Fernandes Alda, Li Yu-Wen
Department of Neuroscience Biology, Research and Development, Bristol-Myers Squibb, Wallingford, CT 06492-7660, United States.
Department of Neuroscience Biology, Research and Development, Bristol-Myers Squibb, Wallingford, CT 06492-7660, United States.
Brain Res. 2017 Sep 1;1670:86-95. doi: 10.1016/j.brainres.2017.05.008. Epub 2017 May 10.
Ketamine produces rapid and long-lasting antidepressant effects in depressive patients. Preclinical studies demonstrate that ketamine stimulates AMPA receptor transmission and activates BDNF/TrkB-Akt/ERK-mTOR signaling cascades, leading to a sustained increase in synaptic protein synthesis and strengthening of synaptic plasticity, a potential mechanism underlying the antidepressant effects. The purpose of this study was to develop an immunohistochemistry (IHC) assay to map the distribution of extracellular signal-regulated kinase (ERK) phosphorylation in the mouse brain in response to systemic ketamine treatment. We established a focused microwave irradiation-assisted IHC assay to detect phosphorylated (phospho) proteins including phospho-ERK, phospho- cAMP-response- element-binding protein (CREB), phospho- glutamate receptor 1 (GluR1) and phospho- calcium/calmodulin-dependent protein kinase II (CaMKII) with greater sensitivity and reproducibility in comparison to conventional IHC methods. A single dose of ketamine produced a robust, dose- and time-dependent increase in phospho-ERK immunoreactive (phospho-ERK-ir) neurons in the medial prefrontal cortex (mPFC) and the central nucleus of the amygdala. Phospho-ERK-ir neurons in the mPFC were primarily located in the prelimbic and anterior cingulate subregions with the morphology resembling pyramidal neurons. An increase in phospho-ERK-ir was also observed in the brainstem dorsal raphe nucleus and locus coeruleus. The NMDA GluN2B subtype receptor antagonist Ro 25-6981 increased phospho-ERK expression in the brain in a similar pattern as ketamine. In summary, we have established a sensitive and reliable focused microwave irradiation-assisted IHC assay, and defined the activation pattern of ERK, in response to systemic ketamine and Ro 25-6981 treatment, in brain regions that are potentially responsible for mediating the antidepressant effects.
氯胺酮对抑郁症患者具有快速且持久的抗抑郁作用。临床前研究表明，氯胺酮可刺激α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体传递，并激活脑源性神经营养因子（BDNF）/酪氨酸激酶受体B（TrkB）-蛋白激酶B（Akt）/细胞外信号调节激酶（ERK）-哺乳动物雷帕霉素靶蛋白（mTOR）信号级联反应，导致突触蛋白合成持续增加以及突触可塑性增强，这是其抗抑郁作用的潜在机制。本研究的目的是开发一种免疫组织化学（IHC）检测方法，以描绘小鼠脑中细胞外信号调节激酶（ERK）磷酸化的分布，作为对全身氯胺酮治疗的反应。我们建立了一种聚焦微波辐射辅助的IHC检测方法，用于检测磷酸化蛋白，包括磷酸化ERK、磷酸化环磷酸腺苷反应元件结合蛋白（CREB）、磷酸化谷氨酸受体1（GluR1）和磷酸化钙/钙调蛋白依赖性蛋白激酶II（CaMKII），与传统IHC方法相比，具有更高的灵敏度和可重复性。单剂量氯胺酮使内侧前额叶皮质（mPFC）和杏仁核中央核中磷酸化ERK免疫反应性（磷酸化ERK-ir）神经元出现强大的、剂量和时间依赖性增加。mPFC中的磷酸化ERK-ir神经元主要位于前边缘和前扣带亚区域，其形态类似于锥体神经元。在脑干背侧中缝核和蓝斑中也观察到磷酸化ERK-ir增加。N-甲基-D-天冬氨酸（NMDA）GluN2B亚型受体拮抗剂Ro 25-6981以与氯胺酮相似的模式增加脑中磷酸化ERK的表达。总之，我们建立了一种灵敏且可靠的聚焦微波辐射辅助IHC检测方法，并确定了在全身氯胺酮和Ro 25-6981治疗后，ERK在可能介导抗抑郁作用的脑区中的激活模式。
