Effect Of Hyperpolarization-activated And Cyclic Nucleotide-gated Cation Channels On Mouse Learning And Memory And Its Mechanism Research

To explore effect of HCN channel on learning and memory and its role in ischemia-reperfusion injury, we studied the changes of learning and memory induced by CsCl, a non selective -HCN channel blocker. And at the same time, with in vivo/vitro hypoxia and ischemia models, effects of CsCl on release of amino acids and activity of enzymes were examed. Three parts of experiment were described.Part one: Effect of CsCl on Learning and Memory in MouseIt has been suggested that HCN is wide expressed in hippocampus, however little is known about its effects on spatial learning and memory. The goal of the current study is to assess whether there is altered expression of the HCN1 channel in hippocampus during hypoxia and whether learning and memory changes if the function of HCN1 channel is altered. A spatial learning task in the Morris water maze was used to assess the cognitive status of hypoxia mouse and CsCl-administrated mouse (168 mg/kg, ip.). Site of expression and amount of HCN1 mRNA were examined by in situ hybridization in CA1, CA3 and DG region of hippocampus. The results showed the mean escape latency of both hypoxia group and CsCl group were longer than that of normal control (P<0.01, n=14) in Morris water maze. In hippocampal tissues of hypoxia group, average gray scale increased, integral optical density decreased compared with that of normal control, which represented lower levels of HCN1 mRNA transcription. In the sections of CsCl group, average gray scale of HCN1 mRNA decreased in DG, CA1 region, integral optical density increased in DG, CA1 and CA3 region which showed higher level of transcription of HCN1 mRNA. Our results showed in hypoxia condition, decreased HCN1 mRNA abundance was correlated with spatial learning impairment. And when HCN1 channel was blocked by CsCl, spatial learning impairment and the feedback promotion of HCN 1 transcription appeared. It suggested that hippocampus HCN channel is involved in spatial learning and memory.Part two: Effect of CsCl on Release of Amino Acids in Mouse Hippocampus SliceLong-term potentiation (LTP) has been extensively studied in hippocampus as a model of learning and memory, LTP of synaptic transmission in hippocampus is thought to be glutamate. To identify the mechanism of that CsCl impairs leaning and memory, release of L-Glutamate, glycine, taurine and GABA from hippocampal slice was detected with high performance liquid chromatoghraphy (HPLC). Hippocampus slice was made from adult mouse brain coronal section, perfused with artificial cerebrospinal fluid (aCSF) buffed with HEPEs. After hippocampal slices stabilized, agents were added in aCSF respectively: cesium chloride (0.2 mM, 2 mM, 20 mM), potassium chloride (50mM) ,TTX(10 uM) to incubate for 30 min or hypoxia model was made by stopping oxy supply for 30min. Then L-Glutamate, glycine, taurine and GABA in aCSF were examined. Result showed CsCl caused a dose-dependent inhibition of glutamate release, and did not alter release of other amino acids. While in hypoxia group only glutamate deduced, and potassium chloride could increase the content of glycine, GABA and taurine respectively 4.6, 4.1 and 9.2 times. It suggested that HCN channel increase the release of L-glutamate.Part three: Effect of CsCl on Learning and Memory in Ischemia-Reperfusion MouseChanges in the expression of HCN channel have been associated with central nervous system diseases, including epilepsy, Huntington's disease, neuropathic pain et al. To explore the effect of HCN channel during ischemia/reperfusion injury, we tested learning and memory in Morris water maze, and assayed activity of SOD and tNOS in hippocampus and cortex in ischemia-reperfusion mouse. Forebrain ischemia was produced by occlusion of both carotid arteries for 15 min, and blood flow was restored by unclamping the carotids. Five days after reperfusion cesium chloride was given intragastrically in dose of 0.2 and 0.4g /kg.d, for 14d. 19 days after reperfusion spacial memory was tested in Morris water maze and then content of MDA, activity of SOD, tNOS and iNOS were detected. Result showed in ischemia-reperfusion mouse, escape latency prolonged; activity of tNOS in hippocampus and SOD in cortex increased. In CsCl-treated mouse, swimming distance to platform was longer than that of ischemia-reperfusion mouse; and the activity of tNOS in cortex decreased. It suggested that 19 d after reperfusion, ischemia-reperfusion injury has not recovered. And when administrated with CsCl, it worsens the spatial learning and memory.Overall, under physiology condition, HCN channel blocker could impair learning and memory, which is related with decreasing release of glutamate. During ischemia/reperfusion injury, HCN channel blocker could worsen learning and memory, and it maybe due to the decrease of activity of tNOS.