KR101881650B1 - A brain derived neurotrophic factor overexpressed animal model for depression disorder and a method for producing thereof - Google Patents

Abstract

The present invention relates to an animal model of depression overexpressing brain-derived neurotrophic factor (BDNF) and a method for producing the same, and more particularly, to a ventral tegmental area (VTA) of a mesolimbic system, To an animal model depressed to overexpress a brain-derived nerve growth factor and a method for producing the animal model. The present inventors can inhibit BDNF secretion by inducing brain-derived nerve growth factor signaling of the septal canthus nucleus which can induce depressive behavior and is increased by chronic defeat stress by administering ANA-12, and by inhibiting BDNF, . Therefore, it is expected to be able to treat neurodegenerative diseases such as depression by inhibiting the expression of BDNF gene in the part of the dorsal side of the midbrain, which is the main producing site of BDNF.

Description

Technical Field [0001] The present invention relates to an animal model of depression that overexpresses brain-derived neurotrophic factor and a method for producing the same,

The present invention relates to an animal model of depression overexpressing brain-derived neurotrophic factor (BDNF) and a method for producing the same. More specifically, the present invention relates to an animal model of depression transformed to overexpress a brain-derived nerve growth factor in a ventral tegmental area (VTA) of the mesolimbic system and a method for producing the same.

The mental impairment that occurs in modern societies is not caused by a great psychological impact or stimulation but rather by the weak but continuous and repetitive stress that occurs in everyday life. These stresses are easily overlooked by the hospital even though the patients themselves are not aware of it. The weak stimuli accumulate and cause depression in the individual. Depression is an emotional pathology that occurs irrespective of the objective situation. All of the patients' lives are covered with depressed moods, decreased interest, anhedonia, deprivation of mental exercise, impatience and despair. It is a disease that leads to suicide prayer because it is motivated suicide. Depression is manifested by a variety of physical symptoms such as decreased appetite, insomnia, constipation, loss of libido, and increased susceptibility to illness caused by impaired immune function.

To date, depression has been largely dependent on drug therapy. However, about 60% of the patients respond to a single drug, and even if the medication is used, the probability of the patient's depression not improving is about 10%. In addition, the medication treatment is delayed because the treatment effect is delayed by starting treatment and waiting until 1 month. In such a case, the physical and psychological distress of the patient is great, . In addition, 10% of patients who did not respond to medication were treated with classical electrical shock therapy or light - cranial magnetic stimulation, but the effect was not significant or significant in a considerable number of patients. In accordance with this trend, efforts have been made to study the mechanism of depression and the behavioral response by manufacturing long-term stress animal models. In particular, mental illnesses such as depression have limited research at the cellular level, so that a suitable animal model is more important.

Accordingly, the present inventors intend to provide an animal model depressing overexpression of a brain-derived neurotrophic factor (BDNF) and a method for producing the same. The animal model according to the present invention is an animal model depressed to overexpress a brain-derived nerve growth factor in the ventral tegmental area (VTA) of the mesolimbic system. It is expected to contribute greatly to the search for methods.

Prim Care Companion J Clin Psychiatry, 3 (6), 244-254 .; Br J Psychiatry Suppl (30), 17-30. Arch Intern Med, 163 (20), 2433-2445 .; J Subst Abuse, 7 (4), 481-497.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and an object of the present invention is to provide an animal model of depression that overexpresses brain-derived nerve growth factor and a method for producing the same. However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In one embodiment of the present invention, " depression disorder " is a type of mood disorder characterized by a mood disorder characterized by bipolar disorder in that there is no history of manic, mixed, . In the DSM-IV-TR, depressive disorder is divided into major depressive disorder, dysthymic disorder, and unclassified depressive disorder (APA, 2000). It is essential that depressive episodes are required for the diagnosis of depressive disorder. These illustrations are not diagnosed as independent psychiatric disorders but provide basic information for the diagnosis of depressive disorders. An essential symptom of depression is a depressed mood for at least 2 weeks or a loss of interest or pleasure in most activities. In addition to these essential symptoms, four or more symptoms should be present for at least 2 weeks in appetite and weight changes, sleep changes, mental motility irritability or retardation, fatigue, no sense of worth or guilt, reduced thinking and concentration, suicidal ideation or attempt APA, 2000). Of the essential symptoms, depressed mood may appear to be easily irritated (irritable) for adolescents and children who are not adults. In addition, activity in interpersonal activities is often reduced due to loss of interest or pleasure in activities, and appetite is reduced or increased. Sleep usually causes insomnia, but some sleep over. In the case of fatigue, it appeals to tiredness without physical exercise or difficult activities. In elderly depression, the reduction of thinking and concentration is distinguished from dementia, which usually distinguishes between progressive decline in cognitive function or sudden appearance with depression. Although all depression has a high risk of suicide, it should be handled very carefully, especially when suicidal or suicidal attempts are reported. Major depressive disorder can manifest itself in a variety of mental illnesses, according to the study, about half of individuals experiencing major depressive disorders suffer from anxiety. Anxiety symptoms adversely affect the recovery and recurrence of major depressive disorder, leading to more severe impairment and increased risk of suicide attempts (Prim Care Companion J Clin Psychiatry, 3 (6), 244-254 .; Br J Psychiatry Suppl (30), 17-30). In addition, individuals with major depressive disorders have a higher rate of substance addiction and dependence, including alcohol, and a higher percentage of attention deficit hyperactivity disorder and post-traumatic stress disorder (Arch Intern Med, 163 (20), 2433-2445. J Subst Abuse, 7 (4), 481-497.).

