Dopamine-induced Apoptosis In Primary Cultured Rat Amygdala Neurons

Objective: In the forensic practice, some suspects can show mental damages and even death after suffering non-lethal mechanical injuries caused by limit position or physical oppresssion.Generally, these injuries are not serious enough to cause death, so it has become a difficult problem to identify such cases for forensic workers.It is not difficult to discover that such cases have one characteristic in common, that is to say, non-lethal mechanical injuries results in the occurrence of psychological stress reaction in parties. Studies have shown that excessive and prolonged psychological stress can cause mental damage, but the underlying molecular mechanism remains unclear. It has a very important scientific significance to study on the scientific issue,which is meaningful for the identification of these cases.Stress is an adaptive response of organism induced by kinds of stressors,which can activate the neuroendocrine system. As a result, locus ceruleus-norepinephrine system and hypothalamus-pituitary-adrenal cortex system are started, which lead to the rapid increase in the levels of stress-related hormones,such as catecholamine( CA), corticosteroids( GC) and glucagon, to keep functions and metabolisms of the important vital organs nomal. But a high level of stress hormones maintaining for a long time can cause oxidative stress, inflammation, apoptosis and many other pathological processes,which make defensive mechanisms turn into an injury to the body.Catecholamine is one of the main stress hormones during the stress process.The study on the damage effects of catecholamine is important to clarify the effects of stress on the body. Dopamine(DA), one kind of catecholamine and an important neurotransmitter, distributes in the brain widely.DA plays a key role in regulating movement, reward, learning, memory and emotion. More and more research results confirmed that the dopamine system plays a key role in the development of psychiatric disorders, such as schizophrenia, major depressive disease and so on, induced by stress. To exploring the damage role of dopamine in the central nervous systems can provide theoretical foundation for clarification the mechanism of mental disorder induced by psychological stress.In recent years, more and more research results confirmed that amygdala play an important role in stress.The amygdala, an part of the limbic system, attaches to the end of the hippocampus and has widely contacts with cortex, hippocampus,hypothalamus, brainstem and olfactory bulb.The amygdala is involved in the react to fear stimulations and play an important role in regulating fear and anxiety. Patients with PTSD show a smaller amygdala volume than that of healthy subjects.In the animal models of PTSD, neuronal apoptosis of the amygdala could be observed. Studies show that human amygdala is riched in both dopamine receptors and dopamine is released from the amygdala in stress.Whether or not the dopamine could cause amygdala neuronal apoptosis is sill unclear.Based on the above research background,the aim of the present study is exploring the role of dopamine on amygdala neurons apoptosis at the cellular level to provide experimental basis to elucidate the mechanism of stress-induced mental disorders.Method:1 The purity of primary culture of rat amygdala neurons were identified by using immunofluorescence cytochemistry method.2 To determining the optimal concentration of DA by MTS assay,amygdala neurons were treated with different concentrations of dopamine(10 ?M?30 ?M?50 ?M?100 ?M ?150 ?M); to determinging the optimal treatment time by MTS assay, amygdala neurons were treated with 50 ?M dopamine for survival rates were tested after different times(24h?48h?72h). 3 The translocation of Bax protein in amygdala neurons was observed after DA treatment by using immunofluorescence cytochemistry method to determine whether or not dopamine can cause apoptosis of the amygdala neurons.4 Amygdala neurons were incubated with 50 ?M DA and DA1 receptor antagonist SCH-23390 to further confirm whether DA could resulte in apoptosis of the primary cultured amygdala neurons.The experiment was divided into four groups:control group;DA group;DA+SCH-23390 group;SCH-23390 group.After 50 ?M DA teatment for 48 h in amygdala neurons, the expression level of Bax(one of proapoptosis genes) m RNA was measured by RT-PCR method; the rates of TUNEL positive cells was measured by TUNEL method;the expression level of ATF-6 protein was measured by western bloting method.5 The data were presented as Means±SD and analyzed with one way ANOVA by SPSS17.0 statistical program,Tamhane'S T2 do the comparison between groups. A level of P < 0.05 was supposed to be statistically significant.Results:1 Result of primary cultured amygdala neuronsAfter 8-10 days, the cultured amygdala neurons had become mature neurons:the soma was compact; nucleus was large and roud;processes formed a filamentous network.And amygdala neuron's purity was more than 90%,which could be used by the next experiments.2 The effect of dopamine on the survival rates of amygdala neurons(1)The number of inoculated cells each well was determined by MTS. The optical density are positively correlated with absorbance at 490 nm. After statistic analyzing,the correlation coefficient of the line was 0.98916.To ensure consistency of the results, the OD was controlled between 0.8-0.9, then 20000 cells can be obtained each well.(2) The effect of different concentrations of DA in the amygdala neuronal viabilityCompared with control group, DA(50 ?M?100 ?M ?150 ?M)gradually decreased cell viability after 48 hours(P<0.05). DA(10 ?M?30 ?M) had no effects on the amygdala neuronal viabilty(P>0.05).Considering the lower cytotoxic concentration, so 50 ?M was used in follow-up work.(3)The effect of different treatment time of DA in the amygdala neuronal viabilityCompared with control group,DA treatment for 48 hor 72h gradually decreased cells viability(P<0.05).Treatment for 24 h had no effects on the amygdala neuronal viabilty(P>0.05).So, appropriate conditions( 50 ?M DA,48 hours) was use in follow-up experiment conditions.3 Results of translocation of Bax protein in amygdala neuronsAfter DA treatment, translocation of Bax protein to the nucleus was observed, which suggested that apoptosis might occur in amygdala neurons.4 Results of expression of Bax m RNA levels in amygdala neuronsCompared with control group, after DA treatment,expression of Bax m RNA levels were significantly increased(P<0.05); while compared with DA group, expression of Bax m RNA levels were significantly reduced(P<0.05) in DA+SCH-23390 group. Above results indicated that DA can induce apoptosis of primary cultured rat amygdala neurons, and dopamine D1 receptors are involved in the process of apoptosis.5 TUNEL results in amygdala neuronsCompared with control group, after DA treatment, rate of positive cells was significantly increased(P<0.05);compared with DA group, the rate of positive cells was significantly reduced(P<0.05) in SCH-23390+DA treatment. The Results indicated that DA can induce apoptosis of primary cultured rat amygdala neurons through dopamine D1 receptors.6 Results of expressions of endoplasmic reticulum transmembrane protein ATF-6 protein levels in amygdala neuronsCompared with controls, after DA treatment,expression of ATF-6 protein levels were significantly increased(P<0.05);compared with DA group, after SCH-23390 treatment,expression of ATF-6 protein levels were significantly reduced(P<0.05).Results indicated that endoplasmic reticulum stress(ERS) may participate in the process of DA-induced amygdala neuron apoptosis.Conclusion:DA can induce apoptosis of primary cultured rat amygdala neurons through D1 receptor.