| Part I Long-lasting spatial learning and memory impairments caused by chronic cerebral hypoperfusion associate with a dynamic change of HCN1/HCN2 expression in hippocampal CA1 regionObjection:Chronic cerebral hypoperfusion(CCH) is a progressive disease and causes memory deficits through several biologically plausible pathways, yet the mechanisms underlying the disease process remained unclear particularly in a temporal manner. The aim of the present study was to investigate the dynamic changes of HCN1 and HCN2 levels in the hippocampal CA1 regions of rats with CCH and to provide a promising drug for ameliorating cognitive defects caused by CCH. Methods: We performed permanent bilateral occlusion of the common carotid arteries(two-vessel occlusion, 2VO) to induce CCH. Adult male SD rats were randomly distributed into sham-operated 4, 8 and 12 weeks group, 2VO 4, 8 and 12 weeks group. Learning and memory performance were evaluated by Morris water maze(MWM) and long-term potentiation(LTP) was used to address the underlying synaptic mechanisms. Expression of Neu N, HCN1 and HCN2 was quantified by immunohistochemistry and western blotting. Results: Our results suggested that CCH induced a remarkable and long-lasting spatial learning and memory deficits in rats although neuronal loss only occurred after 4 weeks of 2VO surgery in CA1. In addition, a significant reduction of HCN1 surface expression in CA1 was observed in the group that suffered 4 weeks ischemia but neither 8 nor 12 weeks. However, HCN2 surface expression in CA1 increased throughout the ischemia time-scales(4, 8 and 12 w). Conclusion: The imbanlance of HCN2/HCN1 surface expression in hippocampal CA1 is implicated in the long-lasting spatial learning and memory impairment caused by brain hypoperfusion. During which both HCN1 and HCN2 surface expressions are disrupted in the early stage(4 w) while the up-regulation of HCN2 surface expression rather attributes to the entire long-lasting procedure.Part II Fluoxetine ameliorates cognitive impairments induced by chronic cerebral hypoperfusion via down-regulation of HCN2 surface expression in the hippocampal CA1 area in ratsObjection:In our previous study, we have found that the long-lasting impairments of spatial learning and memory in rats with CCH may be partially due to the disorder of HCN2 subunit rather than HCN1 subunit. Therefore, in this study we investigated the effect of fluoxetine on cognitive impairments caused by CCH and to clarify the underlying mechanisms of its efficacy relative to HCN2 surface levels in the hippocampal CA1 of rats. Methods: CCH was induced by 2VO. Rats were randomly assigned to one of the three groups: sham-operated group, 2VO group, 2VO + fluoxetine group. Two weeks after surgery, rats were treated with 30 mg/kg fluoxetine(intragastric injection, i.g.) for 6 weeks. Cognitive function was evaluated by MWM and novel objects recognition(NOR) test. LTP was used to address the underlying synaptic mechanisms. Western blotting was used to quantify the protein levels of HCN2. Results: The results showed that fluoxetine treatment significantly improved the cognitive impairments caused by 2VO. Furthermore, 2VO caused an up-regulation of HCN2 surface expressions in the hippocampal CA1 area and fluoxetine also effectively recovered the disorder of HCN2 surface expressions. Conclusion: Fluoxetine could ameliorate cognitive impairments induced by chronic cerebral hypoperfusion via down-regulation of HCN2 surface expression in the hippocampal CA1 area in ratsPart III Baclofen ameliorates spatial working memory impairments induced by chronic cerebral hypoperfusion via up-regulation of HCN2 expression in the PFC in ratsObjection:CCH causes memory deficits and increases the risk of vascular dementia through several biologically plausible pathways. However, whether CCH causes prefrontal cortex(PFC)-dependent spatial working memory impairments and Baclofen, a GABAB receptor agonist, could ameliorate the impairments is still not clear. In this study, we investigated the effect of Baclofen, a GABAB receptor agonist on working memory impairments caused by CCH and to clarify the underlying mechanisms of its efficacy relative to HCN1/HCN2 protein levels in the PFC of rats. Methods: CCH was induced by 2VO. Two weeks after 2VO surgery, rats were treated with 25 mg/kg Baclofen(intraperitioneal injection, i.p.) for 3 weeks. Spatial working memory was evaluated in a Morris water maze using a modified delayed matching-to-place(DMP) procedure. Western blotting and immunohistochemistry was used to quantify the protein levels and protein localization. Results: The results suggested that 2VO caused striking spatial working memory impairment, accompanied with a decreased HCN2 expression in PFC, but the protein levels of HCN1, protein gene product 9.5(PGP9.5, a neuron specific protein), glial fibrillary acidic protein(GFAP), synaptophysin(SYP), brain-derived neurotrophic factor(BDNF), parvalbumin(PV) were not distinguishably changed as compared with sham-operated rats. Baclofen treatment significantly improved the spatial working memory impairments caused by 2VO, accompanied with a reversion of 2VO-induced down-regulation of HCN2. Furthermore, there was a co-localization of HCN2 subunits and parvalbumin-positive neurons in PFC. Conclusion: HCN2 may target inhibitory interneurons that are implicated in working memory processes and Baclofen treatment could relief spatial working memory deficits via upregulating the decreased HCN2 expression in PFC of rats with CCH. |
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