In one embodiment of the present invention, " social defeat stress " means stress caused by continuous experience of defeat and frustration in the social and emotional relationship. In the case of mice, the stress arising from the social sequence difference between male rats causes stress to be continuously attacked by mice with higher sequence than the experimental mice. Recently, the proportion of people experiencing social defeat stresses is increasing due to problems such as severe and persistent competition, interpersonal conflict / violence and social isolation (so-called 'bullying'), which are socially required.

In one embodiment of the present invention, the term "brain-derived neurotrophic factor (BDNF)" is a protein in the brain produced by the BDNF gene, which is one of the neuronal populations that are part of the growth factor. This factor is related to the underlying neural growth factors and can be found in the brain and its periphery. In relation to depression, a mesolimbic dopamine reward circuit consisting of the nucleus accumbens (NAc) and the ventral tegmental area (VTA) plays an important role in depressive behavior However, when the mid-sibling-compensating circuit is activated, BDNF is synthesized with dopamine in the dopaminergic neurons of the VTA and secreted into the septal nucleus. This phenomenon is known to be caused not only by compensatory stimuli such as addictive drugs but also by aversive stimuli such as stress. In particular, the role of dopamine and brain-derived neurotrophic factors in depressive behavior induced by long-term stress has not been fully elucidated and is constantly controversial.

In one embodiment of the present invention, the term " mesolimbic system " refers to a region within the brain that is called a 'compensation circuit' because it regulates mood and emotion through the production and secretion of dopamine. Nucleus accumbens, NAc ) And a ventral tegmental area (VTA).

In one embodiment of the present invention, " nucleus accumbens (NAc) " is a part that constitutes a midbrain in the brain. It controls the mood and emotion of the brain by releasing dopamine, which is responsible for pleasure and compensation by dopamine.

In one embodiment of the present invention, the term " ventral tegmental area (VTA) " It is the part of the brain that produces dopamine that regulates mood and emotion. It sends neural bundles that secrete dopamine into the nucleus of the septum.

In one embodiment of the invention, the term "dopamine" refers to the decarboxylation of dopa (3,4-dihydroxyphenylamine) in L-tyrosine, represented by the formula C ₈ H ₁₁ NO ₂ , Is a neurotransmitter that is produced in the body by Dopamine molecules bind to dopamine receptors in neurons and dopamine receptors bind to GTP-binging proteins to activate the second messenger, or to activate or inhibit specific signaling systems Lt; RTI ID = 0.0 > stimulated < / RTI > or suppressed. It becomes norepinephrine by dopamine hydroxydase. It has been found that the amount of dopamine in the brain is reduced in Parkinson's disease and the lineament of the brainstem contains more dopamine than norepinephrine. It is therefore clear that dopamine itself acts as a neurotransmitter of neurons in the central nervous system. Adrenal medulla, brain, sympathetic nervous system, lung, small intestine, liver are often contained.

In one embodiment of the present invention, " Trk (tyrosine kinase) " is a group of protein kinases that specifically phosphorylate the tyrosine residue of a protein, and is classified into a cytosolic type represented by the cancer gene product Src and an epithelial growth factor receptor EGFR), platelet-derived growth factor (PDGF), and the like. They are specific phosphorylases for tyrosine residues, but there are also weel, an inhibitor of cyclin dependent kinases, and tyrosine phosphorylases, which phosphorylate serine and threonine, such as MAP phosphorylase. TrkA, TrkB and TrkC families are known, among which TrkB is a receptor tyrosine kinase family having neurotrophin as a binding factor. There is a single tyrosine phosphorylase site in the intracellular region with a single membrane penetration structure with a molecular weight of about 140,000. In particular, neurotrophin is an essential receptor for neurons when they exhibit physiological activities such as differentiation, proliferation, and maintenance of survival.

In one embodiment of the present invention, "optogenetics " is a combination of opto- and genetics, which uses a genetic technique to express a light-sensing sensor in a desired cell or neural circuit, Means an experimental methodology for manipulating a desired neuron or neural circuit at a desired point in time using light of a wavelength. It is possible to activate or inhibit neurons depending on the kind of ion channel and light wavelength, and it is possible to change the activity of highly specific nerve cells such as expressing only specific cells in a desired site through combination of various promoters and recombinase.

In one embodiment of the present invention, "social interaction" is an evaluation of sociality as "whether the reaction to the favorable space is maintained constant regardless of whether or not the social target object exists" . Social access can be quantified on the basis of the formula of "time spent in an appealing space x 100%" without a time / social target in contact with the social target object.

In one embodiment of the present invention, the term " stress-vulnerable group or vulnerable group " means a population in which a behavioral pattern is changed by stress stimulation. In the present invention, the vulnerable group means an experimental group in which the social accessibility is changed after the social defeat stress stimulus, and the social accessibility ratio is less than 100, but is not limited thereto.

