Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (^mCherryMCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.

中文翻译：

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用于研究黑色素浓缩激素受体 1 的 N 端融合等位基因

神经元上的纤毛在中枢神经系统 (CNS) 的发育和功能中发挥着关键作用。虽然阐明神经元纤毛的确切作用仍然具有挑战性，但很明显，G 蛋白偶联受体 (GPCR) 的一个子集优先定位于纤毛膜。此外，纤毛 GPCR 信号传导与调节多种行为有关。黑色素浓缩激素受体 1 (MCHR1) 是一种 GPCR，在已知富含纤毛的啮齿动物中集中表达。在这里，我们使用 MCHR1 作为模型纤毛 GPCR 来开发一种策略来荧光标记从体内内源性基因座表达的受体。使用 CRISPR/Cas9，我们将荧光蛋白 mCherry 的编码序列插入Mchr1的 N 端。融合蛋白分析（^mCherry MCHR1) 揭示了其定位于 CNS 中的神经元纤毛，跨越多个发育时间点和成人大脑的各个区域。我们的方法同时产生了偶然的 in/dels，改变了Mchr1起始密码子，导致了新的 MCHR1 敲除线。使用电生理学的功能研究表明 MCHR1 敲除小鼠的突触强度发生了显着变化。在 mCherry 插入纯合子的小鼠中也检测到强度降低，这表明虽然该策略可用于监测受体，但可以改变活性。然而，这两条线都应该有助于研究 MCHR1 的功能，并有助于我们理解大脑中的 MCHR1 信号。此外，这种方法可以扩展以帮助研究其他纤毛 GPCR。