Experimental results

During the modeling process of the social failure model, the model mice are mainly divided into three states (see Figure 1d for the schematic diagram): CD1 retired breed mice attack C57BL/6J mice (Attack); The CD1 retired breeder approached (attempted to approach) the C57BL/6J mouse (Approach); the CD1 retired breeder left the C57BL/6J mouse (Non-approach).

As shown in Figure 1e, the activity of PV-positive neurons in the primary auditory cortex of mice in the depression non-susceptible group (RES) was slightly enhanced when they were attacked in the early stage (2 days), and after 5 days of modeling, CD1 decommissioned The approach of the breeder rats made the mice feel threatening stimuli. When responding to this approach (Approach) stimuli, the activity of PV-positive neurons in the primary auditory cortex of the mice increased significantly and lasted until the end of the last 10 days of modeling.

However, the activity of PV-positive neurons in the primary auditory cortex of mice in the depression-susceptible (SS) group (Fig. Metacells are not involved.

The above results show that when fighting against external continuous or intermittent but long-term adverse stimuli (stress response), organisms can resist depression-like behaviors that may be caused by long-term stimuli by increasing the activity of PV-positive neurons in the primary auditory cortex. Therefore, specifically activating PV-positive neurons through NMR, sound waves, vibration or current can have the effect of treating mental and neurological diseases such as depression.

Example 3 Activation of PV-positive neurons in the primary auditory cortex can produce antidepressant-like effects

1. Experimental method

Based on the experimental results of Example 1 and Example 2, in individuals with the ability to resist stress stimuli and exhibit antidepression (RES), Activation of PV-positive neurons in the primary auditory cortex plays a key role. Based on this conclusion, pharmacological methods were further used to activate PV-positive neurons in the primary auditory cortex for behavioral testing.

Muscimol is an activator of inhibitory receptors (GABA), which can broadly activate inhibitory neurons, but some studies have found that muscimol is selective for PV-positive neurons in inhibitory neurons, so muscimol Can cause increased activity of PV-positive neurons.

(1) Using commonly used experimental models for rapid detection of antidepressant effects: forced swimming test (FST) and open field test (OFT), to study whether pharmacological activation of PV-positive neurons in the primary auditory cortex can exhibit antidepressant effects.

Specifically: the cannula was implanted in the bilateral primary auditory cortex of C57BL/6J mice. Three days later, 1 microliter of 10mM muscimol (medication Muscimol group) was directly administered to the primary auditory cortex through the buried cannula, and the control group was given the same treatment. Dosage of normal saline (control Saline group), 10min after the forced swimming test (FST). Three days later, the drug was administered again (the Muscimol group was given 1 microliter of 10mM muscimol, and the control Saline group was given 1 microliter of normal saline), and the open field test (OFT) was performed 10 minutes later.

Further, C57BL/6J mice were used to establish a 10-day social failure model to study the antidepressant effect of pharmacologically activating PV-positive neurons in the primary auditory cortex.

Specifically: after the 10-day social failure model (CSDS) was modeled, the social interaction behavior was tested, and the mice in the depression-susceptible group were obtained. The bilateral primary auditory cortex of the depression-susceptible group mice was implanted, and randomized three days later. They were divided into two groups and given 1 microliter of 10mM muscimol (model-making drug Muscimol group) and the same dose of normal saline (model-making control group saline group), and social interaction behavior was tested again 10 minutes later.

2. Experimental results

The results are shown in Figure 2a. In the FST experiment, compared with the control Saline group, the muscimol group activated the PV-positive neurons of the primary auditory cortex, and the immobility time (Immibolity) was significantly reduced after ten minutes of administration, indicating that the drug Mice in the muscimol group had obvious antidepressant-like effects, indicating that the targeted activation of GABAergic inhibitory interneurons (PV-positive neurons) in the primary auditory cortex by drugs has a rapid antidepressant effect.

The results are shown in Figure 2b. In the OFT experiment, compared with the control Saline group, the total distance of the mice in the muscimol group did not change, indicating that muscimol did not affect the locomotion ability of the mice. Again, the reduction in immobility time in the FST of the mice in the treatment group was a significant antidepressant phenotype rather than a change in motor capacity. That is, the drug muscimol group has significant antidepressant performance compared with the control Saline group.

The results are shown in Figure 2c. After the social failure model was established, the Interaction Ratio was significantly increased after the primary auditory cortex of the mice in the depression-susceptible group was treated with muscimol, while the mice given normal saline had no behavioral phenotype changes. It shows that after modeling, the mice can maintain a depressed state, and the drug can significantly improve the depression-like behavior of the mice after activating PV-positive neurons in the primary auditory cortex, indicating that drug activation of PV-positive neurons in the primary auditory cortex has a significant effect on treating depression. effect of such behavior.

