高灵敏度体内检测急性应激后脑啡肽的动态变化。

Marwa O Mikati, Petra Erdmann-Gilmore, Rose Connors, Sineadh M Conway, Jim Malone, Justin Woods, Robert W Sprung, R Reid Townsend, Ream Al-Hasani
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摘要

脑啡肽是一种阿片肽,可调节镇痛、奖赏和压力。由于脑啡肽的内源性浓度短暂且较低,再加上其固有的序列相似性,因此脑啡肽的体内检测仍然十分困难。为了着手解决这个问题,我们之前开发了一套系统,将体内光遗传学与微透析和基于质谱的高灵敏度检测相结合,测量自由活动的啮齿动物体内阿片肽的释放(Al-Hasani,2018,eLife)。在这里,我们展示了检测分辨率的提高和脑啡肽检测的稳定,这使我们能够研究急性应激期间脑啡肽的释放。我们提出了一种分析方法,用于在急性应激后实时、同时检测小鼠延脑核壳(NAcSh)中的Met-和Leu-脑啡肽(Met-Enk & Leu-Enk)。我们利用纤维光度法证实,急性应激激活了 NAcSh 中的脑啡肽能神经元,并导致释放 Met- 和 Leu-Enk。我们还展示了Met-和Leu-Enk释放的动态,以及它们在NAc腹侧外壳中如何相互关联,这在以前是很难做到的,因为我们使用的方法依赖于mRNA转录水平而不是翻译后产物。这种方法提高了时空分辨率,优化了通过蛋氨酸氧化检测元脑啡肽的过程,并为了解应激后元脑啡肽和亮脑啡肽之间的关系提供了新的视角。
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Highly sensitive in vivo detection of dynamic changes in enkephalins following acute stress.

Enkephalins are opioid peptides that modulate analgesia, reward, and stress. In vivo detection of enkephalins remains difficult due to transient and low endogenous concentrations and inherent sequence similarity. To begin to address this we previously developed a system combining in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents (Al-Hasani, 2018, eLife). Here we show improved detection resolution and stabilization of enkephalin detection, which allowed us to investigate enkephalin release during acute stress. We present an analytical method for real-time, simultaneous detection of Met- and Leu-Enkephalin (Met-Enk & Leu-Enk) in the mouse Nucleus Accumbens shell (NAcSh) after acute stress. We confirm that acute stress activates enkephalinergic neurons in the NAcSh using fiber photometry and that this leads to the release of Met- and Leu-Enk. We also demonstrate the dynamics of Met- and Leu-Enk release as well as how they correlate to one another in the ventral NAc shell, which was previously difficult due to the use of approaches that relied on mRNA transcript levels rather than post-translational products. This approach increases spatiotemporal resolution, optimizes the detection of Met-Enkephalin through methionine oxidation, and provides novel insight into the relationship between Met- and Leu-Enkephalin following stress.

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