| Alzheimer's disease (AD) is a neurodegenerative disorder, leading to loss of memory, progressive decline in cognitive function, and premature death. One of the hallmarks of AD is the presence of numerous senile plaques in the main brain regions, especially hippocampus, an area known to play a role in memory processing. Beta-amyloid (Aβ) is the main constituent of these plaques. The neurotoxicity of Aβin vivo and in vitro has been widely reported, and thus"Aβhypothesis"has been accepted and used to explain the main cause of AD.Long-term potentiation (LTP) is an activity–dependent increase in the synaptic transmission, which has been long regarded as a cellular model for learning and memory. Considering the serious impairment of learning and memory in AD, LTP may then represent a good model with which to examine the neuronal mechanisms involved in diseases associated with cognitive, such as AD. In the previous study, it has been widely reported that Aβpeptides can impair hippocampal synaptic plasticity in the form of LTP in vitro and in vivo. However, the mechanisms by which Aβand it's fragments inhibit LTP are still mostly uncertain, especially the changes in intracellular signal transduction pathways. Previous research shows that Calcium/calmodulin-dependent protein kinase II (CaMKII) is the most abundant protein kinase in hippocampus. It is reported that the mutation of alpha-CaMKII gene and postsynaptic application of CaMKII inhibitor induced impairment of spatial learning in the Morris water maze and blocked LTP induction. These all show that CaMKII plays an important role in gene expression, synaptic plasticity and permanent memory. Long-term depression (LTD), an opposing process compared with LTP, is an activity–dependent decrease in the synaptic transmission. It is reported that hippocampal LTD may be important for the clearing of old memory traces. Some study further suggested a possible involvement of CaMKII in the induction of LTD.However, it is unclear whether there must be the alteration of phosphrolated level of CaMKII while recording LTP/LTD with electrophysiological method, especially, whether the phosphrolated level of CaMKII has been changed during Aβ-induced suppression of LTP. In the present study, in order to investigate the correlation between the changes in phosphorylated CaMKII and the induction of LTP /LTD in the CA1 region of the hippocampus and to clarify whether phosphorylated CaMKII was involved in Aβ-induced inhibition of hippocampal LTP, we recorded field excitatory postsynaptic potentials (fEPSPs) in the CA1 region of rat hippocampus by using electrophysiological technique at first, and then measured the phosphorylated CaMKII in the CA1 region of hippocampus with immunohistochemistry technique at the same brains of animals after finishing the electrophysiological recordings. The results showed that:(1) On th e basis of stable recording of the basal fEPSPs for 30 min, the amplitude of fEPSP in HFS control group (n=6) increased to (204.38±10.22) % immediately at the end of HFS, and still remained at(162.37±5.5)% at 30 min post HFS, suggesting a successful induction of LTP in hippocampal CA1 region. Simultaneously, the immunoreactive product of p-CaMKII in test stimulation group was weak, with a mean grey value of 165.01±9.43; but in HFS control group, the dye of immunoreactive substance is obviously dark, with a mean grey value of 132.53±8.19 (P<0.01) and indicating a significantly stronger expression of p-CaMKII than that in test stimulation group.(2) HFS-induced hippocampal LTP was significantly decreased by the pretreatment with Aβ25-35 (i.c.v., 25 nmol) for 15 min or 1h. In the 15 min pretreatment group (n=6), LTP values were (171.71±6.74)% and (132.74±3.86)% at 1 min and 30 min following HFS, with significant difference compared to the values of (204.38±10.22) % and(162.37±5.5)% in the HFS control group (p<0.05) at the same time points. In the Aβ25-35 pretreatment 1h group, LTP values were (128.09±5.13)% and (102.54±5.17)% at two time points, indicating an further depression of LTP compared to the HFS control group and 15 min pretreatment groups (p<0.05). At the same time, the results of immunohistochemical experiment show that the mean grey values of immunoreactive product of p-CaMKII in 15 min and 1h Aβ25-35 pretreatment groups were 152.27±7.68 and 163.93±7.14 respectively, significantly higher than the value of 132.53±8.19 in HFS control group (p<0.05). There was also a significant difference between two Aβ25-35 groups (p<0.05). These results show that i.c.v. injection of Aβ25-35 time-dependently reduced the expression of p-CaMKII.(3) low-frequency stimulation (LFS) successfully induced hippocampal LTD. The amplitude of fEPSP evoked by test stimulation decreased to (65.94±5.38)% and (85.41±2.39)% at 1min and 30min following LFS respectively (n=5). The results from immunohistochemical measurement show that the mean grey value of immunoreactive product of p-CaMKII was 149.63±10.72, which was significantly stronger than that in the test stimulus group( P<0.05) and indicating an increase in p-CaMKII during LTD.(4) HFS-induced LTP in the CA1 region of the hippocampus could be effectively reversed by LFS. After LFS was applied, the amplitude of fEPSP decreased from (158.33±6.80) % at 30 min following HFS to (102.17±6.27)% (n=6). Then the phosphorelated CaMKⅡwas observed by immunohistochemistry 30min after LFS. The mean grey value of p-CaMKII immunoreactive product was 138.52±9.84, without significant difference compared to the HFS control group (P>0.05).Our results indicates that (1) the expression of p-CaMKII in the hippocampal CA1 region was up-regulated at different degree during HFS-induced LTP and LFS-induced LTD, with a more stronger enhancement in LTP, suggesting a close relationship between the induction of LTP/LTD and p-CaMKII in the hippocampal CA1 region; (2) Aβ-induced LTP impairment is consistent with the decreas in the level of phosphorylated CaMKⅡin the hippocampal CA1 region, suggesting that reduced phosphorylation of CaMKⅡmay be involved in the Aβ25-35-induced LTP impairment; (3) LFS could effectively reverse HFS-induced LTP, but phosphorylated CaMKⅡdid not return to the level during LTP. Therefore, our results further confirmed the participation of CaMKⅡduring LTP/LTD induction electrophysiologically and immunohistochemically, and show that reduced phosphorylation of CaMKⅡplays an important role in the Aβ25-35-induced suppression of LTP. Accordingly, we suppose that increasing the phosphorylation of CaMKⅡmay promote or resume learning and memory in AD patient by protecting hippocampal LTP against Aβ25-35-induced neurotoxicity. Meanwhile, the enhancement of CaMKⅡactivity also facilitates the inductin of LTD, which will help to strengthen the clearing of old memory and make relearning more easy.
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