In summary, the use of GABA receptor agonists (muscimol) to activate the inhibitory neurotransmitter GABA receptors in the primary auditory cortex (including PV-positive neurons) can cause rapid antidepressant effects in mice; in addition, GABA receptor agonists Targeted activation of GABA receptors in the primary auditory cortex (including PV-positive neurons) also reversed the behavioral phenotype of depression-susceptible mice. It is suggested that activation of GABA neurons (including PV-positive neurons) in the primary auditory cortex by using drugs such as GABA agonists can rapidly treat depression.

Example 4 Chemical Genetic Technology Specific Activation of PV-positive Neurons in the Primary Auditory Cortex Can Produce Antidepressant-Like Effects

Chemical genetics technology (or pharmacogenetics technology) is a receptor activated by specific drugs (Designer receptors exclusively activatedby designer drugs, DREADDs) technology, using it to change the structure of the G protein-coupled receptor-acetylcholine receptor, so that it can only be treated by a specific compound, clozapine-N-oxide (Clozapine-N-oxide, CNO) activate or inhibit. The modified hM3Dq was induced by CNO to depolarize neurons, promote the firing activity of neurons, and enhance the excitability of neurons.

1. Specific activation of PV-positive neurons in the primary auditory cortex can produce antidepressant-like effects.

By using a chemogenetic approach to specifically activate PV-positive neurons in the primary auditory cortex, we explored whether this approach can produce antidepressant effects.

1. Experimental method

The transgenic mice using PV-cre were randomly divided into two groups, and the chemical genetic virus rAAV-Ef1a-DIO-hM3D(Gq )-mCherry-WPRE-pA (Product No. PT-0042) (hM3Dq group) and the control virus rAAV-Ef1a-DIO-mCherry-WPRE-pA (Product No. PT-0013) (mCherry group) that only expresses red fluorescent protein after infection of cells . After 21 days, 2 mg/kg CNO was injected intraperitoneally, and the forced swimming test (FST) was carried out 35 minutes later. After 3 days, 2 mg/kg CNO was injected intraperitoneally, and the open field test (OFT) was carried out after 35 minutes. Exhibits the effect of an antidepressant phenotype.

2. Experimental results

The results in Figure 3a show that after the injection of CNO in the hM3Dq group, the PV-positive neurons are activated, and the immobility time (Immobility) of the mice in forced swimming is significantly reduced; while the results in Figure 3b show that the total distance of the movement shows The locomotor capacity of the mice was not affected and locomotor activity was unaffected. It indicated that specific activation of PV-positive neurons in the primary auditory cortex could produce antidepressant-like effects.

2. The effect of specifically activating PV-positive neurons in the primary auditory cortex to prevent depression

1. Experimental method

The chemical genetic virus rAAV-Ef1a-DIO-hM3D(Gq)-mCherry-WPRE-pA (product number PT-0042) (hM3Dq group) and the control virus rAAV- Ef1a-DIO-mCherry-WPRE-pA (Cat. No. PT-0013) (mcherry group).

The social failure model was established for 10 days. During the 10-day modeling process, 2 mg/kg CNO was injected intraperitoneally twice a day in the morning and evening, so that the PV-positive neurons in the primary auditory cortex were always activated. After 10 days of modeling, the social interaction behavior test was carried out. Compare the proportion of depression non-susceptible mice in the hM3Dq group injected with chemical genetic virus and the mcherry group injected with control virus after the same modeling.

2. Experimental results

The results in Figure 3c show that the social interaction test after social failure model modeling shows that the proportion of non-susceptible group (Interaction Ratio greater than 100) in the mcherry group injected with control virus is 55%, while the non-susceptible group (Interaction Ratio greater than 100) in the hM3Dq group injected with chemical genetic virus The proportion of the susceptible group was 82.7%, showing an obvious upward trend, and the total Interaction Ratio of the hM3Dq group was significantly higher than that of the mcherry group.

The above shows that when receiving adverse stimuli and maintaining in a stressful environment, the continuous activation of PV-positive neurons in the primary auditory cortex can significantly increase the proportion of mice in the non-depression-susceptible group, that is, reduce the occurrence of stress-induced depression and prevent depression. Depression after stress. Activating the PV-positive neurons in the primary auditory cortex can resist the occurrence of depression caused by long-term and continuous stress stimulation.

3. The therapeutic effect of activating PV-positive neurons in the primary auditory cortex after the onset of depression

1. Experimental method

The chemical genetic virus rAAV-Ef1a-DIO-hM3D(Gq)-mCherry-WPRE-pA (Cat. No. PT-0042) (hM3Dq group) and the control virus were injected into the primary auditory cortex of two groups of randomly assigned PV-cre mice. rAAV-Ef1a-DIO-mCherry-WPRE-pA (Product No. PT-0013) (mcherry group).

After 21 days, the social failure model was established, and the depression-susceptible mice with Interaction Ratio less than 100 were obtained. According to the virus injected 21 days ago, they were divided into the depression-susceptible hM3Dq group and the depression-susceptible mcherry group. All were given intraperitoneal injection of CNO to activate PV-positive neurons in the primary auditory cortex of depressed mice in the hM3Dq group. The social interaction behavior detected 35 minutes after the intraperitoneal injection, the depression-like behavior of the mice was observed.

