Antidepressant-like Effects Of Rg1 Are Due To Astrocyte Gap Junctions In The Prefrontal Cortex

At present, affective disorders such as major depressive and bipolar disorders are the most common psychiatric disorders. About 16% of the population are estimated to be affected by major depressive disorder(MDD) one or more times during their life time. The main presence of MDD are includes a long- lasting depressed mood, low self-esteem, feelings of guilt, anxiety, cognitive decline, and tendency to suicide in severe cases. Undoubtedly, MDD is the maximum expression of people suffering, and it has become the leading cause of death by suicide. About 15% of patients with MDD die as a consequence of the disease, and MDD accounts for more than 90% of all die by suicide. MDD is considered as a risk factor for other diseases and is one of the leading causes of non-fatal disorders, imposing considerable economic burden worldwide. To date, although the pathogenesis of depression remains unclear, the onset of the disease is certainly related to many factors such as biological, psychological and social factors. Actually, it has been estimated that genetic contribute 40-50% to depression. Other non-genetic factors such as stress, viral infection, emotional harm and neurodevelopmental abnormalities increased the complexity of the disease. The emotional and cognitive disorders of MDD are the result of the interaction of the whole brain network system, rather than an individual protein, a receptor, or a sudden neuronal disease. Large number of experimental results have led to a variety of molecular mechanisms of depression hypotheses, for example, monoamine neurotransmitter hypothesis, the hypothalamus-pituitary-adrenal(HPA) axis dysfunction hypothesis, neurotrophin hypothesis, the hypothesis receptors, etc., but a certain pathogenic mechanism has not yet clear.The current antidepressant drugs are primarily focused on serotonin(5-HT), dopamine and norepinephrine, three monoamine neurotransmitter systems, such as tricyclic antidepressants, selective 5-HT reuptake inhibition agents(SSRIs), monoamine oxidase inhibitors(MAOIs), etc. These drugs are often focus on a certain kind of depression symptoms, for instance, changes in appetite, neurological motor, interest. Drugs acting on one of the crucial systems, like 5-HT system, may have obvious effects within a certain time, but the brain is regulated by multiple neural circuits system. So, Drugs act on multiple systems simultaneously may more effective for the recovery of MDD or chronic diseases. Although current first-line clinical antidepressants SSRIs, like fluoxetine, has been greatly improved their effect on depression compared with tricyclic antidepressants, the tolerability and delayed efficacy in many patients have seriously affected the treatment of depression. Therefore, to explore the pathogenesis of depression and develop new drugs with quickly onset and better efficacy has become an urgent problem.There is a substantial findings suggest that the prefrontal cortex(PFC) is closely related to depression. PFC have many neuronal circuits connected with several subcortical structures such as hippocampus, amygdala, nucleus accumbens. The incoming or efferent neurons delivery disorders between PFC and these brain regions is associated with depression. Glial cells have supporting, protection, nutrition and repairing effects on neurons. Increasingly studies have showed that astrocyte dysfunction are associated with depression. Activities of astrocyte are depend on gap junctional communication. Adjacent astrocytes exchange material through gap junction channels(GJs). Precious studies have found that gap junction dysfunction in the PFC is closely related to MDD.Panax ginseng C. A. Mey is the root of Araliaceae perennial herb, widely distributed in Northeast of China, the Korean peninsula, Russia, etc. And it has been used as a medication for more than 2, 000 years in East Asian countries for the activities on the cardiovascular system, immune system, central nervous system, cancer, and diabetes. So far, ginseng has become one of the most widely used traditional Chinese medicine in the word and has the reputation of "panacea". The active ingredients of ginseng include polysaccharides, saponins, peptides, fatty acids, etc. Ginsenosides Rg1(Rg1), with a variety of pharmacological activities, is one of the main active ingredient of ginseng. Accumulating evidence have demonstrated that Rg1 has beneficial effects on neuron, for instance, neurotrophic and neuroprotective activities. It can increase the differentiation of neural progenitor cells to promote neurogenesis. Rg1 lessened amyloid β1-40 induced neuron apoptosis in rat. Rg1 can also play a neuroprotective effect by activating insulin-like growth factor-1(IGF-1) in the study of central nervous system degenerative disease, such as Alzheimer’s disease and Parkinson’s disease. Additionally, Rg1 can significantly alleviate the cerebral ischemia-reperfusion injury. Recent studies have found that Rg1 have antidepressant-like activities by promoting neurogenesis, improving synaptic plasticity, regulating the HPA axis and so on. Whether Rg1 can alleviate MDD through astrocyte GJ in the PFC has not yet been reported.The chronic unpredictable stress(CUS) model, originated in the late eighties of last century, was used in both clinical and preclinical studies for the etiology of depression. Rodents exposure to a series of stressors will resulting in dysfunction in rewarding system and appearing reduction in sucrose consumption, which was similar to symptoms of anhedonia in depression. And this performance can be reversed by chronic conventional antidepressant drug treatment, which validated the CUS model. At present, this model has been widely used for a variety of neurobiological studies related to depression.Based on the above backgrounds, we determined to investigate the relationship between Rg1 and the GJ of PFC by CUS induced depressed rat models. Specific contents are as follows:Part 1 Depressed rat model were induced by CUSObjective: To observe the effect of Rg1 on depressive- like