Targeting Morphine-Responsive Neurons: Generation of a Knock-In Mouse Line Expressing Cre Recombinase from the Mu-Opioid Receptor Gene Locus.
Targeting Morphine-Responsive Neurons: Generation of a Knock-In Mouse Line Expressing Cre Recombinase from the Mu-Opioid Receptor Gene Locus.
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DOI：
10.1523/eneuro.0433-19.2020
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发表时间：
2020-05-01
期刊：
影响因子：
3.4
通讯作者：
Kieffer, Brigitte L
中科院分区：
文献类型：
--
作者：
Bailly, Julie;Del Rossi, Natalie;Kieffer, Brigitte L
μ-阿片受体（莫尔）调节伤害性通路和奖赏加工，并介导药用以及滥用阿片类药物的强镇痛和成瘾特性。莫尔功能已被广泛研究，并且可获得操纵或可视化受体蛋白的工具。然而，电路机制的基础MOR介导的影响是鲜为人知的，因为遗传访问MOR表达的神经元是缺乏的。在这里，我们报告了一个敲入Oprm1-Cre小鼠系的产生，它允许靶向和操纵莫尔阿片类反应神经元。将编码与增强型绿色荧光蛋白（EGFP/Cre）融合的T2A可切割肽和Cre重组酶的cDNA插入Oprm 1基因序列的下游。所得Oprm1-Cre系显示完整的Oprm1基因转录。莫尔和EGFP/Cre蛋白在相同的神经元中共表达，并分别定位于细胞质和细胞核。莫尔信号传导未改变，通过维持DAMGO诱导的G蛋白活化来证明，并且体内莫尔功能被保留，如正常吗啡诱导的镇痛、过度运动和致敏所示。Cre重组酶有效地驱动Cre依赖性报告基因的表达，这通过在内侧缰中的局部病毒介导的表达和在与tdTomato报告小鼠交配时的全脑荧光显示，后者显示典型的莫尔表达的分布模式。最后，我们证明了Oprm1-Cre小鼠腹侧被盖区的莫尔神经元的光遗传学激活引起强烈的回避行为，正如文献中所预期的那样。因此，Oprm1-Cre细胞系是一个很好的工具，用于MOR阳性神经元的定位和功能研究，并将对阿片类药物，疼痛和成瘾研究产生广泛的兴趣。
The mu-opioid receptor (MOR) modulates nociceptive pathways and reward processing, and mediates the strong analgesic and addictive properties of both medicinal as well as abused opioid drugs. MOR function has been extensively studied, and tools to manipulate or visualize the receptor protein are available. However, circuit mechanisms underlying MOR-mediated effects are less known, because genetic access to MOR-expressing neurons is lacking. Here we report the generation of a knock-in Oprm1-Cre mouse line, which allows targeting and manipulating MOR opioid-responsive neurons. A cDNA encoding a T2A cleavable peptide and Cre recombinase fused to enhanced green fluorescent protein (EGFP/Cre) was inserted downstream of the Oprm1 gene sequence. The resulting Oprm1-Cre line shows intact Oprm1 gene transcription. MOR and EGFP/Cre proteins are coexpressed in the same neurons, and localized in cytoplasmic and nuclear compartments, respectively. MOR signaling is unaltered, demonstrated by maintained DAMGO-induced G-protein activation, and in vivo MOR function is preserved as indicated by normal morphine-induced analgesia, hyperlocomotion, and sensitization. The Cre recombinase efficiently drives the expression of Cre-dependent reporter genes, shown by local virally mediated expression in the medial habenula and brain-wide fluorescence on breeding with tdTomato reporter mice, the latter showing a distribution patterns typical of MOR expression. Finally, we demonstrate that optogenetic activation of MOR neurons in the ventral tegmental area of Oprm1-Cre mice evokes strong avoidance behavior, as anticipated from the literature. The Oprm1-Cre line is therefore an excellent tool for both mapping and functional studies of MOR-positive neurons, and will be of broad interest for opioid, pain, and addiction research.