2. Experimental results

The results in Figure 3d show that Pre-SI is the Interaction Ratio measured for the first time after the model was established to separate the depressed mice (Interaction Ratio is less than 100), and Post-SI is the Interaction Ratio measured after 35 minutes of administration of CNO.

The depressive phenotype of the mice in the mcherry group injected with the control virus did not improve after administration of CNO, indicating that the mice would maintain depression-like behavior after the model was established, while the injection of the virus itself had no effect, and CNO itself had no antidepressant effect. In the group injected with the chemical genetic virus hM3Dq, the Interaction Ratio of SS mice in Pre-SI was increased after CNO was given to activate PV-positive neurons in the primary auditory cortex (Post-SI after CNO injection), and the depression-like phenotype was significantly improved, indicating that specific Sexual activation of PV-positive neurons in the primary auditory cortex significantly reversed the depressive phenotype, leading to an antidepressant phenotype.

In summary, in the case of long-term or frequent exposure to external stress stimuli in life, specific activation of PV-positive neurons in the primary auditory cortex by chemical genetics can prevent and reduce depression-like neurons after stress stimuli. The generation of behavior can greatly reduce the incidence of depression; if the PV-positive neurons in the primary auditory cortex are activated after depression occurs, it can also relieve and treat depression symptoms. This provides a new way for clinical disease treatment, and finds an effective means to prevent the occurrence of mental and neurological diseases.

Example 5 PV-positive neurons in the primary auditory cortex are mainly regulated by neurons in the MG brain region

A neural circuit tracing method for retrograde transmonosynaptic rabies virus (Rhabdoviridae, RV). After RV infects the central system, it mainly labels neurons and barely labels glial cells. The replication-deficient recombinant RV constructed based on the infectious clone of the RV vaccine strain Sad-B19 has low toxicity and high safety, can clearly mark the fine morphology of neurons, and realizes the neural network through the reverse complementation strategy Retrograde tracing of connections across single-level synapses. The use of Cre transgenic mice combined with Cre-LoxP to control the expression of TVA and G protein AAV helper virus can realize the expression of TVA and G protein only in specific types of neurons in specific regions, so that RV-EnvA-ΔG can be used to achieve specific types of neurons. Reverse labeling across single-level synapses.

Determining how PV-positive neurons in the primary auditory cortex are regulated can increase the pathways and means of activating PV-positive neurons in A1.

1. PV-positive neurons in the primary auditory cortex are mainly regulated by neurons in the MG brain area

1. Experimental method

By using a retrograde transmonosynaptic rabies virus (Rhabdoviridae, RV) neural circuit tracing method, the brain regions and neurons controlling the PV in the primary auditory cortex were identified.

Specifically, on day 1, PV-cre mice were stereotaxically microinjected into the primary auditory cortex the cre expression-dependent AAV virus rAAV-Ef1a-DIO-His-EGFP-2a- TVA-WPRE-pA (Cat. No. 9-21-K180918), and AAV virus rAAV-CMV-DIO-RVG-WPRE-pA (Cat. No. 9-991-K180726) expressing RVG for assisting rabies virus retrograde across monosynapses .

On the 15th day, the same location was injected with the modified pseudorabies virus RV-ENVA-ΔG-dsRed (Product No. R01002), which recognizes TVA and transports it retrogradely along the axon of RVG to the upper neurons to express the red fluorescent protein reporter gene carried .

On the 21st day, perfusion, postfixation, sugar precipitation and whole brain cryosection were performed.

By analyzing the brain area where the red fluorescence is located, we can know which brain area the PV-positive neurons in the primary auditory cortex receive projections from.

2. Experimental results

As shown in Figure 4a, the brain areas of PV-positive neurons projecting (input) to the primary auditory cortex mainly include the primary auditory cortex (A1) (41.17%), and the surrounding secondary auditory cortex, including: ventral Secondary auditory cortex (AuV) (16.69%), dorsal secondary auditory cortex (AuD) (15.86%). In addition, among other brain regions, the medial geniculate nucleus (MG) was the main projection brain region, accounting for 16.58%, and the rest totaled 9.7%.

Thus, it was found that, except for the internal projections of the auditory cortex, most of the external projections of PV-positive neurons to the primary auditory cortex came from the MG.

Therefore, by enhancing the projection of MG to the primary auditory cortex or enhancing the projection of MG to the PV-positive neurons of the primary auditory cortex, the excitability and activity of PV-positive neurons can also be enhanced to achieve the purpose of antidepressant or treatment of depression .

2. The neurons in the MG brain area that regulate the primary auditory cortex are excitatory neurons

1. Experimental method

The characteristic protein of excitatory glutamatergic neurons - glutamate transporter 2 gene (Vglut2, also known as Slc17a6) is expressed in multiple brain regions. Slc17a6-cre mice were used to label excitatory neurons, Gad2-cre mice were used to label GABAergic inhibitory interneurons, and retrograde-projecting AAV2/2Retro-CAG-FLEX-Flpo was injected into the primary auditory cortex of two kinds of cre mice -WPRE-pA (Cat. No. S0273-2/R), and inject AAV2/9-hEF1a-fDIO-mCherry-WPRE-pA (Cat. No. S0553-9) in MG at the same time, perfuse the slices 21 days later, and observe the red fluorescent labeling of MG.