In one embodiment of the present invention, " a stress tolerant group or a resistant group " means a population in which a behavioral pattern is not changed by a stress stimulus. In the present invention, the tolerable group means an experimental group that does not change the social accessibility after the social defeat stress stimulus, and the group having the social accessibility ratio of 100 or more, but is not limited thereto.

In one embodiment of the present invention, the term " fixation of the brain fixation "refers to fixation of the head using a stereotactic plate with X, Y, and Z coordinates inscribed in order to find the exact location of the skull and brain, , And for framelink stereotactic surgery. Because the size and position of the lesion are displayed three-dimensionally on the film when the brain fixation is performed and the CT or MRI is taken, the surgeon designs the safest and most effective surgical plan to reach the lesion based on these coordinates, .

&Quot; Pharmaceutical composition "in one embodiment of the present invention means a composition to be administered for a specific purpose. For purposes of the present invention, the pharmaceutical compositions of the present invention are administered to an animal for the purpose of expressing an animal model of depression, or for the treatment of depression, and the protein and pharmaceutically acceptable carrier, excipient or diluent . ≪ / RTI > The above "pharmaceutically acceptable" carrier or excipient as referred to above means approved by the regulatory agency of the government or listed in government or other generally approved pharmacopoeias for use in vertebrates, and more particularly in humans do.

Compositions suitable for parenteral administration may be in the form of a suspension, solution or emulsion in an oily or aqueous carrier and may be prepared in the form of a solid or semi-solid and may be in the form of a suspension, stabilizer, solubiliser and / And may include formulating agents. This form can be sterilized and liquid. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria or fungi. Alternatively, the compositions of the present invention may be in the form of sterile powders for reconstitution with suitable carriers before use. The pharmaceutical composition may be in unit-dose form, in a micro needle patch, in an ampule, or in other unit-dose containers, or in multi-dose containers. Alternatively, the compositions of the present invention may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, e. G., A vehicle immediately prior to use. Immediately, the injection solutions and suspensions may be prepared from sterile powders, granules or tablets.

In some non-limiting embodiments, the compositions of the present invention may be formulated as a liquid, or may be included in the form of microspheres in a liquid. In certain non-limiting embodiments, the compositions of the present invention comprise a pharmaceutically acceptable compound and / or mixture at a concentration of between 0.001 and 100,000 U / kg. In certain non-limiting embodiments, compositions of the present invention may also contain suitable excipients such as preservatives, suspending agents, stabilizers, dyes, buffers, antibacterial agents, antifungal agents, and isotonic agents, for example, sugar or sodium chloride. As used herein, the term "stabilizer" refers to a compound that is optionally used in a pharmaceutical composition of the invention to increase shelf life. In a non-limiting embodiment, the stabilizer is a sugar, amino acid, . The pharmaceutical compositions may comprise one or more pharmaceutically acceptable carriers. The carrier may be a solvent or a dispersion medium. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycols), oils, and suitable mixtures thereof.

Parenteral formulations can be sterilized. Non-limiting examples of sterilization techniques include filtration through a bacteria-inhibiting filter, terminal sterilization, incorporation of a sterile preparation, irradiation, sterile gas irradiation, heating, vacuum drying and freeze-drying.

In one embodiment of the present invention, "administration" means introducing the composition of the present invention to a subject by any suitable method, and the administration route of the composition of the present invention may be administered via any conventional route ≪ / RTI > Intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intradermal, intradermal, intraocular, , To be administered to an animal for the expression of a depressed animal model, or to be administered for the treatment of depression. The kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, administration time, administration route and fraction of the composition, And the like.

In one embodiment of the present invention, there is provided a method of producing an animal model of depression comprising over-expressing a brain-derived neurotrophic factor in a ventral tegmental area, the present invention further provides a method for producing an animal model of depression comprising the step of transplanting a vector comprising a channel rhodopsin gene and further comprising the step of transplanting an optical fiber, Wherein the overexpression of the brain-derived nerve growth factor is an expression of an mRNA or protein of brain-derived nerve growth factor, wherein the depression is selected from the group consisting of social defeat depression, social anxiety depression, social phobia depression, , Panic disorder depression, postpartum depression, post traumatic stress disorder, menstrual syndrome, dysthymia, treatment-resistant depression And treatment - and provides at least one method of producing an animal model of depression is selected from the group consisting of resistant bipolar disorder, the depressive provides a method for producing a depression in animal models of chronic social defeat depression.

In another embodiment of the present invention, there is provided an animal model of depression transformed to overexpress a brain-derived neurotrophic factor in the ventral tegmental area, There is provided a depression animal model transformed with a vector comprising a rhodopsin gene, wherein the depressed animal model further comprises an optical fiber, wherein the brain derived neural nerve Wherein the overexpression of the growth factor provides an expression model of depressed mRNA or protein of brain derived nerve growth factor, wherein the depression is selected from the group consisting of social defiant depression, social anxiety depression, social phobic depression, Post-traumatic stress disorder, menstrual syndrome, dysthymia, treatment-resistant depression, and treatment-resistant Bipolar disorder, wherein the depression provides an animal model of depression that is chronic social defiant depression.

