Effect Of Chronic Stress On Spatial Learning And Memory In Adult Rat

Objective: Combining isolation with chronic unpredictable mild stress (CUMS) establish the chronic multiple stress rat model, using the behavioral testing, histological observed and molecular biology method, study the effect of chronic stress on the rat spatial learning and memory and the expression of neural cell adhesion molecule (NCAM) and growth-associated protein (GAP-43) in hippocampus and frontal brain, as well as the relationship between the change of expression of NCAM, GAP-43 and stress behavior, to explore the mechanism of the effects of chronic stress on spatial learning and memory.Methods: (1) Animal model: a continuous 35 days of chronic unpredictable mild stress with isolated raised establish the chronic multiple stress rat model. 16 male Sprague-Dawley rats were randomly divided into normal control group and stress model group (n=8). The control group rats were normal fed during the period of establish model, while the model group were given 35 days of the stress stimulus. (2) Animal behavior indicators: Observing the body weight of the two group rats during experiment process, using the Open-field method and double bottles of sugar water consumption to evaluate the changes of behavior after stress, as the standard of the model whether to establish. Using Morris water maze and Y maze examine the capability of learning and memory in rat. (3) Brain tissues morphology observation: at the basis of the success in establishing chronic stress rat model, all rats were deeply anesthetized and anatomized, whole brain were fixed with cold 4% paraformaldehyde, cut the bilateral hippocampus and frontal brain areas, HE staining, using light microscope (×400) observe two group rats of morphological changes of neurons in various hippocampus sub-regions and frontal cortex, count the number of neurons, and analysis of the correlation with the number of neurons in brain regions and the behavior indicators of Morris water maze and Y maze. (4) Protein expression assay: Using immunohistochemical staining technique determined the changes of NCAM, GAP-43 expression in sub-region of rat hippocampus, prefrontal brain regions, then using IPP image analysis system, measured the mean optical density of the immune reaction products, and analysis of the correlation with the expression levels of NCAM, GAP-43 and the behavior indicators of Morris water maze and Y maze. Results:(1) Effects of chronic stress on spatial learning and memory and other behavior: weight growth and the percentage of the amount of sugar intake in total water intake in model group were significantly reduced (p<0.01); in open-field experiments, compared with the control group, the model group of the horizontal move distance and standing times were significantly reduced (p<0.01), the residence time in central area, dejecta grains had increased (p<0.05), the difference was significant, indicating that the chronic multiple stress rat model building successful; in Morris water maze test, the model group, the average latency period of 5 days training obviously delayed (p<0.01), the number of through platforms significantly reduced (p<0.01); in Y maze experiment, the model group of the number of active avoidance response at the next day reduced (p<0.01), compared with control group, the differences were statistically significant.(2) The impact of chronic stress on the morphology and the number of neural cells of rat hippocampus sub-region, frontal cortex brain:Light microscope (×400) morphology observation: model group, the positive hippocampal CA1 pyramidal cell layer growth thinner, arrangement were sparse and disorder, the cell body shrink, cell gap large; while the control group of rats hippocampus CA1 pyramidal cells were clear and complete, the distribution were tight and order, there are obviously a large number of nerve fibers. Model group, hippocampal CA3 pyramidal cells, arrangement were sparsely scattered, cell gap widened, cell loss, cytoplasmic staining were weak; while the control group of rats hippocampus CA3 pyramidal cells, arranged in order, the cytoplasm stained dark. Model group, hippocampal granule cells in DG area edema, showed vacuolar and degeneration, deletion, cell layer is thinner, Cell gap widened, arrangement sparse; while the control group of rats hippocampus granule cells in DG area clear and complete, the distribution of area were large and dense. Model group the nuclei of granules cells in rat frontal cortex were densely stain, condensation, accompanied by proliferation of glia cells gathered, near by the necrotic neurons; while the control group of frontal cortex granules cells showed normal morphology and distribution.As the number of neurons, model hippocampal pyramidal cells in CA1 and CA3 area were reduced (p<0.05), granule cells in dentate gyrus decreased (p<0.01), compared with the control rats, the difference was significant; the prefrontal cortex granule cell, two groups were no significant difference. (3) Effect of chronic stress on the expression of NCAM, GAP-43 in hippocampus sub-region and frontal cortex brain regions: Model group, the NCAM protein expression level in hippocampal CA3 and DG areas were low, namely both negative reaction, NCAM immunohistochemical results in the frontal brain areas showed a weak positive reaction, while the control group were both positive reaction; measuring the immune reaction products average optical density make semi-quantitative analysis, the difference between the model group and the control group were marked (p<0.01). Model group, the immunohistochemistry results of GAP-43 in hippocampal DG area were weak positive reaction, while the control group showed strong positive reaction; measuring the immune reaction products average optical density make semi-quantitative analysis, the model group compared with control group difference was significant (p<0.01).(4) The correlation analysis of the number of neuron cells, expression level of NCAM, GAP-43 in the two brain regions and the behavior indicators of Morris water maze and Y maze, as well as the expression level between NCAM and GAP-43:The relation between the cell number in hippocampal CA1, CA3, DG area and the frequency of active avoidance response the next day in Y maze were both significantly positively correlated (p<0.01; p<0.05; p<0.05), the relation between the cell number in hippocampal DG and the times of learning during the first day in Y maze was significantly negatively correlated (p<0.05); the relation of NCAM expression in the hippocampal CA3 as well as in the frontal brain and the average latency period of 5 days water maze training were both significant negative correlation (p<0.05), the relation between NCAM expression in hippocampal CA3 area and the frequency of active avoidance response the next day in Y maze was significantly positively correlated (p<0.01); the relation between GAP-43 expression level in hippocampal DG area and the times of through platform in water maze also was significantly positive correlation (p<0.05).The relation between the expression level of GAP-43 in hippocampal DG area, expression of NCAM in hippocampus DG area as well as frontal cortex and the expression levels of NCAM in hippocampus CA3 area were both significantly positive correlation (p<0.05); the relation between the expression level of GAP-43 in hippocampal DG area and the expression level of NCAM in hippocampus DG area was significantly positive correlation (p<0.01). Conclusion:(1) Chronic stress damage spatial learning and memory ability of rats, not only disturb the learning process (the acquisition of spatial memory consolidation), but also affect the extraction of spatial memory.(2) Chronic stress change the morphology and number of neuron cells in rat hippocampus and frontal cortex, and there is a correlation with the number of neuron cells in the hippocampus sub-regions and the indicators of spatial learning and memory, suggesting that chronic stress damaged spatial learning and memory of rat may be cause by the neuronal degeneration or apoptosis in hippocampus and frontal brain regions.(3) Chronic stress model of rat NCAM, GAP-43 protein expression decreased in hippocampus and frontal, there is a correlation between the expression of two proteins; and the relation between expression level of the two proteins and the indicators of spatial learning and memory of rat was strongly correlation, speculated that the reduction of expression level of NCAM, GAP-43 in rat hippocampus and frontal may be one of the molecular mechanisms in chronic stress damage spatial learning and memory of rats.