2. Experimental results

As shown in Figure 4b, there is no red fluorescence in the MG brain area of Gad2-cre mice, indicating that the neurons projecting to the primary auditory cortex are not inhibitory neurons; while the MG brain area of Slc17a6-cre mice has strong red fluorescence, indicating that Neurons projecting to the primary auditory cortex are excitatory neurons.

In summary, the MG is the main outer projection (non-auditory cortex) brain area of PV-positive neurons in the primary auditory cortex, and those projecting to the primary auditory cortex are excitatory neurons.

Example 6 Specific activation of neurons in the MG brain region projecting to the primary auditory cortex can produce antidepressant-like effects

1. Experimental method

The neurons projecting to the MG of the primary auditory cortex were specifically activated by chemical genetic techniques, and their antidepressant effects were detected.

Specifically, C57BL/6J mice were randomly divided into two groups, and the retrovirus rAAV2/R-hSyn-Cre-WPRE-hGH-pA was stereotaxically injected into the primary auditory cortex. Strong, and at the same time retrograde transport along the axon to the synaptic terminal receiving the projection. The retrovirus can infect primary auditory cortex neurons and transport retrogradely along the dendrites to the synaptic terminal, and the Cre enzyme can be transported across the synapse to the upper-level neurons that receive the terminal.

It can be known from Example 5 that the external projection brain area received by the PV-positive neurons of the primary auditory cortex is mainly MG. Further, while injecting the retrovirus, the two groups of mice were stereotaxically microinjected into the MG brain area with the chemical genetic virus rAAV-Ef1a-DIO-hM3D(Gq)-mCherry-WPRE-pA (product number PT-0042) (hM3Dq group ) and the control virus rAAV-Ef1a-DIO-mCherry-WPRE-pA (product number PT-0013) expressing only red fluorescence (mcherry group). 21 days after the virus injection, all mice were intraperitoneally injected with CNO 2mg/kg, and the forced swimming test (FST) was performed 35 minutes after the intraperitoneal injection. After 3 days, the intraperitoneal injection of CNO was performed again. Carry out modeling of social failure model (refer to embodiment 4 for specific experimental process).

2. Experimental results

The results are shown in Figure 5a. Due to the activation of MG neurons projecting to the primary auditory cortex, the mice in the hM3Dq group significantly decreased the immobility time (Immobility) in the forced swimming test. This rapid detection method showed an increase Neuronal activity in the MG projecting to the primary auditory cortex has antidepressant effects.

In addition, the results are shown in Figure 5b, there is no difference in the total distance of the two groups of mice, indicating that the spontaneous activity of the mice has not changed, that is, this chemical genetic method will not affect the organism while antidepressant and demonstrated that the reduced immobility time in 5a was an antidepressant phenotype rather than a change in motor capacity.

Figure 5c shows that during the modeling process of the social failure model, the mcherry group of the control virus and the hM3Dq group of the chemical genetic virus were intraperitoneally injected with CNO once a day in the morning and evening. The social interaction test after modeling showed that the proportion of the non-susceptible group (Interaction Ratio greater than 100) in the normal control mcherry group was 50%, and the proportion of the non-susceptible group in the hM3Dq group was 68.4%, showing a significant upward trend, and the hM3Dq group The overall Interaction Ratio was significantly increased compared with the control group.

The above shows that when receiving adverse stimuli and maintaining in a stressful environment, the continuous activation of MG neurons projecting to the primary auditory cortex can significantly increase the proportion of mice in the depression non-susceptible group, indicating that continuous or intermittent frequent response In the stimulating environment, the activation of MG neurons projecting to the primary auditory cortex can resist the generation of depression after external stimulus stress.

Pre-SI is the Interaction Ratio measured for the first time after the model was established to separate the depressed mice (Interaction Ratio is less than 100), and Post-SI is the Interaction Ratio measured after 35 minutes of administration of CNO. The results in Figure 5d show that the depressive phenotype of the mice in the mcherry group injected with the control virus was not improved after administration of CNO, indicating that the control injection of the virus had no effect and CNO itself had no antidepressant effect. In the hM3Dq group, after CNO was given to activate the MG neurons projecting to the primary auditory cortex, the Interaction Ratio of SS mice in Pre-SI was significantly improved (Post-SI after CNO injection), and the depression-like phenotype was significantly improved, indicating that the activation projected to the primary auditory cortex. MG neurons in the primary auditory cortex can significantly reverse the depressive phenotype, making them exhibit an antidepressant phenotype.