In another embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of depression comprising tyrosine kinase B inhibitor as an active ingredient, wherein the tyrosine kinase B inhibitor is ANA-12 ( 2-oxoazepan-3-yl) carbamoyl] phenyl] -1-benzothiophene-2-carboxamide), wherein the depression is selected from the group consisting of social defeat depression, social anxiety depression, The present invention provides a pharmaceutical composition for the treatment of depression selected from the group consisting of depression, depression, obsessive-compulsive depression, panic disorder depression, postpartum depression, post-traumatic stress disorder, menstrual syndrome, dysthymia, treatment-resistant depression, and treatment- And wherein said depression is chronic social defiant depression.

In another embodiment of the present invention, there is provided a method of inducing overexpression of a brain-derived nerve growth factor in a first and a second individual, (b) measuring the expression of a brain-derived nerve growth factor in the septal side of the first individual; (c) administering a candidate substance for treating depression to said second subject; And (d) re-assaying the expression of brain-derived nerve growth factor in the second individual, wherein expression of brain-derived nerve growth factor in the second individual is lower than expression of nerve growth factor in brain of the first individual (B) to (d), the expression level of the mRNA or protein of the brain derived nerve growth factor is measured. Wherein the depression is selected from the group consisting of social defiant depression, social anxiety depression, social fear depression, obsessive compulsive depression, panic disorder depression, postpartum depression, post-traumatic stress disorder, dysmenorrhoea, A screening room for one or more candidate depressive candidates selected from the group consisting of treatment-resistant depression, and treatment-resistant bupropion And provides the depression provides a screening method for depression, chronic social defeat of candidates for the treatment of depression. Hereinafter, the present invention will be described in detail.

The present inventors can inhibit BDNF secretion by inducing brain-derived nerve growth factor signaling of the septal canthus nucleus which can induce depressive behavior and is increased by chronic defeat stress by administering ANA-12, and by inhibiting BDNF, . Therefore, it is expected to be able to treat neurodegenerative diseases such as depression by inhibiting the expression of BDNF gene in the part of the dorsal side of the midbrain, which is the main producing site of BDNF.

Figure 1 shows the results of a social accessibility study of the role of BDNF in NAc of mice during CSDS, according to one embodiment of the present invention. More specifically, FIG. 1-A is a schematic diagram showing an experimental schedule for injecting a drug into NAc of a mouse and performing CSDS, FIG. 1-B is a schematic diagram showing details of a daily drug injection and a social defeat stress schedule, FIG. 1C is a graph showing the position of drug injection in NAc, and FIG. 1-D is a result of measuring social accessibility in drug-injected rats.
Figure 2 is a result of confirming the photogenic genetic phase stimulating effect in the mouse VTA-NAc pathway during CSDS, according to one embodiment of the present invention. More specifically, FIG. 2-A is a schematic diagram showing a schedule of experiments in which VTA is activated by performing CSDS and phase stimulation, FIG. 2-B is a view showing in detail the position of vector implantation in the NAc region, FIG. 2-D is a diagram showing the results of staining of cells expressing ChR2-EYFP and TH in the VTA region, and FIG. 2-E is a diagram showing the results of staining of cells expressing ChR2- FIG. 2-F is a schematic diagram showing a schedule of experiments giving a light stimulus immediately after inducing CSDS in a mouse, and FIG. 2-G is a schematic diagram showing a schedule of an experiment in which a CSDS is induced, This is the result of measuring the social approach by light stimulus in mice transplanted and CSDS induced.
Figure 3 shows the results of confirming the role of BDNF in the NAc region by repeated optical genetic stimulation in the CSDS and VTA-NAc pathways, according to one embodiment of the present invention. More specifically, FIG. 3-A is a schematic diagram illustrating a schedule of ANA-12 injection and phase stimulation experiments during CSDS, and FIG. 3-B is a schematic diagram detailing the order and time difference of drug injection, social defeat stress, , FIG. 3-C is a diagram showing the position where the vector and ANA-12 are injected into the NAc and the optical fiber is implanted into the VTA. FIG. 3-D shows that the CSDS is induced in the mouse for 5 days, FIG. 3-E shows the results of showing the social avoidance response when the CSDS was induced in the mouse for 10 days and given the phase stimulus. FIG. 3-F shows the result of repeated photogenetics to the VTA-NAc pathway during CSDS FIG. 3-G is a model eh showing the schedule of experiments in which the BDNF gene is silenced in the VTA region and the photodynamic stimulation is applied during the CSDS, and FIG. 3-H The FIG. 3-I shows the results of the measurement of the social accessibility in the mice inducing the silencing of the BDNF gene.
FIG. 4 shows the results of assessing the extent of dopamine (DA) secretion occurring in the NAc region after stimulation with the optical genetics in the CSDS and VTA regions, according to one embodiment of the present invention. More specifically, FIG. 4-A shows the result of measuring CSDS and social accessibility by repeated light stimulation. FIG. 4-B shows the result of color plots of the degree of DA secretion in each group, FIG. 4-D shows the degree of DA secretion in each group according to the number and size of stimulated pulses. FIG. 4-D shows the degree of DA secretion caused by the NAc shell by single pulse or 5 multiple pulses, This is a result.
FIG. 5 shows cell-specific pERK expression by CSDS according to an embodiment of the present invention. More specifically, FIG. 5-A is a graph showing the similarity in social accessibility of the stress-resistant group compared to the control group, and FIG. 5-B is a graph showing the result of decreasing the social accessibility of the stress- FIG. 5-C is a photograph showing cells expressing green by GFP at the cell counting position, FIG. 5-D is a photograph showing pERK-positive cells (red) and D2-GFP positive nerve FIG. 5-E shows the ratio of pERK-positive cells to D2-negative cells in the NAc shell. FIG. 5-F shows the results of confocal microscopy photographs of the cells (green) Compared with the proportion of pERK-positive cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Experimental Example

