Effects Of Chronic Multiple-Stress On Learning And Memory And The Role Of Fyn, BDNF/TrkB Signal Pathway In Its Mechanism

The effect of chronic stress on learning and memory functions has been one of the hot topics in neuroscience. Hippocampus is a key region highly involved with leaning and memory and it is the target site of stress. Therefore,stress induced functional impairment of the hippocampus must impact the learning and memory function. But the investigations found that different stress stimulation has different effect to learning and memory,and there has been much controversy over its mechanism. Many studies show that chronic body,mental,or multiple stress can leads to the morphology changes,even the death of the hippocampal neuron. Meanwhile it impacted the brain cognitive functions and induced to learning and memory impairment. There are a few studies indicated that chronic multiple stress could enhance the learning and memory function. Such single stressor as restraint,foot shock,induced pain,sleeping deprivation or noise,which impaired learning and memory has been applied to make animal models in the past. It is an innovation of this topic that animals were given chronic multiple stresses formed by four kinds of stressors at the same time to perform the following experiments:①Observe the changes of rats'learning and memory after six-week stresses.②Observe the expression changes of Synapsin I,Fyn,BDNF and TrkB protein levels after six-week stresses.③Observe the changes of Fyn mRNA and TrkB mRNA levels after six-week stresses.④The rats were injected PP2,a tyrosine kinase inhibitor in lateral cerebral ventricle to study the effects of PP2 on function of learning and memory and the expression of Fyn,BDNF,TrkB in the hippocampus of the rat whose leaning and memory was enhanced by chronic multiple stress.⑤In addition,PP2 was added to culture media to observe the effect of PP2 on the free calcium concentrations ([Ca2+]i) and Fyn expressions in hippocampal neurons by culturing primarily hippocampal neurons in vitro. The results in vivo were in agreement with those in vitro.This experiment includes three parts:Part I. The objective of the first part is to investigate the effects of chronic multiple-stress on learning and memory and on the levels of Synapsin I,Fyn, BDNF, TrkB protein and Fyn, TrkB mRNAs.Adult male Wistar rats were randomly divided into control and chronic multiple-stressed groups. The latter were administered four kinds of stressors,including vertical rotation,sleeping deprivation,restraint and illumination irregularly and alternately for 6 weeks. The body weights were examined once every week during the period of the stress. In order to examine the scores of leaning and memory,two groups of animals were given 3-day Morris Water Maze (MWM) test before the stress and MWM tests successively after completing the stress. The animals were divided into the following three groups:①Morphological group:Observe the distributions and expressions of Synapsin I,Fyn and BDNF in the hippocampal CA1,CA3 and DG using immunohistochemical technique. Examine the nerve cell densities of hippocampal CA1,CA3 and DG using Cresyl violet stain.②Western Blotting groups: Measure the Fyn,BDNF,TrkB protein expressions of hippocampal total protein extracted.③RT-PCR groups:Measure the Fyn mRNA and TrkB mRNA levels of the total mRNA isolated from hippocampus.These results were showed as follows: After stress,①Learning and memory scores of chronic multiple-stressed rats excelled those of the control group in the MWM.②The results of mmunohistochemical technique indicated that Synapsin I , Fyn and BDNF-immunostaining of the chronic multiple-stressed rats was harder than that of the control group. Respectively,the distributions of them are in the dendrites and axons of the pyramidal and granule cells of hippocampal (synapsin I),or in the stratum radiatum of hippocampal CA3(Fyn),or in the nucleus of the pyramidal and granule cells of hippocampal CA1,CA3,DG (BDNF).③The nerve cell densities in hippocampal CA1,CA3 and DG of chronic multiple-stressed animals were markedly higher than that of the control group.④The Fyn,BDNF,TrkB proteins of hippocampal total protein in chronic multiple-stressed group enhance significantly as compared with the control one from the Western Blotting.