The above results show that activation of MG neurons projecting to the primary auditory cortex has an antidepressant effect; in a continuous or intermittent but long-term stress environment, activation of MG neurons projecting to the primary auditory cortex can reduce external stress stimuli eventually lead to depression. Therefore, the method of activating MG neurons projecting to the primary auditory cortex can effectively treat depression and other mental diseases, and early application before the disease can prevent the occurrence of depression and other mental and neurological diseases.

Example 7 Specific activation of PV-positive neurons in the primary auditory cortex receiving MG brain region projections can produce antidepressant-like effects

1. Experimental method

Combining the LP/FRT recombinase system with the Cre/loxP recombinase system specifically activated only PV-positive neurons in the primary auditory cortex that received MG projections.

The specific method is as follows:

Stereotaxic microinjection of the chemogenetic virus AAV2/9-hEF1a-fDIO-hM3D(Gq)-mCherry-ER2-WPRE-pA (Catalog No. S0335-9) dependent on Flp enzyme expression in the primary auditory cortex using PV-cre mice hM3Dq group) or the control virus AAV2/9-hEF1a-fDIO-mCherry-WPRE-pA expressing only red fluorescence (product number S0553-9) (mcherry group). At the same time, the two groups of mice were stereotaxically microinjected into the MG brain area with Cre enzyme-dependent expression of Flp enzyme virus AAV2/1-CAG-FLEX-Flpo-WPRE-pA (Cat. No. S0273-9) , the expression of Cre enzyme in PV-positive neurons of PV-cre mice can lead to the expression of Flp enzyme projected from MG, thereby causing the expression of hM3D (Gq) protein in the primary auditory cortex, and the intraperitoneal injection of CNO activates the projection of MG in the primary auditory cortex PV-positive neuronal cells.

21 days after the virus injection, all mice were intraperitoneally injected with CNO 2mg/kg, and the forced swimming test (FST) was performed 35 minutes after the intraperitoneal injection. After 3 days, the intraperitoneal injection of CNO was performed again. After 35 minutes, the behavior of the open field test (OFT) was tested. Carry out modeling of social failure model (refer to implementation case 4 for specific experimental process).

2. Experimental results

As shown in Figure 6a, activation of PV-positive neurons that receive MG projections in the primary auditory cortex (hM3Dq group) can significantly reduce the immobility time (Immobility) of mice in the forced swimming test. In addition, the two groups of mice There was no difference in total distance (Figure 6b), indicating that the exercise ability of the mice was not affected, that is, this chemical genetic method did not affect the exercise ability of the organism while it was anti-depressant. It was shown that only activation of the PV in the part of the primary auditory cortex that receives MG projections can have a significant antidepressant effect.

In the Pre-SI group, the depressed mice were detected for the first time after modeling (Interaction Ratio was less than 100), and in the Post-SI group, the Interaction Ratio was measured after 35 minutes of administration of CNO. As shown in Figure 6c, all mice were subjected to the social defeat model after virus injection, and intraperitoneal injection of CNO in the detected SS mice activated PV-positive neurons in the part of the primary auditory cortex that received MG projections. The depressive phenotype of mice in the control mcherry group was not improved after administration of CNO, indicating that injection of the control virus had no effect and that CNO itself had no antidepressant effect. In the hM3Dq group, the Interaction Ratio of SS mice in Pre-SI was significantly improved after CNO was given to activate PV-positive neurons in the primary auditory cortex that received MG projections (Post-SI after CNO injection), and the depression-like phenotype was significantly improved, indicating that Activation of PV-positive neurons in the MG-projecting portion of the primary auditory cortex significantly reversed the depressive phenotype, rendering it antidepressant.

To sum up, specific activation of PV-positive neurons that accept MG projections in the primary auditory cortex can have an antidepressant effect, and can reverse the established depressive symptoms. This precise regulation method can be used clinically. Greatly reduce the possibility of side effects.

Example 8 Early antidepressant individuals receive stress stimulation and reduce the activity of neurons in the MG brain area projected to the primary auditory cortex

1. Experimental method

Further, in order to explore the dynamic process of neuron changes in the MG brain region in the observed social failure model of mice, C57BL/6J mice were used to stereotaxically microinject retrovirus rAAV2/R-hSyn-Cre-WPRE into the primary auditory cortex - hGH pA (Cat. No. PT-0136).

Simultaneously in the MG stereotaxic micro-injection of the virus rAAV-Ef1a-DIO-GCaMp6f-WPRE-hGH pA (Cat. No. PT-0106-9) expressing calcium ion indicator dependent on Cre enzyme and the optical fiber plug for optical fiber recording. The GCaMP6f calcium indicator protein with green fluorescence is expressed by cleavage with Cre enzyme, excited by excitation light with a wavelength of 470nm, and its fluorescence signal is collected and recorded by the corresponding program in the computer.

21 days after virus injection, a 10-day social failure model was started, and the activity of MG neurons projecting to the primary auditory cortex was recorded while the social failure model was being modeled.

After 10 days of modeling, the depression-susceptible group (SS) (the Interaciotn Ratio of the mice who had experienced social failure was less than 100) and the depression-non-susceptible group (RES) (the Interaciotn Ratio greater than 100).

