Spatial Learning And Memory Deficit In Chronic Stress-induced Depressive Rats And The Mechanism

Background:Depression, the most common neuropsychiatric disorders, is the fourth major disease in the word and has durable and significant mood disorder as its main feature. Patients with depression shows symptoms of emotional disorders, sleep disorders and cognitive dysfunctions (including dysfunctions of attention, learning, memory, decision, performance and so on). The World Health Organization reported that there are about350million patients with depression all over the world, among them the number of suicides is estimated at100million per year. Additionally, depression is a risk factor for Alzheimer’s disease (AD), which is an important neurodegenerative disease.Depression is a multifactorial disease caused by social-physical-psychological factors. Stress is an important inducement in the pathogenesis of depression. Neuroimaging studies shows in the brains of depression patients the prefrontal cortex, amygdala and hippocampus become smaller, and the lessen of these regions will be more obvious with the disease progressing. The atrophy of these regions were also showed by autopsy studies. Because of the important role in learning and memory, and also in emotion regulation, hippocampus is largely concerned in recent studies on depression. Studies have shown spatial learning can increase neurogenesis and improve hippocampal-dependent cognition. Disorders of hypothalamic-pituitary-adrenal (HPA) axis together elevated serum glucocorticoids were reported in depression patients. Glucocorticoids are a large class of stress-related hormone, whose short term increasing may help to resist external stimuli. However, long term elevation of glucocorticoids may bring adverse effects. Hippocampus has large distribution of glucocorticoid receptors (GR). The mRNA and protein levels of GR were reported decreased in the hippocampus of depression patients. All above studies suggest the important role of hippocampus in depression, but the mechanism remains unclear. Some key problems need to be further studied such as whether the improvement of hippocampal function could improve symptoms of depression.Death-associated protein kinase1(DAPK1) is activated and involved in various neuronal injury diseases (such as stroke, AD) by different ways. During the stroke, activation of DAPK1induces apoptosis of neron; in AD, activation of DAPK1causes neurodegeneration by hyperphosphorylating microtubule-associated protein tau. Extracellular signal-regulated protein kinase/ribosomal S6kinase1(ERK/RSK1) are important signaling molecules antagonizing DAPK1activation induced apoptosis. Whether DAPK1is involved in the pathogenesis of depression, and the relationship between hippocampal damage and DAPK1activation, have not yet been reported.Aim:To study whether hippocampal damage appeared in chronic stress-induced depressive rats and its mechanism. To investigate whether enhancement of the hippocampal function can improve the symptoms of depressive rats.Methods:In this study, SD rats received chronic stress for21days, including restraint for2hours, night lighting, food deprivation for24hours, water deprivation for24hours, high temperature (45℃), ice water swimming (4℃), clip the tail, foot shock (current intensity is0.5mA). After stress, behavior changes were detected by open field test, forced swimming test, sucrose preference test and Morris water maze test. Then, the Bruker Magnetic Resonance Imager (MRI) were used to detected hippocampal atrophy of chronic stressed rats. Then the number of neuron, the number and type of dendritic spines of rat hippocampus were detected and analyzed through Nissl staining and Golgi staining respectively. Enzyme-linked immunosorbent assay (ELISA) was used to detect the corticosterone levels of serum and hippocampus after stress. Western blotting and immunohistochemical were used to detect and analyze the level and distribution of glucocorticoid receptor and other proteins. To observe the improvement of depressive behavior of hippocampal function, we give7days of water maze learning to depressive rats, depression-related behavior, the biochemical and morphological examination conduct after the end of learning. To study the mechanisms of reduced number of neurons in hippocampus of depressive rats, we examined changes in ERK, RSK1activity and apoptosis and autophagy-related proteins in N2a cells treated with GC (10-4M), reduced DAPK1of N2a cells treated with GC (10-4M), and upregulation of DAPK1in N2a cells.Result:After stress, rats’ spontaneous activity weakened in the open-field experiments, the immobility time was significantly longer in forced swimming test, and the percentage of sucrose consumption reduced31.6%, indicating that chronic stress can induce depression-like behavior in rats. In the Morris water maze, the latency to find the platform of the fifth and sixth day were13.1. and12.8seconds, and on the seventh day of the test, the latency for the first time across the platform region was13.2seconds, and the number of cross-platform within60seconds was3.1times in the control group. In the depressive rats, the latency to find the platform of the fifth and sixth day increased to28.4and26.2seconds, and on the seventh day of the test, the latency for the first time across the platform region increased to23.8seconds, and the number of cross-platform within60seconds decreased to1.6times. On the14th day of the test, the lantency (36.1seconds) is still higher than that of normal rats (12.5seconds). And the number of cross-platform within60seconds (1.6times) is still lower than in the control group (3.9times). The above data shows that not only short-term memory ability is impaired in depressive rats, long-term memory is also damaged. Long term potentiation (LTP) is the functional basis of learning and memory. In hippocampal LTP experiments, stimulation of the