Experimental Example 1. Preparation of an animal model for social defeat stress test

room Hum Example 1-1. Experimental animals and breeding environment used

Experimental animals were male C57BL / 6J mice (25-30 g, Jackson rap) 7-12 weeks old, male floxed BDNF mice (floxed BDNF, 25-32 g, BL6 / sv129 background) 25-32 g, C57BL / 6J background transformed to express green fluorescent protein (GFP) in dopamine receptor 2 (DRD2) at 12 weeks of age) and CD-1 mice (CD-1 retired breeders, 35-45 g; Charles River Laboratories, Mass.) Were used. Mice were fed a free diet and were raised at a temperature of 22 to 25 ° C for 12 hours / cancer cycle. CD-1 mice were accommodated alone except when the social defeat test was performed. The genetic sequence of the Drd2-GFP mouse used the known information (Gene Expression Nervous System Atlas [GENSAT], Project; www.gensat.org ). All tasks related to animal testing were performed according to the guidelines recommended by the Mount Sinai Commission and the Animal Welfare Organization.

room Hum Example 1-2. Stereotactic fixation for pharmacological treatment and photodynamic experiments

During CSDS (chronic social defeat stress), stereotaxic surgery was performed for the optical activity of the VTA (double skin zone) -NAc (septal cortex) pathway. 0.5 μl of AAV2.5-hsyn-ChR2-eYFP vector (a retrograde traveling adeno-associated virus vector engineered to yellow fluorescence upon channelrodothin-2 expression, Pennsylvania Vector Center, Pennsylvania) And an angle of 101 ° from the contact point between the sagittal and tubular sutures) at a rate of 1 μl / min. After 3 weeks, the optical fibers were implanted in the right and left fixed positions of the VTAs on both sides (anteroposterior axis 23.2; lateral axis 61.0; backaxis 24.6; angle from the contact point of sagittal and tubular suture; 71 °). When necessary for drug injection, a 4-mm long 26-gauge guide catheter was inserted into the NAC's anchoring position on both sides (anterior-posterior axis 11.5; lateral axis 1.75; backaxis 23.9; angle from contact with sagittal and tubular suture; ). Since the precise target point of the drug injection is the medial shell of NAc or the brain of the mouse is too small, the operation of the surgery was designed toward the center of the NAc. Stereotactic fixation for the optical activity of the VTA-NAc pathway during ASDS (Acute Social Defeat Stress) was performed in the following manner. First, the AAV-DIO-ChR2-EYFP vector (DIO (double floxed) Cre-dependent AAV vector, North Carolina Vector Center, North Carolina) was injected into both VTAs. Two weeks later, duplicate-defective PRV-Cre (retrograde traveling pseudorabies virus engineered to express Cre, Rockefeller University, New York) was injected into both sides of the NAc. AAV-Cre or AAV-GFP (North Carolina Vector Center) and AAV2.5-hsyn-ChR2-EYFP were incubated with Bdnf (Bacillus thuringiensis) in order to cause local Bdnf gene knockdown for optogenetics experiments on the VTA- Mice were injected into the VTA and NAc regions, respectively. Two weeks after the primary and secondary surgeries, the optical fibers were implanted into the fixed position of the two side VTAs in the manner described above.

Experimental Example 2: Drug injection

Mice were injected with 1 mg / 0.5 μl of SCH 23390 (D1 receptor antagonist), eticlopride (D2 receptor antagonist), or ANA-12 (D2 receptor antagonist) on the inner NAc of both sides of the mouse about 15 minutes before performing defeat stress (TrkB inhibitor) was injected using a microinfusion pump (Harvard Apparatus, Mass.) To maintain a rate of 1 μL / min. In the control group, sterile saline or 50% DMSO (dimethyl sulfoxide), which is a drug, was injected. The injection needle of the pump was removed after 5 minutes of completion of the drug injection. The drug - injected mice were allowed to relax slowly for about 5 minutes before performing defeat stress. One-time defeated stress mice were injected with SCH 23390, ethicloprid, or vehicle of drug (control) in the inner NAc on both sides of the mouse, one hour before the social interaction test.