By analyzing the changes in the cell activity of the MG neurons projecting to the primary auditory cortex in the depression-susceptible group (SS) and the depression-non-susceptible group (RES) mice in the three states of modeling (embodiment 2, Figure 1d), It can be known that when such neurons experience social defeat as a stress stimulus, the activity of MG neurons increases or decreases when the mice are stimulated by the challenge mouse CD1.

2. Experimental results

During the modeling process of the social failure model, the model mice are mainly divided into three states (see Figure 1d for the schematic diagram): CD1 retired breed mice attack C57BL/6J mice (Attack); CD1 retired species approached (attempted to approach) C57BL/6J mice (Approach); CD1 mice left C57BL/6J mice (Non-approach).

As shown in Figure 7a, the neurons in the MG brain area projecting to the primary auditory cortex in SS mice did not have large ups and downs during the modeling process, indicating that the neurons in the MG brain area did not participate in the process of depression.

However, as shown in Figure 7b, the activity of MG neurons of mice in the depression non-susceptibility group (Interaciotn Ratio greater than 100) decreased in the early days (2 days) when they were attacked (Attack), and all the modeled mice were attacked at the second day. The calcium signal when attacked (Attack) was negatively correlated with the Interaciotn Ratio value after 10 days of modeling (Figure 7c). After 5 days of modeling, the approach of CD1 retired breed mice made mice feel threatened Stimulation, in response to this (Approach) stimulation, the activity of MG neurons projecting to the primary auditory cortex of the mice increased significantly, and continued until the end of the modeling on the 10th day (Figure 7b).

The above results show that when first exposed to continuous or intermittent but long-term adverse external stimuli (when stress occurs), the organism will reduce the activity of neurons in the MG brain area, and further in the subsequent continuous stimulation environment (stress response) Increased MG neuronal activity, thereby activating projected PV-positive neuronal activity in the primary auditory cortex, counteracts depression-like behavior that may result from chronic stimulation. Therefore, by simulating this short-term inhibition of neuron activity, the purpose of enabling organisms to resist stress can be achieved, and the occurrence of depression and other mental and neurological diseases can be greatly reduced.

Example 9 Short-term inhibition of neurons in the MG brain region projecting to A1 can activate PV-positive neurons in A1

1. Experimental method

From Examples 5 and 6, it can be seen that by interfering with the MG neurons projecting to the primary auditory cortex, the PV-positive neurons of the primary auditory cortex can be activated, thereby preventing the occurrence of depression and treating depression. Simultaneously, Example 8 shows that in the early stage when individuals with antidepressant ability face external stimuli, MG neurons will be temporarily inhibited when the organism is stimulated (Attack). Subsequent anti-stress stimulation (Approach) can increase the activity of MG neurons, thereby increasing the activity of PV-positive neurons in the primary auditory cortex to achieve antidepressant effects.

Optogenetics (Optogenetics) refers to the combination of optical and genetic means to precisely control the activity of specific neurons. The activity of cells regulated by optogenetics depends on the type of light-sensitive channel protein, that is, excitatory light-sensitive channel and inhibitory light-sensitive channel. If the channel transferred into the cell is NpHR, when the cell is irradiated by yellow light, the channel will open, and a large number of anions will flow inward, resulting in hyperpolarization, which will make it difficult to emit action potentials and inhibit cell activity.

Therefore, using the method of optogenetics, the virus AAV2/2Retro-CAG-FLEX-Flpo-WPRE-pA (Cat. No. S0273-2/R) reversely expressing the FLP protein was simultaneously stereotaxically microinjected into the primary auditory cortex of PV-Cre mice. and the virus rAAV-Ef1a-DIO-GCaMp6f-WPRE-hGH pA (catalogue number PT-0106-9) expressing calcium ion indicator, and at the same time injected into MG stereotaxically dependent on Flp enzyme expression inhibitory light-sensitive channel protein Virus rAAV-nEF1a-fDIO-eNpHR3.0-mCherry-WPRE-hGH polyA (Cat. No. PT-1261), the virus expresses NpHR3.0 protein after being cleaved by Flp enzyme after injection, and can be activated by excitation light with a wavelength of 589nm Inhibits neuronal activity.

In order to simulate the attack time of mice in social defeat, the mouse MG brain area was given 8 times of 10-second photoinhibition every day, with an interval of 25 seconds between each stimulation, within 5 minutes of the total daily duration, for three consecutive days, and the total duration of the daily suppression 80 s of inhibitory projections to MG neurons in the primary auditory cortex.

On the fourth day, the mice were placed in the cage of the social failure model, and the CD1 challenge was carried out for one day: first, the mice were placed in a glass baffle with small ventilation holes and a CD1 decommissioned mouse cage, and the PV of the primary auditory cortex of the mice was detected The normal calcium activity of the positive neurons when facing the CD1 retired breed mouse approach (Pre-approach) without stimulation, then remove the baffle CD1 retired breed mouse to attack the mouse (Attack), and finally put the baffle The two mice were separated by a board, and the PV of A1 was recorded under the isolation of the baffle (Approach) and when there was no interaction (Non-approach) calcium activity.

