氯胺酮抑制HCN1离子通道参与抗抑郁作用的研究

Inhibition of HCN1 Channels by Ketamine Accounts for Its Antidepressant Actions

目的探讨超极化激活的环核苷酸门控的离子通道亚型1（HCN1）是否参与了亚麻醉剂量氯胺酮（KET）的抗抑郁作用。方法选择全身HCN1敲除（HCN1^-/-）及其相对应的野生型（HCN1^＋/＋）8～12周龄雄性C57BL6小鼠。在行为学实验中,长期低剂量饮水口服皮质酮（CORT）使小鼠产生慢性抑郁状态,采用强迫游泳实验（FST）中小鼠不动时间来评估小鼠抑郁状态。小鼠随机一次性腹腔注射5mg/kg KET（KET组,n=7）或等体积生理盐水（NS组,n=7）,并且记录比较4组抑郁小鼠给予注射后30min,24h,7d在强迫游泳实验中的不动时间。另外选取正常HCN1^-/-和HCN1^＋/＋小鼠,各小鼠给予腹腔注射5-溴脱氧尿嘧啶核苷（BrdU）后24h,随机一次性腹腔注射5mg/kg KET（KET组,n=5）或等体积生理盐水（NS组,n=5）,24h后断头取脑,采用免疫荧光染色,观察4组小鼠海马中BrdU标记的新生神经元的数目。结果 CORT处理前HCN1^-/-小鼠的不动时间短于HCN1^＋/＋小鼠（P〈0.01）,抑郁状态较HCN1^＋/＋小鼠轻;CORT处理后各基因型小鼠的不动时间均较基础值增加（P〈0.01）,抑郁状态加重。比较30min内、24h内及7d内不动时间的减小幅度,HCN1^＋/＋KET组在任一时间点均大于其余3组（P〈0.05）,HCN1^-/-KET、HCN1^＋/＋NS组及HCN1^-/-NS组在上述任一时间段不动时间变化幅度差异均无统计学意义。NS处理后,HCN1^-/-小鼠海马中新生神经元数目多于HCN1^＋/＋小鼠（P〈0.05）;HCN1^＋/＋小鼠给予KET 24h后,其海马中新生神经元数目较生理盐水组增多（P〈0.05）,而HCN1^-/-小鼠给予KET后,其海马神经元数目与生理盐水组比较,差异无统计学意义。结论KET抗抑郁作用可能是通过抑制HCN1离子通道实现的。

Objective To investigate the roles of hyperolarization-actived cyclic nucleotide-gated channels 1（HCN1）in antidepressant actions of ketamine（KET）.Methods Male HCN1 knock out（HCN1^-/-）and wildtype（HCN1^＋/＋）C57BL6 mice（8-12 weeks,20-25g）were chosen.The depression model of mice was developed by continuously oral administration of low dosage of corticosterone（CORT）.The immobility time in forced swimming tests（FST）was used to assess the depressive state of mice.Then the two genotype depressive mice were treated with single intraperitoneal injection of 5mg/kg ketamine（KET group,n=7）or same volume of normal saline（NS group,n=7）respectively.After treatment,the immobility time at 30 min,24hand 7dafter the intraperitoneal injection of ketamine or normal saline in CORT-treated mice were compared.In addition,normal HCN1^-/-and HCN1^＋/＋mice were intraperitoneally injected of BrdU and then treated with 5mg/kg ketamine（KET group,n=5）or same volume of normal saline（NS group,n=5）by single intraperitoneal injection.Each group was euthanized for immunohistochemical processing of 5-Bromo-2-deoxyuridine（BrdU）-labeled cells in hippocampus at 24 hafter the intraperitoneal injection of saline or ketamine.Results The immobility time in FST of HCN1^-/-mice was less than the HCN1^＋/＋mice before administration of CORT.It shows that the depressive state of HCN1^-/-mice is less intensive than that of HCN1^＋/＋mice.And the immobility time in both HCN1^-/-and HCN1^＋/＋mice was increased after oral administration of low dose corticosterone,with an increase in depression.In addition,the comparisons were also made to the reduction of immobility time within 30 min,24 hand 7 d.At any time point,the reduction of immobility time in HCN1^＋/＋KET group was higher than those in the other three groups（P〈0.05）.Furthermore,there were no statistical significances among the three groups including HCN1^-/-KET group,HCN1^＋/＋NS group,HCN1^-/-NS group at any point.The number of newborn neurons were more in HCN1^-/-mice than HCN1^＋/＋mice after the treatment of normal saline.Compared with the NS group,the number of neonatal neurons labeled by BrdU were increased after the intraperitoneal injection of ketamine in HCN1^＋/＋mice but not in HCN1^-/-mice.Conclusion Inhibition of HCN1 channels by ketamine accounts for its antidepressant actions.