Induced Mini-stroke In Hippocampal CA1 Region Regulates The Subsequent Synaptic Plasticity

Stroke has become a leading cause of death in the world wide. Studies of its dysfunction and recovery mechanism are becoming increasingly fierce. Research shows that, long-term enhancement in glutamate receptor mediated excitatory responses has been observed in stroke model. This pathological form of plasticity, termed post-ischemic long-term potentiation(i-LTP), points to the functional reorganization after stroke. This kind of plasticity plays an important role in the ischemic dysfunction and recovery process. Therefore, to enhance the comprehension of i-LTP will contribute to the cerebral ischemia disease treatment. A lot of studies have proved that the i-LTP was caused by the activation of NMDA receptors following the excessive Ca2+ flow. However, when the i-LTP occurs in the ischemia models, whether and how the subsequent synaptic plasticity will be influenced? The answer remains unclear.The subsequent synaptic plasticity after i-LTP termed as metaplasticity, is proposed by Wickliffe C.Abraham and Mark F.Bear, defined as plasticity of synaptic plasticity. They think that synaptic plasticity can be modulated by prior synaptic activity. Therefore, this kind of plasticity plays a very important role in cognitive functions of the brain. We hypothesized that metaplasticity was important for regulating the dysfunction and recovery in the infarct regions. However, the metaplasticity regulation in ischemia regions are seldom reported.Moreover, loca field potential(LFP) which represents electrical property of cell ensembles in more restricted regions(50-350 ?m from the tip of the electrode), and reflects the neural network in the local area. To study the effect on LFP in ischemia models may provide new ideas for the treatment of stroke.At present, the effects on the metaplasticity and LFP in ischemia models are seldom reported. Therefore, we performed a series of experiments to study the effects on metaplasticity and local field potential in CA1 region of hippocampus by ET-1-mediated focal ischemia, and aimed to promote the treatment or functional recovery of cerebral ischemia.First of all, we established the dorsal hippocampal CA1 region local mini-stroke models by stereotaxic injection, and using magnetic resonance imaging and TTC staining to evaluate the models. Secondly, we performed Western blotting to test the molecules associated with i-LTP to confirm whether the i-LTP occurred. Thirdly, we recorded the fEPSP on the acute hippocampal slices by patch clamp, and we observed how the LTP was effected after the i-LTP. Otherwise, we used an 8-channel microelectrode to record the local field potential(LFP) in vivo, which can represent the real process after ischemia. Finally, we applied fear conditioning experiments to observe whether this local mini-stroke would impair the dorsal hippocampus dependent learning and memory.Results:(1) We used the MRI and TTC staining to examine and confirm the establishment of the mini-stroke model at 6h, 12 h and 24 h. We found that the region subjecting to ischemic attack was getting larger progressively as time went on.(2) We first directly confirmed the i-LTP can be induced in ET-1-induced ischemia model as described previously in other in vitro models.(3) Then we employed western blotting assays to examine the expression level of NR2 B, CaMKII and p-Ca MKII at 6 h, 12 h and 24 h after ischemia. We found that there was no changes of these protein molecules at 6 h after ischemia, and 12 hoursafter ischemia, the expression of NR2 B and Ca MKIIshowed a slight increase, but this increase was not statistically significantwhen compared with control while at this time there was no increases of p-Ca MKII. However, at 24 hoursafter ischemia, the expression level of NR2 B, Ca MKII and p-Ca MKII increasedsignificantly. These results reminisce that ischemic attack can increase the expression level of these i-LTP-associated molecules and indirectly demonstrate the occurrence of i-LTP.(4) After the occurrence of i-LTP, we found LTP induced by HFS decreased slightly but significantly in CA1 hippocampal neurons at 12 h after ischemia, and at 24 h after ischemia, LTP was totally reversed. In this part of experiments we found that LTP of fEPSPs induced by high-frequency stimulation(HFS) displayed a progressive impairment.(5) To examine possible alterations in LFP, we employed in vivo multi-channel recording to study the effect of ischemia on LFP in hippocampal CA1 pyramidal neurons. We continuously recorded LFP signals with 8-channel electrodes at differenttime-points after ischemiaon anesthesia rats. We found that the amplitude of the LFP decreased dramatically at both 12 h and 24 h time-points after ischemia.(6) The power spectral density(PSD) analysis and spectrogram analysis of LFP showed that the low frequency rhythms were also impaired significantly at these two time-points after ischemia(12 h and 24 h).(7) We perfomed the fear condition experiments on the ET-1-induced mini-stroke animals at 24 h after ischemia, and found that the context fear condition was significantly impaired.Conclusions: In this study, we found that LTP of fEPSPs induced by high-frequency stimulation(HFS) displayed a progressive impairment at 12 hours and 24 hours after ischemia. These results point to the notion that i-LTP elevates the induction threshold of subsequent synaptic plasticity. Moreover, using in vivo multi-channel recording, we found that the local field potential(LFP), which represents electrical property of cell ensembles in more restricted regions, was also dampened at these two time-points. Otherwise, in the ET-1-induced local mini-stroke models, the context fear condition was significantly impaired, whereas the tone-trace fear condition was not affected. Because of the synaptic plasticity, metaplasticity and learning and memory shares a similar molecular mechanisms, we inferred that the impairment of context fear condition might be related with the changes of synaptic plasticity and metaplasticity. These findings enrich the comprehension of metaplasticity in cerebral ischemia, and provide some new ideas for clinical treatment of the ischemia.