The Mechanism Of Psychological Stress's Effects On Working Memory And Dopaminergic Neuron Of Rats And The Intervention Effects Of Tyrosine

Psychological stress is a common situational response. US army's researches indicate that psychological stress and various military circumstance stress are to decrease the thinking capability and mental analysis capability, and apt to decline work memory and efficiency. The etiological significance of psychological stress in brain's cognitive function becomes the focus of research in this area.Dopamine (DA), a pivotal neurotransmitter of human being, is known to relate nearly to cognitive functions such as work memory, cognitive adaptation, draw-up function, and so on. Resent researches suggest that DA system of central diencephalons- limbic system-forehead cortex (known as VTA-NAc-mPFC) plays a pivotal role in the procedure of cognitive and learning. D1 and D2 receptors take part in the regulation of memory procedure, and DA lack and excessive have affinitive relation to function.Otherwise, some studies indicated that tyrosine, the precursor of catecholamine neurotransmitter, is administrated to prevent the cognitive dysfunction induced by stress. However there are no relative reports about the dosage of Tyr administration, its effects and relationship with the system of VTA-NAc-mPFC, and dopaminergic neuron protective effects of Tyr administrated in vitro. There is practice significance to study into the biological effects and dosage-effects relationship of Tyr administrated in vivo and in vitro.OBJECTIVEAnimal model of psychological stress was interfered with Tyr administration in different dosage, to probe into its effects and mechanism on rats' spatial memory, central VTA-NAc-mPFC DA system. And to study Try's dopaminergic neuron protective effects in corticosterone injuring primer culture of VTA neuron.METHODS1. The animal model of learning and memory was established by training rats with eight arm rediatiform labyrinth box, and was induced psychological stress by communication box method. It was probe into the effects of intervention with tyrosine (administrated by feed in different dosage 250mg/Kg, 500mg/Kg, 1000mg/Kg) on work memory.2. effects and its mechanisms of psychological stress and tyrosine administration on the VTA-NAc-mPFC dopamine system: (1) The maxim bonding volume (Bmax) and the maxim dissociation constant (Kd) were assayed by radioactive ligand-bonding assay. (2) The content of DA and DOPAC, and the turnover rate of DA (DOPAC/DA) were determined by high pressure liquid chromatogram-electrochemical detector (HPLOECD). (3) The TH-positive neuron and Fos protein density in VTA-NAc-mPFC system were observed with immunohistochemistry (IHC) staining. (4) The concentrations of tyrosine, Excitatory and inhibitive amino acid in separate area of VTA-NAc-mPFC system were determined with HITACHI-835 amino acid auto-analyzer. (5)The concentrations of adrenocorticotropin(ACTH) and corticosterone (CORT) in blood plasma were detected with ELISA kit.3. The primer culture of VTA neuron was derived from neonatal SD rats, evaluated by morphologic observation and immuno-f luorescence, andtreated with CORT. After intervention with various dosage of tyrosine (10"°, 10"6, lO'W/L), the neuron survive ratio was determined with MTT colorimetry.4. statistic analysis: data, showed as x+S. E.M or x±SD, wereanalyzed with SPSS 11.0 software pack by the anova and t test of the mean of specimen.RESULTS1. effects of psychological stress and tyrosine intervention on the work memory: there were significant increase in errors of work memory of psychological stress group at the time of Id, 7d, lOd, 13d compared to control. Errors of reference memory at all time-point were high too. There were significantly declined in errors of work memory of Tyr lOOOmg/kg intervention group at 7d and 13d, and in reference errors at 4d and lOd. Tyr 500mg/kg intervention group also appeared statistical significantly declining in errors of work memory and reference memory at lOd.2. Effects and its mechanisms of psychological stress and tyrosine administration on the VTA-NAc-mPFC dopamine system(1) detect of Dl and D2 receptors: Comparing to control, there was significant decreasing in Bmax of Dl receptor in mPFC and NAc areas of psychological stress group, significant decreasing in and Bmax of D2 receptor in mPFC area, there were significant increasing in Bmax of Dl receptor in mPFC area of Tyr 500mg/Kg and lOOOmg/Kg intervention group Comparing to that of psychological stress group, while no difference in Bmax of D2 receptor in three cerebral areas. There was no difference in Kd of Dl and D2 receptors between various groups.(2) TH and Fos protein immunohistochemistry staining: There wassignificant decreasing in amount of TH immuno-positive neuron of various slices of VTA of psychological stress group than that of control. The amounts of TH immuno-positive neuron at the slice 5.1, 5. 4, 5. 7mm apart from anterior fontanelle of Tyr 500mg/kg intervention group were higher than that of psychological stress group, while that of the slice 4.8N 5.1, 5.4, 5.7mm apart from anterior fontanelle of Tyr lOOOmg/kg intervention group were increased too. There was no significant difference in the amounts of TH immuno-positive neuron between Tyr 250mg/kg intervention group and psychological stress group. The count densities of Fos protein immunoreaction substance of psychological stress group in VTA, NAc and mPFC areas were significantly ascended than that of control, while that of Tyr high and middle dosage intervention group in VTA and mPFC area were higher than that of psychological stress group.(3) the content and the turnover rate of DA: the contents of DA in mPFC , NAc , VTA areas of psychological stress group were all significantly decreased, and the turnover rate were increased than that of control. Intervention with Tyr, the contents of DA in various areas increased than that of psychological stress group, and had significant relationship with the dosage.(4) concentration of ami no acid in separate cerebral areas: in mPFC, NAc and VTA area, Asp, Glu and GABA of psychological stress group were increased, EAA/IAA was inclined, Tyr were decreased, and there was no difference in Gly. while intervention with Tyr, Asp of mPFC, NAc were declined, the degree of changes of EAA and IAA levels in central VTA —NAc—mPFC system was lowed. There had significant linear relationship between concentration of Tyr in various cerebral areas and dosage of Tyr.(5) The concentrations of ACTH and CORT in blood plasma: CORT ofpsychological stress group were significant increased in Id and 7d than that of control. CORT of Tyr lOOOmg/kg intervention group was decreased in 7d than that of psychological stress group. ACTH had the same change trend as CORT.3. Effects of Tyr intervention on the prime culture of dopaminergic neuron injured by CORT: CORT in 10"° mol/L induced damage of VTA neuron in vitro, the survive rate after 24h and 72h CORT administration was 64. 7% and 58. 6% of that of normal culture. Tyr in 10"* mol/L efficiently reduced the damage of VTA neuron in vitro induced by C0RT(24h and 72h, P<0. 01), while no effects in 10"5 mol/L 24h, but protective effects was significant in72h(P<0. 05). Tyr in 10"7 mol/L had significant protective effects in 24h, but not in 72h.CONCLUSIONS1. Psychological stress impairs the spatial work memory of rat; lOOOmg/kg tyrosine improved the spatial memory grade of psychological stress rats in the rather early stage; 500mg/kg tyrosine had certain protective effects on the detriment of long-term spatial memory.2. The central DA mechanism of psychological stress impairing the work memory is in several aspects showed as below:(1) Psychological stress impairs spatial work memory of rats by a DA mechanism mediated by Dl receptor. Dl and D2 receptor has cooperative effects, and has area specificity in their effects on VTA—mPFC—NAc system.(2) Psychological stress courses damages of TH immuno-positive neuron in VTA, lows product of TH or the staining character of TH immuno-positive neuron.(3) Psychological stress decreases content of DA in mPFC -. VTA, while hoists turnover rate of DA.