2. Experimental results

The results in Figure 8a show that before the mice were challenged by the CD1 retired breed mice, there was no obvious activity change in the PV-positive neurons of the primary auditory cortex when the CD1 retired breed mice approached, indicating that the primary auditory cortex was safe and free of external threats. The PV-positive neurons in the auditory cortex did not produce obvious excitation or inhibition, and the transient MG neuron inhibition did not produce obvious excitation or inhibition on the PV-positive neurons in the primary auditory cortex.

However, when mice were attacked by CD1 retired breed mice, when CD1 retired breed mice approached the mice again, the mice would be stimulated by the threat of being attacked, and the mice that had experienced short-term inhibition of MG neurons in advance Under this Approach stress, mice learned to enhance the PV-positive neuron activity of the primary auditory cortex in advance, so after only being attacked once, the PV-positive neuron activity of the primary auditory cortex of the mouse was rapidly enhanced, which is consistent with Example 2 The PV calcium signal enhancement results in RES mice experienced at least 5 days were consistent (Figure 1e), that is, the mice had the ability to resist depression when they were first exposed to external stimuli.

The above results show that early short-term inhibition of MG neurons projecting to the primary auditory cortex can enable individuals to have the ability to resist external adverse stimuli in the early stage, thereby reducing the mental and neurological diseases such as depression caused by continuous stimulation in the later stage. This short-term prevention method can effectively reduce the generation of mental and neurological diseases caused by the continuous stress response of organisms.

Example 10 Short-term inhibition of neurons in the MG brain region projecting to the primary auditory cortex can treat depression

1. Experimental method

According to Example 9, before exposure to external stimuli, short-term inhibition of MG neurons projected to the primary auditory cortex can activate PV-positive neurons of the primary auditory cortex, thereby having an antidepressant effect. Whether short-term suppression has therapeutic effects in reversing depressive-like behaviors in depressed individuals.

C57BL/6J mice were randomly divided into two groups, and both groups were stereotaxically injected into the primary auditory cortex with the virus rAAV2/R-hSyn-NLS-FLP-bGH pA (product number PT-0133) reversely expressing Flp protein: photoinhibition group At the same time, stereotaxic microinjection of virus rAAV-nEF1a-fDIO-eNpHR3.0-mCherry-WPRE-hGH polyA (product number PT-1261) (eNpHR3.0 group) dependent on Flp enzyme expression inhibitory channelrhodose protein was performed in MG. After virus injection, NpHR3.0 protein was expressed by cleavage by Flp enzyme, and neuronal activity could be inhibited after being activated by excitation light with a wavelength of 589nm; the control group was microinjected with a control virus AAV2/9-hEF1a that only expressed red fluorescence in MG stereotaxic - fDIO-mCherry-WPRE-pA (Cat. No. S0553-9) (mcherry group).

21 days after the virus injection, the social failure model was established for 10 days, and the depression-susceptible (SS) mice were detected by the social interaction experiment. For the SS mice, an optical fiber was embedded in the MG brain area, and 8 times a day was given after 3 days 10 seconds of photoinhibition, 25 seconds between each stimulation, within 5 minutes per day, for three consecutive days, after the photoinhibition was given every day, the mice were put back into the cage with CD1, and CD1 was separated by a glass baffle of the air hole On, so that the mice still face the stimulation of CD1 proximity every day.

Three days after photoinhibition, the social interaction experiment was performed on the mice again to detect the depressive state of the mice.

2. Experimental results

As shown in Figure 8b, the mice in the mcherry group did not undergo the process of inhibiting MG neurons, and still maintained a depression-susceptible phenotype (Interaction Ratio was less than 100), indicating that the depressed mice after modeling could still maintain depression-like behaviors. In the eNpHR3.0 group, after 3 days of photoinhibition of MG neurons projecting to the primary auditory cortex, the social avoidance depression-like behavior of mice was significantly reversed. It shows that for individuals who have developed into depression, even if they are still exposed to the stressful stimulus environment, a short-term (3 days in this embodiment) method of inhibiting the projection of MG neurons to the primary auditory cortex for less than 14 days can produce Significant effect of treating/improving depression-like behavior.

