Study On The Effects Of Zinc Deficiency On Learning And Memory Function In Rats And The Cell Signaling Mechanisms Involved

Objective:In the present study, the typical zinc deficient rat model was set up at first; and then learning and memory function, efficacy of synaptic transmission, function of antioxygen and the apoptosis of hippocampal nerve cells were evaluated, and the expression of several signals involved in the cAMP/PKA and MEK/ERK signaling pathways in learning and memory function under zinc deficient condition were explored. The results can provide us with important insights into the molecular mechanisms of cognitive dysfunction induced by zinc deficiency. Methods: (1) Establishment of zinc deficient rat model. Ninety-six male weanling Wistar rats (60g～80g) were randomly assigned to zinc-deficient (ZD), pair-fed (PF) and zinc-adequate (ZA) groups. Rats of ZD group were given the basal diet(1.5 mg Zn/kg). Rats of ZA group and PF group were fed with the basal diet supplemented with zinc sulfate(30 mg Zn/kg). Rats of PF group consumed the same intakes as the corresponding ZD partner on the previous day. All rats were given free access to deionized water. The period of experiment is one month. (2) Assessment of zinc status. Zinc levels in plasma, hippocampus and cortex were determined with flame atomic absorption spectrophotometry. The distribution and level of zinc in rat hippocampal CA3 region were visualized and assayed by TSQ fluorescence method. The activities of plasma alkaline phosphatase (ALP) were measured by kinetic colorimetric assay. (3) Evaluation of cognitive function. Learning and memory function of rats were examined by passive avoidance performance and Y maze experiment . (4) Induction of LTP. The efficacy of synaptic transmission was observed by the induction of LTP in rat hippocampal dentate gyrus. (5) Measurement of several biochemistry indexes. The levels of Ach , NE, DA and 5-HT in serum, hippocampus and cortex were determined by kinetic colorimetric assay and fluorospectrophotometry respectively. The content of malondialdehyde (MDA) in serum, hippocampus and cortex was assayed by thiobarbituric acid (TBA) method. The activity of superoxide dismutase (SOD) and the total antioxidative capacity (T-AOC) in serum, hippocampus and cortex were assayed with commercial reagent kits. The apoptosis rate of hippocampal nerve cells was measured by Flow cytometry (FCM). (6) Detection of the changes of signals in signaling pathways. The content of cAMP was determined by radio immunoassay (RIA); The activity of PKA was analyzed by PepTag detecting system . the expression levels of p-MEK, p-ERK1/2, p-CREB and BDNF were examined by Western blot, and the transcriptive levels of CREB and BDNF by RT-PCR. Results: (1) Rats in ZD group displayed typical symptoms of zinc depletion such as anorexia, growth retardation, severe hair loss and skin lesions. (2) Compared with ZA and PF groups, the body weight, food intake, plasma zinc concentration and ALP activity and the content of zinc in hippocampal CA3 region of rats in ZD group have obviously reduced(P<0.05). But there was no significant difference of zinc concentration in the hippocampus among the three groups after 1 month treatment. (3) Compared with ZA and PF groups, the latency period of ZD group was shortened(P<0.05), while the number of wrong response in Y maze test increased predominantly(P<0.05). (4) LTP was induced in rats of the three groups. However, compared with ZA and PF groups, the amplitude of LTP in hippocampal dentate gyrus in ZD group decreased significantly (P<0.05). (5) In comparison to ZA and PF groups, the whole blood Ach concentration in ZD group increased significantly(P<0.05), while Ach levels in hippocampal and cortex had the trend of reduction(P<0.05). The concentrations of NE, 5-HT and DA in hippocampal and cortex increased in ZD group compared with the other two groups(P<0.05). (6) Compared with ZA and PF groups, the MDA content in serum, hippocampal and cortex in ZD group increased remarkably(P<0.05). While the activity of SOD and the T-AOC were lower(P<0.05) in ZD group. And the apoptosis rate of hippocampal nerve cells increased remarkably in ZD group(P<0.05). (7) Compared with ZA group, the cAMP content of hippocampus in ZD group and PF group significantly increased (P<0.05), and ZD group tended to be higher than PF group. The cAMP content of plasma and cortex in ZD group was also significantly higher than ZA and PF groups(P<0.05). While the PKA activity of hippocampus and cortex in ZD group was lower than the other two groups (P<0.05). (8) The p-MEK,p-ERK1/2 protein expression level of hippocampus and cortex in ZD group strikingly decreased compared with ZA and PF groups(P<0.05). (9) Compared with ZA and PF groups, the p-CREB,BDNF protein expression levels and CREB,BDNF mRNA levels were remarkably lower(P<0.05).Conclusions: (1) Typical zinc deficiency model of rat was established successfully. (2) Learning and memory dysfunction in rats was induced by zinc deficiency. (3) Under zinc deficient condition, the efficacy of synaptic transmission in hippocampal dentate gyrus lowered, the contents of neurotransmitter abnormally changed, the function of antioxidant defense system impaired and the apoptosis of hippocampal nerve cells increased. All those maybe the factors that affecting learning and memory. (4) Zinc deficiency affected cAMP-PKA and the MEK-ERK signaling pathways of hippocampus and cortex in rats,then decreased the p-CREB,BDNF protein expression levels, inhibited the formation of LTP and impaired the learning and memory function. This may be the important molecular mechanism of learning and memory dysfunction caused by zinc deficiency .