⑤The Fyn mRNA levels in chronic multiple-stressed group rose significantly as compared with the control one too from the RT-PCR.These results maked clear that chronic multiple stress can enhance spatial learning and memory function of rats. The cell density of hippocampal subregions is increased in the stessed rat. The expression of Synapsin I,Fyn,BDNF and TrkB proteins and the level of Fyn mRNA are increased in the stessed rat hippocampus respectively. Its suggest that BDNF/ TrkB signal transduction pathway and Fyn may participate in the process of the enhanced learning and memory by chronic multiple stress.PartⅡ. The second part aimed to investigate the effects of PP2,a tyrosine kinase inhibitor,on function of learning and memory and the expression of Fyn,BDNF,TrkB in the hippocampus of the rat whose leaning and memory was enhanced by chronic multiple stress with the method of lateral cerebral ventricle injecting.Adult male rats were randomly divided into three groups:chronic multiple stressed group (control group),chronic multiple stressed with saline group (saline group) and chronic multiple stressed with PP2 group (PP2 group). All rats were exposed to chronic multiple stress for 6 weeks,then rats of saline group and PP2 group were injected normal saline or PP2 in lateral cerebral ventricle(11d with one time / d). After that,the performance of spatial learning and memory of all rats was measured using Morris water maze. The expression of Fyn,BDNF and TrkB proteins in the hippocampus of rats were detected by immunohistochemical method.The Results showed:①Compared with control group and saline group,the performance of spatial learning and memory of rats was decreased in PP2 group;②The immunoreactivities of Fyn and BDNF in the hippocampus were weakened in rats of PP2 group;③But no difference of TrkB protein level in the hippocampus was observed between the three groups.These results maked clear that PP2 with intracerebro-ventricular may degrade the function of rat's learning and memory,and downregulated expression of Fyn and BDNF in the hippocampus of the rat whose leaning and memory was enhanced by chronic multiple stress. It suggests that Fyn and BDNF/TrkB signal transduction pathway may participate in the process of the enhanced learning and memory by chronic multiple stress.PartⅢ. The third part aimed to explore the effects of PP2,a tyrosine kinase inhibitor,on primary cultured hippocampal neurons and their free cytoplasmic calcium concentration ([Ca2+]i ) and Fyn expressions and its'possible mechanisms.The hippocampi were taken from 24 to 48-hour postnatal Wistar rats and hippocampal neurons were primarily cultured. 10 days later,the purity of neurons was detected using the antibody to neuron specific enolase (NSE) and the cells were divided into control group (with only serum-free DMEM-F12) and groups treated with PP2,which final concentration is 12.5μmol/L. All groups were performed the following experiments after the cells were cultured for 4d;①Observe the morphological changes of hippocampal neurons in different time;②Observe the morphological changes of hippocampal neurons in control group and PP2 group;③Explore the apoptosis of hippocampal neurons induced by PP2 using annexinV/PI by flow cytometry;④Measure the hippocampal neurons [Ca2+]i of two groups under the confocal laser microscope;⑤Examine the distributions and expressions ofFyn in hippocampal neurons,treated with PP2 by adopting the method of immunocytochemistry and Western blotting.These results were showed as follows:①The hippocampal neurons could adhere to the bottom of culture plate in time,grow well and form neural network.②Purity of hippocampal neurons in this experiment exceeded 70%.③The morphological change of hippocampal neurons was appearanced in PP2 group. Compared with the control neurons,the neurite of hippocampal neurons retracted and the neural network became rare. The primary apoptosis was caused in the PP2 groups.④There was a marked increase in hippocampal neurons [Ca2+]i at the PP2 group.⑤Fyn expressions were decreased in hippocampal neurons with PP2 in the immunocytochemistry and Western blotting technique. Their differences were statistically signifcant. There was a negative correlation between hippocampal neurons [Ca2+]i and Fyn expressions.The primary apoptosis occurred in the hippocampal neurons treated with PP2. The increase of hippocampal neurons [Ca2+]i might both result from an impairment of the hippocampal neurons induced by PP2 and result in the apoptosis of hippocampal neurons and low expressions of Fyn.To sum up,the changes in these indexes of chronic multiple-stressed rats and primarily cultured hippocampal neurons were observed at the whole and cellular level using in-vivo and vitro model system in this study. They were both independent and closely interrelated depending on PP2,a tyrosine kinase inhibitor. Chronic multiple stress can enhance spatial learning and memory function of rats. The cell density of hippocampal subregions is increased in the stessed rat. The expression of Fyn,BDNF and TrkB proteins and the level of Fyn mRNA are increased in the stessed rat hippocampus respectively. PP2 with intracerebro-ventricular may degrade the function of rat's learning and memory,and downregulated expression of Fyn and BDNF in the hippocampus of the rat whose leaning and memory was enhanced by chronic multiple stress. The morphological change of hippocampal neurons was appearanced in PP2 group on primary cultured hippocampal neurons. Primary apoptosis was caused and there was a marked increase in hippocampal neurons [Ca2+]i at the PP2 group. Fyn expressions were decreased in hippocampal neurons with PP2. Fyn and BDNF/ TrkB signal transduction pathway may participate in the process of the enhanced learning and memory by chronic multiple stress.