Combining the descriptions of Examples 8 to 10: currently commonly used antidepressants take at least 4 weeks to take effect, short-term rapid application lasts for 6-8 consecutive weeks, and then long-term use for at least 6 months to maintain the effect. However, the present invention suppresses the activity of MG neurons by simulating the short-term stress stimulation less than 14 days (3 days in this embodiment), which can quickly make the organism have the ability to resist depression, and quickly reverse the depression-like behavior . This short-term suppression intervention has a better acceptance in clinical application, and patients do not need to insist on taking medicine for a long time; and for potential high-risk groups who have not yet developed the disease, giving short-term suppression intervention in advance can be effective Reduce the occurrence of mental and neurological diseases such as depression in the future.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions and ideas, they can also make There is no need to and cannot exhaustively list all the implementation manners for other changes or changes in different forms. All modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

Products that detect the activation of PV-positive neurons in the primary auditory cortex are used in the preparation of diagnosis of psychiatric diseases and/or neurodegenerative diseases, assessment of the risk of psychiatric diseases and/or neurodegenerative diseases and/or evaluation of mental diseases and/or neurodegenerative diseases Application of products for the treatment of degenerative diseases.
A system for evaluating the treatment effect of mental illness and/or neurodegenerative disease, characterized in that it includes an information collection module, a data processing module and a result output module;

The information collection module is used to obtain the activation of PV positive neuron cells in the primary auditory cortex of the patient before and after treatment, or to obtain the activation of PV positive neuron cells in the primary auditory cortex of the treatment group and the control group;

The data processing module is used to compare the activation degree of PV positive neuron cells in the primary auditory cortex before and after the treatment, or to compare the activation degree of PV positive neuron cells in the primary auditory cortex of the treatment group and the control group;

The result output module is used to output the treatment effect:

The activation degree of PV-positive neurons in the primary auditory cortex after treatment is higher than that of PV-positive neurons in the primary auditory cortex before treatment, and the treatment is effective; otherwise, the treatment is ineffective;

Alternatively, the activation degree of PV-positive neuron cells in the primary auditory cortex of the treatment group is higher than that of the primary auditory cortex in the control group, and the treatment is effective; otherwise, the treatment is ineffective.
Use of the product for activating or enhancing the activation of PV-positive neuron cells in the primary auditory cortex in the preparation of products for antipsychotic and/or neurodegenerative diseases.
Use of a product that activates or enhances activation of neurons of the medial geniculate nucleus projecting to the primary auditory cortex in the preparation of a product for antipsychotic and/or neurodegenerative diseases.
Use of a product that activates or enhances the projection of the medial geniculate nucleus to the primary auditory cortex in the preparation of a product for antipsychotic and/or neurodegenerative diseases.
The use according to any one of claims 3 to 5, characterized in that the anti-mental disease and/or neurodegenerative disease is treatment and/or prevention of mental disease and/or neurodegenerative disease.
A tangible computer-readable medium encoded with a program, wherein said program, when executed, enables a device to perform a treatment, the treatment method comprising at least one of the following methods:

Activation or enhanced activation of PV-positive neuronal cells in the primary auditory cortex;

Activation or enhanced activation of medial geniculate nucleus neurons projecting to the primary auditory cortex;

Activation or enhanced activation of MG projections to primary auditory cortex;

The treatment of neuron activity suppression of the medial geniculate nucleus for less than 14 days is a course of treatment;

To treat the subject's psychiatric disease and/or neurodegenerative disease or reduce the possibility of the subject developing a psychiatric disease and/or neurodegenerative disease.
The computer-readable medium according to claim 7, wherein the device is an apparatus for stimulating neurons through nuclear magnetic resonance, sound waves, vibrations or electric currents.
A method for antipsychotic and/or neurodegenerative diseases, characterized in that the method achieves the purpose of antidepressant by at least one of the following methods:

Activation or enhanced activation of PV-positive neuronal cells in the primary auditory cortex;

Activation or enhanced activation of medial geniculate nucleus neurons projecting to the primary auditory cortex;

Activation or enhanced activation of projections of the medial geniculate nucleus to the primary auditory cortex;

To inhibit the activity of neurons in the medial geniculate nucleus, one course of treatment is less than 14 days, but not limited to one course of treatment.
The method according to claim 9, characterized in that said antipsychotic and/or neurodegenerative disease is treatment and/or prevention of mental disease and/or neurodegenerative disease.
A method for evaluating the effect of antidepressant treatment, characterized in that the experimental animals are modeled with a social failure model, and after successful modeling, they are randomly divided into two groups, a control group and a treatment group, and the experimental animals in the treatment group are treated with antidepressants. After treatment, compare the activation degree of PV positive neuron cells in the primary auditory cortex of experimental animals in the control group and the treatment group, and the activation degree of PV positive neuron cells in the primary auditory cortex of the treatment group is higher than the activation degree of PV positive neuron cells in the primary auditory cortex of the control group. Improve, the treatment is effective, otherwise the treatment is ineffective.
A method for treating and/or preventing depression, characterized in that one or more of the following methods are used for treatment:

Activation or enhanced activation of PV-positive neuronal cells in the primary auditory cortex;

Activation or enhanced activation of medial geniculate nucleus neurons projecting to the primary auditory cortex;

Activation or enhanced activation of projections of the medial geniculate nucleus to the primary auditory cortex;

To inhibit the activity of neurons in the medial geniculate nucleus, one course of treatment is less than 14 days, but not limited to one course of treatment.
The method according to claim 12, wherein the activation or enhanced activation is performed by one or more of transcranial magnetic stimulators, brain wave recorders, vibrators, sound frequency generators, and current stimulators .