EXPERIMENTAL EXAMPLE 3. Social defeat stress accompanied by photo-genetic stimulation

To induce chronic stress and acute stress in the mice, male C57BL / 6J mice were exposed to male CD1 versus untreated mice for 5 minutes and then allowed to rest in a familiar cage for 15 minutes. Exposure to CD1 large intestine mice was performed twice at 15 minute intervals. After the last defeat stress and after 24 hours, the social accessibility test was conducted. Based on social interaction ratios (X100% of time spent in the friendly space without the time / social target in contact with the social target object), the mice were given a resilient (above 100 ratio above) Susceptible (above 100%). Phasic stimulation of the VTA-NAc pathway was performed for 5 minutes during or immediately after the stress period, allowing the mice to undergo CSDS (chronic social defeat stress) for 10 days. ASDS (acute social defeat stress) experimental CD-1 mice were subjected to 2.5-minute phase stimulation during social accessibility testing.

Experimental Example 4. Performing Voltage and Current Test on Socially Deficient Stressed Animals

In order to screen vulnerable group mice after CSDS, the mice were subjected to the social accessibility test, and after 18-22 hours, pre-synaptic dopamine secretion, absorption, and terminal response to phasic stimulation patterns in the NAc shell The voltage and current test was performed as a method to measure the ability. First, in order to obtain baseline recordings from a 400 mm thick coronal brain tissue section containing NAc shell region, a single-pulse stimulus (350 μA, 4 ms, monophasic) was given every 5 minutes to cause DA secretion Respectively. Phasic stimulation curves were plotted after obtaining a stable baseline value (3 times within 10% variability) and single-pulse and multi-pulse (5 times in various sizes within the range of 5-20 Hz) The DA secretion was evaluated.

EXPERIMENTAL EXAMPLE 5. Place Preference Test with Optical Genetics Stimulation Test

A conditioned place preference (CPP) test was performed. For this purpose, mice were allowed to freely navigate in a three chambered CPP box for 20 minutes, and place preference was measured. In the following two days, mice were given saline or cocaine (10 mg / kg, intraperitoneal injection), saline / dark environment and cocaine / blue light (473 nm, 20-Hz frequency, 5 flashes, 40- Each) environment was trained for 30 minutes. On the day of the CPP test, the mice were allowed free cage exploration for 20 minutes. The CPP score is expressed as the residence time in the preferred or non-preferred space.

Experimental Example 6 Immunohistochemical staining

After 24 hours of sociability testing, mice were anesthetized with a lethal dose of chloral hydrate and perfused with PBS and 4% paraformaldehyde solution. Brain tissue was obtained and fixed at room temperature with 30% sucrose solution one or more days later. The suspension was washed with PBS and blocked with a solution containing 3% bovine serum albumin and 3% Triton X-100 (T8787; Sigma-Aldrich, Missouri) for 1 hour.

Anti-TH (1: 4000, T1299; Sigma-Aldrich) and chicken-derived anti-GFP (1: 1000, GFP-1020; Abbess Laboratories, Oreg.). The next day, the donkey anti-mouse Cy3, the secondary antibody of anti-TH, and the donkey anti-chicken Cy2 (1: 500, Jackson Immunology Laboratory, Pennsylvania). For the double fluorescent staining of GFP and pERK of D2 GFP mouse tissues, tissue sections were incubated with anti-GFP (1: 1000, Abbes Laboratories) of chicken origin and anti-pERK (1: 1000, 4370S; cell signaling, The next day, the donkey anti-chicken Cy2, the secondary antibody of anti-GFP, and the donkey anti-mouse Cy3 (1: 500, secondary antibody of anti-pERK, Jackson Immunology Laboratory ). All stained tissues were encapsulated with an encapsulating agent (VectaShield, H-1000; Vector Laboratories, Calif.) And observed with a confocal phase contrast microscope (LSM 710 confocal microscope, Callaze, Germany). The counts of cells stained by GFP and pERK in the NAc shell or central region were performed by the double blind method in the area of 200 탆 X 200 탆.

Experimental Example 7. Western blotting

After 24 hours of sociability testing, 1 mm of NAc coronary tissue was obtained from both sides of the NAc of the mouse with a 14-gauge punch and the tissue was lysed in 30 l of milling buffer (320 mmol / L sucrose, 5 nmol / L HEPES buffer, 1% SDS, phosphatase inhibitor, protease inhibitor). The concentration of the extracted protein was analyzed with a protein analysis kit (Bio-Rad DC Protein Assay, Bio-Rad, Calif.) And a total of 25 μg of protein was separated by size in a concentration gradient polyacrylamide gel, transferred to nitrocellulose membrane (Odyssey Blocking Buffer, LI-COR Bioscience, Nebraska) for 1 hour. After blocking, the membrane was incubated with anti-BDNF (1: 500, sc-546; Santa Cruz Biotechnology, Calif.) And anti-beta-tubulin (1: 10,000, , Washed with TBST buffer, and reacted with a secondary antibody (1: 10,000; LI-COR Bioscience) containing a dye (IRDye) for 1 hour at room temperature. The protein-stained image was photographed (Odyssey Infrared Imaging system, LI-COR Bioscience) and quantified with Densitometry and Image Program (ImageJ, National Institutes of Health, Maryland). The quantified protein values were re-estimated based on the tubulin quantification value.

