Table_3_Transcriptional Activation, Deactivation and Rebound Patterns in Cortex, Hippocampus and Amygdala in Response to Ketamine Infusion in Rats.XLSX

Ketamine, an N-methyl-D-aspartate (NMDA)-receptor antagonist, is a recently revitalized treatment for pain and depression, yet its actions at the molecular level remain incompletely defined. In this molecular-pharmacological investigation in the rat, we used short- and longer-term infusions of high dose ketamine to stimulate neuronal transcription processes. We hypothesized that a progressively stronger modulation of neuronal gene networks would occur over time in cortical and limbic pathways. A continuous intravenous administration paradigm for ketamine was developed in rat consisting of short (1 h) and long duration (10 h, and 10 h + 24 h recovery) infusions of anesthetic concentrations to activate or inhibit gene transcription in a pharmacokinetically controlled fashion. Transcription was measured by RNA-Seq in three brain regions: frontal cortex, hippocampus, and amygdala. Cellular level gene localization was performed with multiplex fluorescent in situ hybridization. Induction of a shared transcriptional regulatory network occurred within 1 h in all three brain regions consisting of (a) genes involved in stimulus-transcription factor coupling that are induced during altered synaptic activity (immediate early genes, IEGs, such as c-Fos, 9–12 significant genes per brain region, p < 0.01 per gene) and (b) the Nrf2 oxidative stress-antioxidant response pathway downstream from glutamate signaling (Nuclear Factor Erythroid-Derived 2-Like 2) containing 12–25 increasing genes (p < 0.01) per brain region. By 10 h of infusion, the acute results were further reinforced and consisted of more and stronger gene alterations reflecting a sustained and accentuated ketamine modulation of regional excitation and plasticity. At the cellular level, in situ hybridization localized up-regulation of the plasticity-associated gene Bdnf, and the transcription factors Nr4a1 and Fos, in cortical layers III and V. After 24 h recovery, we observed overshoot of transcriptional processes rather than a smooth return to homeostasis suggesting an oscillation of plasticity occurs during the transition to a new phase of neuronal regulation. These data elucidate critical molecular regulatory actions during and downstream of ketamine administration that may contribute to the unique drug actions of this anesthetic agent. These molecular investigations point to pathways linked to therapeutically useful attributes of ketamine.

氯胺酮（Ketamine）作为N-甲基-D-天冬氨酸（NMDA）受体拮抗剂，近年来重新成为疼痛与抑郁症的治疗手段，但其分子层面的作用机制仍未完全阐明。本研究以大鼠为对象开展分子药理学研究，通过高剂量氯胺酮的短期与长期输注激活神经元转录过程。我们提出假说：随着给药时长增加，大脑皮层与边缘通路中的神经元基因网络将受到逐步增强的调控。本研究建立了大鼠静脉持续给药模型，设置1小时短期输注、10小时长期输注以及10小时输注+24小时恢复三组方案，以麻醉剂量的氯胺酮通过药代动力学可控的方式激活或抑制基因转录。我们通过RNA测序（RNA-Seq）检测了额叶皮层、海马体与杏仁核三个脑区的转录水平，并采用多重荧光原位杂交技术完成细胞水平的基因定位分析。给药1小时内，三个脑区均激活了共同的转录调控网络，包含两类基因：其一为突触活动改变时诱导表达的刺激-转录因子偶联相关基因，即可即刻早期基因（immediate early genes, IEGs，如c-Fos），每个脑区包含9~12个显著差异基因（单基因p<0.01）；其二为谷氨酸信号下游的Nrf2氧化应激-抗氧化应答通路（核因子红细胞系2相关因子2，Nuclear Factor Erythroid-Derived 2-Like 2），该通路包含12~25个表达量逐步升高的基因（单基因p<0.01）。给药10小时后，早期的调控效应进一步强化，出现更多且更显著的基因表达变化，反映出氯胺酮对脑区兴奋与可塑性的持续增强调控。在细胞水平，原位杂交结果显示，可塑性相关基因脑源性神经营养因子（Bdnf）以及转录因子Nr4a1与Fos在皮层第三、第五层出现上调表达。经过24小时恢复后，我们观察到转录过程出现超调而非平稳回归稳态，提示在向神经元调控的新阶段过渡时，可塑性会发生振荡。本研究阐明了氯胺酮给药期间及其下游的关键分子调控机制，这或可解释该麻醉剂独特的药理作用，同时为其治疗相关的潜在通路提供了分子层面的科学依据。