Establishment Of Animal Model Of Depression And Change Of Cerebral PIP₂ Metabolism

Depression is a complex mental disorder. Depressed mood, anhedonia,guilt and valueless, and suicidal thoughts are the main symptoms of depression. In a recent study of global disease, depression was ranked as the second leading contributor to global disease burden on social economy and family. Depression is one of the medical conditions that result disability. With the prevalence of this disease, depression is heterogeneous and the relapse rate is high. The precise etiology of depression is not well understood. The beneficial effects of antidepressants are slow and some patients are resistant to the treatment. It is thus important to further study the mechanism of depression and to discover new drugs and therapeutic interventions.Phosphatidylinositol-4,5-bisphosphate（PIP₂） is enriched in the inner leaflet of the plasma membrane, comprising about 1% of plasma membrane phospholipids. PIP₂ plays important roles in regulating endocytosis and exocytosis of synapatic vesicle, and on neurotransmitter release. PIP₂ is closely related to the neuron synaptic plasticity. Neuron dysfunction will lead to mood disorder and other metal diseases. Dysfunction of PIP₂ metabolism may lead to Alzheimer’s disease; bipolar disorder is connected with enzymes which are related to PIP₂ metabolism. Chronic administration of antidepressant agents has been associated with increased levels of the Phosphatidylinositols（PIs） in human platelets. All of these studies indicate a possible role for PIP₂ metabolism in depression, which is the aim for this study.There are two parts in this study:In Part 1, we established animal models of depression and evaluated the depression-like behaviors and the effects of antidepressants; In Part 2, we studied the relationship between the PIP₂ metabolism in the brain of social defeated mice, with a newly established method of HPLC/MS measurement of PI（4,5）P2.Part one Establishment of animal models of depressionObjective: To establish social defeat animal model and unpredictable chronic mild stress animal model of depression and evaluate the animal models by behavioral test.Methods:1 Social defeat model of mice. C57 mice were subjected to social defeat stress stimulation for 10 days（10 min/day）. Then sucrose preference test and social interaction test were conducted. We also examined the changes of body weight and food consumption. Those mice with depression-like behaviors after the social defeat stress stimulation were named susceptible mice which were divided into 3 groups, and injected intraperitoneally with solvent saline（NS）, imipramine（20 mg/kg） and paroxetine（20 mg/kg）, respectively for 4weeks. Mice without social defeat stimulation stress were also used, as a normal control group which was treated with NS. Social interaction test was used to evaluate the social behavior changes.2 Unpredictable chronic mild stress model of mice（UCMS）. C57 mice were divided into 4 groups, namely the control group, the NS treated group,the imipramine treated group and the paroxetine treated group. The last 3groups of mice were subjected to different types of mild stressors for 5 weeks.Mice were subjected to 3 different stressors each day, and the same stressors were not used in the adjacent two days. At the beginning of the forth week,mice were treated with different drugs for 2 weeks. Sucrose preference test was conducted before, at the first, the third and fifth week of the experiment.We also examined the changes of body weight, food consumption, behaviors in the open field and the immobility time in the forced swimming test.3 Unpredictable chronic mild stress model of rats. Sprague-Dawley rats were divided into the control group and the model group. Rats in model group were subjected to stressors for 4 weeks. Rats were subjected to 2 different stressors each day, and the same stressors were not used in the adjacent two days. Then sucrose preference test, the open field test and the measurement of body weight were conducted.Results: 1 Mice after the social defeat stimulation were segregated into susceptible and unsusceptible populations based on their behavioral test results:susceptible mice displayed social avoidance, a significant decrease in sucrose preference and body weight, and a significant increase in food consumption;unsusceptible mice displayed social behavior and sucrose preference with no difference reference to the normal control mice. However unsusceptible mice also displayed a significant decrease in body weight and a significant increase in food consumption. After treated with imipramine for 4 weeks, susceptible mice displayed increased social behavior; 2 Compared with the control group mice, UCMS-exposed mice displayed a decrease in sucrose preference, body weight, locomotor activity, and exploratory activity. The consumption of food and immobility time in forced swimming test were increased. Depression-like symptoms were not reversed by antidepressants treatment for 2 weeks; 3UCMS-exposed rats displayed a decrease in sucrose preference, body weight,and exploratory activity.Conclusions: We successfully established the social defeat animal model, and the unpredictable chronic mild stress（CUMS） animal model need to further be evaluated.Part two PI（4,5）P2 analysis in the brain of animal model of depressionObjective: To establish a new method（HPLC/MS） for PI（4,5）P2 analysis in brain tissue, and to study the relationship between changes of PIP₂ metabolism and the development of depression-like behavior in the social defeated mice model of depression.Methods: The concentration of synaptosomal PIP₂ in the midbrain and the prefrontal cortex of mice was detected by the HPLC/MS method. Liquid chromatography conditions: For PIP₂ analyses, samples were separated on a Waters X-Bridge C8 column（100 mm × 2.1 mm,3.5 mm）. Flow rate was 0.4ml/min, and the chromatography was performed at 35℃. Typically, 2 ml of sample was injected. Mobile phase A was acetonitrile/ethylamine（100 : 0.05）.Mobile phase B was 0.05% ethylamine solution. The gradient was 30% A to80% A over 3 min, after which it was held for 2 min, followed by holding for5 min for reequilibration. Mass Spectrometry: The detection was accomplished by MSM scan with ESI source operating in the negative ionization mode.Results: HPLC/MS method was proved to have high specificity. The calibration curve was linear over the investigated concentration range（12.5²00 ng/ml）（correlation coefficient is 0.9989）. The lower limit of quantization is 2 ng/ml. Precision and accuracy was satisfactory. PI（4,5）P2 was found to be stable under the current condition of analysis. The concentration of synaptosomal PIP₂ in the midbrain of social defeat mice was significantly decreased; the concentration of synaptosomal PIP₂ in the prefrontal cortex of social defeat mice was slightly increased.Conclusion: The newly established HPLC/MS method is a valuable tool for brain PIP₂ analysis. The development of depression-like behavior in mice model of depression is possibly related with an altered PIP₂ metabolism in the brain.