entorhinal area perforant path (PP), and record the LTP of dentate gyru (DG). In the first55-60minutes field excitatory postsynaptic potential (fEPSP) slope of the stress group decreased to67.9%of the normal group, in the first85-90minutes decreased to69.4%of the control group, indicating that chronic stress can hinder the induction of LTP, and damage the learning and memory. The volume of hippocampas decrased18.9%compared with control rats detected by Bruker magnetic resonance imager. By Nissl staining the neurons of hippocampal CA1, CA3, DG reduced by18.8%,21.2%,18.9%, while the Golgi staining showed the number of dendritic spines in CA1, CA3and DG region decreased by31.1%,37.1%,41.6%, and the proportion of mushroom dendrites in CA1, CA3, DG area decreased by46.7%,48.1%,52.4%. These results suggest that the hippocampal structure and function abnormalities emerged in depressive rats.After the stress the corticosterone levels of serum and hippocampus increased by169.7%and116.5%respectively compared to control rats detected by ELISA. Western blot found that the glucocorticoid receptor of rat hippocampal tissue decrease by21.9%, and pS211-GR decreased by30.4%. Meanwhile, immunohistochemical staining showed the same trend, indicating the decreased activation of GR.To study the role of DAPK1in hippocampal neurons of depressive rats and its mechanism, by Western blot of rat hippocampus, we found that DAPK1increased193%; ERK reduced by27.3%, ERK phosphorylation (p-ERK) decreased by88.2%, indicating the decreased activation of ERK; RSK1decreased by37.8%, pS380-RSK decreased by64.5%, indicating the decreased activation of RSK1; caspase3increased by60.8%, cleaved caspase3increased by193.2%, indicating the activation of apoptosis; Beclinl increased by52.5%, Microtubule-associated protein light chain3Ⅱ (LC3Ⅱ) increased by45.7%, indicating the increased formation of autophagosome. Immunohistochemical staining found that the levels of DAPK1in hippocampal CA1, CA3and DG region increased by143.7%,72.9%,91.5%respectively. And distribution of DAPK1in neurons have changed, the DAPK1transferred from the neurite to cell body. In the hippocampus of depressive rats, the levels of ERK in CA1, CA3region reduced by18.7%and28.9%respectively; and p-ERK levels were reduced by47.6%and38.4%respectively.The N2a cells were treated with GC (10-4M) for24h. Compared with the control cells, DAPK1increased by56.5%, total caspase3increase by180.3%, cleaved caspase3increased by119.9%, Beclinl increased by192.6%, total LC3increased by127.3%, LC3II increased by50.3%, total ERK decreased by40.6%, ERK phosphorylation decreased by68.6%, RSK1decreased by28.2%, pS380-RSK decreased by68.6%.After transfection of siDAPK1in N2a cells for48hours, glucocorticoids (10-4M) was added in the medium for24hours. Compared with the control cells, DAPK1expression decreased by70.2%, ERK increased by46.4%, ERK phosphorylation increased by134.6%, RSK1increased by119.0%, pS380-RSK increased by49.1%, caspase3decreased by68.9%, cleaved caspase3decrease by68.6%, Beclinl decrease by77.5%, total LC3decreased by26.4%, LC3II decreased by49.3%.After overexpressing DAPK1in N2a cells for48h, compared with the control cells, DAPK1expression increased by70.4%, total ERK decreased by75.5%, ERK phosphorylation decreased by63.7%, RSK1decreased by59.0%, pS380-RSK decreased by81.5%. These results showed that:stress-increased high level of glucocorticoid acitivated DAPK1expression and then induced apoptosis and autophagosome formation; DAPK1inhibited ERK and RSK1activity, which reduced antagonism for apoptosis induced by DAPK1of ERK/RSK1.To further confirm the importance of the hippocampus in depression, seven days of water maze learning and training was given to the depressive rats, the latency to find the platform decreased significantly, in the open field test, the total number of crossing zone (horizontal score) increased to66.2, the number of climbing the walls of the box (vertical score) increased to15.08, and the total distance in the box increased to14.37m, which increased significantly after learning. In forced swimming test, the immobility time decreased from154.3s to74.8s, which decreased significantly after learning.. In sucrose preference test, the sucrose consumption percentage increased from46.5%to75.9%. These suggested that the depression-like behavior had clearly improved. However the rats without learning showed no significant change.By Golgi stain, the density of dendritic spines in hippocampal CA1, CA3, DG regions of rats with spatial learning were4.97/10μm,5.01/10μm,5.12/10μm, compared with the rats without learning, which increased significantly. The mushroom-type dendritic spines proportion of learning group were91.2%,89.3%,85.7%, compared with the rats without learning, which increased significantly. After one week of spatial learning, learning group compared with the non-learning group, DAPK1reduced by81.4%, ERK increase by36.2%, ERK phosphorylation increased by42.4%, RSK1increased by63.6%, p380-RSK increase by73.8%, caspase3decreased by126.7%, cleaved caspase3increased by236.3%, Beclinl decreased by91.6%, LC3Ⅱ decreased by92.4%.These showed that spatial learning can significantly improve depressive symptoms of rats after stress. The key mechanism may be reduced activity of DAPK1and activation of ERK and RSK1. Conclusion:Chronic stress-induced depressive rats showed spatial learning and memory deficit with decreasing of neurons and dendritic spines in hippocampus. Elevated corticosterone levels after stress and apoptosis increasing after DAPK1activation may be parts of the key mechanisms. Spatial learning improved depressive symptoms may be related to enhanced hippocampal ERK/RSK1activities and inhibition of DAPK1. These studies have shown the key role of hippocampus in depression. Whether autophagy activated by DAPK1, autophagy in the pathogenesis of depression and other issues need to be further studied.