Example

Example 1. Identification of BDNF function in stress mechanism

Induced neurotrophic factor (BDNF) in the NAC (septal cortex) during CSDS, inducing drug injection and CSDS (chronic social defeat stress) in mice by the methods described in Experimental Examples 2 and 3, ) Was evaluated by the social interaction test described in Experimental Example 3. The results are shown in Fig. 1-A is a schematic diagram showing a schedule of experiments in which CS 23390 (D1 receptor antagonist), eticlopride (D2 receptor antagonist), or ANA-12 (TrkB inhibitor) drug was injected into the NAc of mice and CSDS was performed , Figure 1B is a schematic diagram detailing daily drug injection and social defeat stress schedules. The drug was injected 15 minutes before the daily social defeat stress. FIG. 1-C is a diagram showing the position of drug injection in the NAc (septal cortex), and FIG. 1-D is a result of measuring the social interaction ratios in the drug-injected rats. In the graph, the numbers in the bars indicate the percentage of tolerable mice in the whole mouse as a percentage. Experimental results showed that D1 or D2 receptor antagonist injected into NAc did not affect social avoidance by CSDS. However, mice that received the TrkB inhibitor, ANA-12, had a reduced social avoidance response.

Example 2. Confirmation of photodynamic stimulation result during stress

In accordance with the method described in Experimental Example 1-2, a vector matching each condition was injected into a mouse, and stereotaxic surgery was carried out for the application of the optical user experiment. Thereafter, mice were subjected to social defeat stress accompanied with photogenetics stimulation by the method of Experimental Example 3. The optogenetic phasic stimulation effect of the VTA-NAc pathway during the above CSDS is shown in FIG. FIG. 2-A is a schematic diagram showing a schedule of experiments in which VTA was activated by performing CSDS and 20-Hz phase stimulation, and FIG. 2-B shows the positions at which the AAV2.5-hsyn-ChR2- (Scale bar = 100 탆). Fig. 2-C is a view showing in detail the position of transplanting the AAV2.5-hsyn-ChR2-eYFP vector in the NAc region and the optical fiber transplantation into the VTA. D is a figure showing the result that ChR2-EYFP is expressed in green in the VTA region and the cells expressing tyrosine hydrate enzyme (TH) are stained in red (Scale bar = 50 탆). Fig. FIG. 2-F is a schematic diagram showing a schedule of experiments in which a light stimulus is given for 5 minutes immediately after inducing a social defeat stress in a mouse, while FIG. 2-F is a schematic diagram showing an experimental schedule for giving a 5-minute light stimulus. Fig. 2-G is a result of measuring the social accessibility of the AAV2.5-hsyn-ChR2-eYFP vector transplanted into the CSDS-induced rats by light stimulation. In the graph, the numbers in the bars indicate the percentage of tolerable mice in the whole mouse as a percentage. As a result of the experiment, it was found that there was no influence on the social avoidance by CSDS in the mice that received the light deficit stress and the social defeat stress. However, after the CSDS, the social avoidance of the mice that received the light stimulus immediately after the social defeat stress was exacerbated.

Example 3. Identification of BDNF function in the brain with stress and photogenetics stimulation

BDNF in the NAc region by repeated optical genetic stimulation on the CSDS and VTA-NAc pathways in the CSDS-induced mice injected with the drug by the methods described in Experimental Examples 2 and 3, , And the results are shown in Fig.

FIG. 3-A is a schematic diagram showing a schedule of ANA-12 injection and 20-Hz phase stimulation experiments in NAc during CSDS, and FIG. 3-B shows the order and time difference of drug injection, social defeat stress, 3-C is a diagram showing a position where an AAV2.5-hsyn-ChR2-eYFP vector and ANA-12 are injected into NAc and an optical fiber is implanted into VTA, and Fig. 3-D shows the stress 5 days, and the social avoidance reaction when the phase stimulus was given. Experimental results showed that social avoidance was not affected regardless of injected vector or drug. FIG. 3-E shows the social avoidance response when inducing social stress for 10 days in a mouse and giving a phase stimulus. In the graph, the numbers in the bars indicate the percentage of tolerable mice in the whole mouse as a percentage. As a result of the experiment, social stress for 10 days exacerbated the social avoidance reaction. The CSDS Vehicle1EYFP group showed worse social avoidance response than the control group, and the group injected with NAc into ANA-12 was completely opposite to the result of the social avoidance by the optical genetic stimulation. 3-F are graphs showing the results of BDNF protein expression in NAc by repeated photoperiodic stimulation on the VTA-NAc pathway during CSDS. BDNF protein was increased by photodynamic stimulation. More specifically, the expression of BDNF protein during the CSDS of the CSDS Vehicle1EYFP group was increased compared to that of the unstressed control group and the CSDN Vehicle1ChR2 group showed the highest BDNF expression. FIG. 3-G is a schematic diagram illustrating a schedule of experiments in which a local Bdnf gene is silenced in the VTA region and a 20-Hz photomagnetic field stimulus is applied to the VTA region during CSDS, and FIG. Figure 5 shows the location of transplanting AAV2.5-hsyn-ChR2-eYFP vector into NAc for genetic stimulation. For local BDNF gene silencing in the VTA region, the AAV-Cre vector was injected into the VTA region of the plastost BDNF mouse. FIG. 3-I is a result of measuring the social accessibility of mice inducing BDNF gene silencing. In the graph, the numbers in the bars indicate the percentage of tolerable mice in the whole mouse as a percentage. Experimental results showed that the BDNF gene silencing mice in the VTA region showed increased social access.

