突触前膜HCN通道在氯胺酮快速抗抑郁中的作用

Evidence from clinical trials and animal experiments shows a single subanesthesia dose of ketamine produces rapid antidepressant responses. However, the psychotomimetic properties and abuse potential of ketamine necessitate caution in promoting this compound to be used as a general treatment of depression. The mechanisms underlying the rapid-acting antidepressant effects of ketamine have not been fully identified. Particularly, it remains uncertain which molecular events mediate ketamine’s action in enhancing excitatory synaptic transmission. In our preliminary experiments, we observed that ketamine or MK-801 strongly potentiated excitatory synapses formed by Schaffer collateral pathway with CA1 pyramidal cells in acutely prepared hippocampal slices. Bath application of ketamine on hippocampal slices or a single dose of ketamine injection (i.p.) potently increased GluR1 expression and phosphorylation on Ser845 site. H89, a PKA inhibitor, totally abolished ketamine’s effect on SC-CA1 synaptic transmission and GluR1 Seer845 phosphorylation. These effects were not occluded by blocking GABAA and GABAB receptors, but were mimicked and occluded by bath application of HCN channel blockers when postsynaptic HCN channel was blocked. Furthermore, ketamine-induced potentiation of excitatory synaptic transmission and the fast antidepressant action of ketamine were absent in GluR1 S845A knock-in mice. Our preliminary data suggests phosphorylation of AMPA receptor GluR1 Ser845 may play an important role in ketamine’s rapid antidepressant actions; ketamine inhibits presynaptic HCN channels, promotes glutamate release at SC-CA1 synapses, and therefore potentiates GluR1 phosphorylation and expression. In the proposed project we have designed experiments to determine the molecular basis of ketamine-induced phosphorylation of GluR1 Ser845. We will combine electrophysiological techniques, including whole-cell patch clamp with cell biological technique such as Western blotting, CA1 cell- or CA3 cell-specific NR1 knockout mice as well as behavioral measurements to achieve the designed experiments. The proposed project will advance our knowledge in understanding the cellular mechanisms of fast-acting antidepressant actions of ketamine. A better understanding of ketamine’s fast-acting antidepressant effects will promote the design of a new generation of rapid-acting antidepressant to offer a better therapy to depression.

氯胺酮的快速抗抑郁机制仍不清楚。对氯胺酮敏感的NMDA受体所存在的部位及从拮抗NMDA受体到增强兴奋性突触传递之间所发生的事件存在很大争议。预实验结果显示氯胺酮增加CA3-CA1突触前膜的谷氨酸释放，这一作用不依赖于去抑制。胞外给予HCN通道的阻断剂模拟了氯胺酮的这一作用，但选择性地阻断突触后膜的NMDA受体或HCN通道对CA3-CA1突触传递没有明显影响。我们推测氯胺酮通过阻断突触前膜的NMDA受体下调HCN通道，增加谷氨酸释放，并引起突触后膜的电压依赖性钙通道开放，增强GluR1 Ser845磷酸化而增加AMPA受体向膜上转运。本项目拟采用GluR1 S845A点突变小鼠、pH敏感的GluR1转染、NMDA受体条件敲除小鼠等方法研究突触前膜NMDA受体和HCN通道在氯胺酮快速抗抑郁中的作用。本项目旨在阐明氯胺酮的快速抗抑郁机制，为开发快速、安全且副作用小的新型抗抑郁药物提供理论依据。

氯胺酮的快速抗抑郁机制一直是近年来抑郁症和抗抑郁药研究领域的一个热点。在本研究项目立项时，氯胺酮的抗抑郁机制研究领域存在多种假说和观点。其中最主要以下三个假说：1. 去抑制假说,认为氯胺酮敏感的NMDA受体存在于抑制性中间神经元，氯胺酮通过抑制这类NMDA受体，降低GABA的释放，对兴奋性突出前神经元产生去抑制，从而加强兴奋性突触传递；2. eEF2假说，这一假说认为，氯胺酮敏感的NMDA受体存在于兴奋性突触后膜， 氯胺酮通过抑制这类受体引起eEF2的去磷酸化，增强突触传递；3.代谢产物假说，这一假说认为氯胺酮的抗抑郁作用由氯胺酮的代谢产物产生，氯胺酮的代谢产物通过NMDA受体非依赖的机制产生抗抑郁作用。这些假说存在很大的分歧且各自有一定的实验依据，但都不能很好的解释氯胺酮在抗抑郁中表现出来的所有现象。.本项目通过四年的研究，经过大量的实验，明确回答了：1.通过运用NMDA受体条件性敲除小鼠，分别在兴奋突触前细胞和突触后细胞敲除NMDA受体，我们发现氯胺酮敏感的NMDA受体的表达亚细胞定位，认为氯胺酮敏感的NMDA受体存在于兴奋性突触前细胞； 2. 明确回答了氯胺酮从阻断NMDA受体后到产生抗抑郁作用过程中所发生的事件，即阻断突触前细胞的HCN通道，增强谷氨酸的释放引起突触后膜的去极化而增加AMPA受体的表达。3. 通过运用GluA1 S845A点突变小鼠，我们发现AMPA受体GluA1S845位点的磷酸化为氯胺酮引起的突触后膜AMPA受体表达及抗抑郁作用所必须。我们的研究提出了氯胺快速抗抑郁作用的新机制，为开发快速安全副作用小的新一代抗抑郁药提供了理论依据。.在本项目实行过程中发表SCI论文 5篇， 国际会议邀请报告1次，国际会议墙报展示6次，国际学术交流邀请报告2次，国内学术交流邀请报告3次。.在本项目实施过程培养博士后研究人员1名，研究生7名，其中硕士研究生6名，博士研究生2名。