behavioral changes in animal modelsMethods: Animals were exposed to tail pinch, noise, food deprivation, cage tilt 45° overnight, ice water swim stress, restraint, water deprivation, crowding overnight, hot water swim stress, wet bedding overnight, isolation, light on 24 h and light off 24 h for depression modeling except for normal reared control group. Two kinds of stressors were scheduled at random per day and conducted for consecutive 35 days, and three doses of Rg1, 5, 10, 20 mg/kg, were administered orally for 4 weeks with the positive control group of venlafaxine. After the stress, the behavioral indicators, line crossings, rears, sucrose preference, novelty suppressed feeding time within 12 min and immobility time within 6 min, were detected by open field test(OPT), sucrose preference test(SPT), novelty suppressed feeding test(NSFT) and forced swimming test(FST)were detected successively to compare and evaluate the antidepressant effects of Rg1.Results: before the CUS, OPT experimental results showed that the time of line crossings(P = 0.9108) and the stand times(P = 0.9603) were not significant different in each group. Sucrose preference were all above 85%, and there was no significant difference between the groups in the SPT baseline test(P = 0.9986). After 5 weeks of CUS, the sucrose preference decreased more than 15% in CUS exposed animals, and long term Rg1 5,10,20 mg/kg administration made the sucrose preference higher than 85%. There was a statistically significant difference between Rg1 and model group(P<0.05). The NFST results showed that the time of latency to feed is 220.7 seconds in normal control group while 461.5 seconds in model group. CUS obviously delayed the time of latency to feed(P<0.01). Although Rg1, 5, 10, 20 mg/kg and venlafaxine shorted the incubation period, no statistically significant difference were detected(P>0.05). In the FST experiment, the immobility time of model group is more than 1 times longer than the normal control group. long term Rg1, 5,10, 20 mg/kg administration significantly reduced the immobility time of CUS exposed rats in the FST(P<0.01).Conclusion: 5 weeks of CUS exposer can induce significant depression-like behavioral changes in rats. Long term Rg1, 5, 10, 20 mg/kg administration significantly improved the sucrose preference index in SPT and shorted the immobility time in FST with good antidepressant activities. Besides, Rg1 20 mg/kg administration has better effect.Part 2 Effect of Rg1 on the function of astrocyte gap junction and the gap junction- related proteins in the PFC of CUS exposed ratsObjective: To study the relationship between Rg1 and the astrocyte gap junction function in the PFC of CUS exposed rats.Methods: Lucifer yellow(LY), a small molecule fluorescent yellow dye, was injected to the PFC of CUS exposed rats and then the frozen brain slice were observed by fluorescence microscopy. To observe the effects of Rg1, the diffusion distance of LY and the coupling number of astrocytes of CUS exposed rats were detected after long term Rg1 5, 10,20 mg/kg administration. To see the ultrastructure changes and the effect of long term Rg1 20 mg/kg administration in the PFC of CUS exposed rats, we removed the brains and separated PFC from them to observe the astrocyte gap junctions in the PFC by Transmission electron microscopy(TEM). To study the effects of long term Rg1 5, 10, 20 mg/kg administration on gap junction protein levers, some of the fresh PFC tissue were used to investigate the Cx43 protein levels by WB. In addition we analyzed the correlation between Cx43 levels and the behavioral changes in SFT and FST in Rg1 treated rats.Results: The diffusion distance of LY in the model group was reduced by 60% compared with normal control. Long term Rg1 10, 20 mg/kg administration, compared with model group, significantly increased the diffusion distance of LY(P<0.001). The detection result of coupling cell number was consistent with the above results. The EM result showed that the ultrastructure of astrocyte gap junctions between two neighboring astrocytes in the PFC of the CUS exposed group was damaged and the width between them increased more than two times with statistically significant difference when compared to the control group(P< 0.001). At the same time, long term Rg1 20 mg/kg treatment significantly improved the gap junction ultrastructure lesions(P<0.001). WB results showed that CUS can significantly reduce the protein levels of Cx43(P < 0.01), while long term Rg1 20 mg/kg administration obviously reversed it(P<0.001). And Cx43 expression levels were significantly correlated with the behavioral changes in SPT and FST.Conclusion: Antidepressant-like effects of Rg1 are due to the regulation on the Cx43 of astrocyte gap junctions in the PFC.Part 3 Animals were injected with CBX, a gap junction blocker, in the PFC, and the effect of Rg1 on animal behavior and Cx43 were observedObjective: To further validate the relationship between Rg1 and astrocyte gap junctions.Methods: Animals were injected with CBX(100 mmol/L) or PBS buffer as a control in the PFC with Rg1 20 mg/kg or water treatment twice per day for consecutive three days, respectively. And then SPT, NSFT and FST were detected as before. After that, rats were sacrificed the brains were removed, the tissue of PFC was then detected by WB like before.Results: The sucrose preference was significant lower in animals injected with CBX. Compared with solvent group, Rg1 can significantly improve animal`s sucrose preference(P<0.001). The average time of latency to feed in the CBX injected animals was 285.3 seconds while Rg1 greatly shortened it in the NSFT(P<0.001). The FST results showed that CBX prolonged the immobility time to about 40%, Rg1 administration significantly reduced the immobility time(P<0.01). CBX significantly decreased the protein levels of Cx43 in the WB experiment(P<0.05), and Rg1 administration greatly reversed the changes above(P<0.05).Conclusion: Rg1 can significantly reverse the depression-like behavior changes and upregulate the expression of Cx43 in animals which were injected with gap junction blocker CBX in the PFC.