Example 4. Confirmation of dopamine secretion in the brain with stress and photo-genetic stimulation

The secretion of dopamine (DA), which occurs in the NAc domain after stimulation with the optical genetics into the CSDS and VTA regions, was evaluated in vitro by a voltage-current method test, and the results are shown in FIG. Figure 4-A shows the result of measuring the CSDS and the social approach by repeated light stimuli. Repeated CSDS induced social avoidance compared to unstressed controls, and this avoidance was accelerated by repeated phase photon genetic stimulation to the VTA-NAc pathway. FIG. 4-B shows the result of DA plot in the CTRL1EYFP, CSDS1EYFP, or CSDS1ChR2 group by color plots. FIG. 4-C shows the DA secretion degree caused by the NAc shell by a single pulse or five multi- Fig. FIG. 4-D shows the results of experiments on the degree of DA secretion in the CTRL1EYFP, CSDS1EYFP, or CSDS1ChR2 groups according to the number and size of stimulated pulses. There was no statistically significant difference in the ratio of the resistance group to the vulnerable group among the groups depending on the number and size of pulses.

Example 5 Confirmation of Expression of pERK by Stress

Cell-specific pERK expression by CSDS is shown in Fig. FIG. 5-A is a graph showing the results of a decrease in the social accessibility of the stress-resistant group compared to that of the stress-resistant group compared to the non-stressed control group. FIG. 5-B is a schematic view showing the coronal section of NAc and the position of cell count, FIG. 5-C is a photograph showing cells expressing green by GFP at the cell counting position (Scale bar = 100 μm) D is a photograph (Scale bar = 100 μm) showing the results of confocal microscopy photographs of pERK-positive cells (red) and D2-GFP-positive nerve cells (green) in the NAc shell. Figure 5-E shows the ratio of pERK-positive cells to D2-negative cells in the NAc shell. The proportion of pERK-positive cells in vulnerable group mice was higher than that of control or stress-resistant groups that were not stressed. FIG. 5-F shows the ratio of pERK-positive cells to D2-positive cells in the NAc shell. The ratio of pERK-positive cells to D2-positive cells was not significantly different in all groups.

As a result of Examples 1 to 5, the BDNF hypersecretion can induce depressive behavior, and the Brain-derived neurotrophic (BDNF) of the nucleus accumbens (NAc) in the mesolimbic system, which is increased by chronic defeat stress, factor, brain-derived nerve growth factor) signaling can be inhibited by administering the TrkB inhibitor ANA-12 to the septal nucleus and inhibiting BDNF may reduce depressive behavior. Therefore, it is expected to be able to treat neurodegenerative diseases such as depression by inhibiting the expression of BDNF gene in the ventral tegmental area (VTA), which is the main producing site of BDNF in the midbrain.

The present invention provides an animal model of depression that overexpresses a brain-derived neurotrophic factor (BDNF) and a method for producing the same. Therefore, the present invention can greatly contribute to clarify the causative mechanism of depression and to search for a therapeutic method.

Claims (20)

A method for producing an animal model of depression by overexpressing a brain-derived neurotrophic factor in a mesolimbic system of an animal other than a human, comprising the step of transplanting a vector containing a channel rhodopsin gene ≪ / RTI > delete The method of claim 1, further comprising implanting an optical fiber. The method of claim 1, wherein the overexpression of the brain-derived nerve growth factor is an increase in the expression of brain-derived nerve growth factor mRNA or protein. The method of claim 1, wherein the depression is selected from the group consisting of social defiant depression, social anxiety depression, social phobia depression, obsessive compulsive depression, panic disorder depression, postpartum depression, post traumatic stress disorder, menstrual syndrome, dysthymia, - < / RTI > resistant bupivacaine. 6. The method of claim 5, wherein the depression is chronic social defiant depression. An animal model of depression transformed to overexpress a brain-derived nerve growth factor in a midgut of an animal other than a human, wherein said animal model of depression is transformed with a vector comprising a channel rhodopsin gene. delete 8. The animal model of depression according to claim 7, wherein the depressed animal model further comprises an optical fiber. 8. The animal model of depression according to claim 7, wherein the overexpression of the brain-derived nerve growth factor is an increase in expression of brain-derived nerve growth factor mRNA or protein. 8. The method of claim 7, wherein said depression is selected from the group consisting of social defiant depression, social anxiety depression, social phobic depression, obsessive compulsive depression, panic disorder depression, postpartum depression, post traumatic stress disorder, menstrual syndrome, dysthymia, - depressive animal model, at least one selected from the group consisting of depressive mood disorders. 12. The animal model of depression according to claim 11, wherein said depression is chronic social defiant depression. delete delete delete delete delete delete delete delete

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