Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization.

TitleKnock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization.
Publication TypeJournal Article
Year of Publication2015
AuthorsOzawa A, Brunori G, Mercatelli D, Wu J, Cippitelli A, Zou B, Xie XSimon, Williams M, Zaveri NT, Low S, Scherrer G, Kieffer BL, Toll L
JournalJ Neurosci
Volume35
Issue33
Pagination11682-93
Date Published2015 Aug 19
ISSN1529-2401
KeywordsAnimals, Cells, Cultured, Gene Knock-In Techniques, Green Fluorescent Proteins, Male, Mice, Mice, Transgenic, Microscopy, Fluorescence, Molecular Imaging, Neurons, Receptors, Opioid, Recombinant Fusion Proteins, Subcellular Fractions, Tissue Distribution
Abstract

UNLABELLED: The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [(35)S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with μ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function.SIGNIFICANCE STATEMENT: The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These knock-in mice have NOP receptors that function both in vitro and in vivo and have provided a detailed characterization of NOP receptors in brain, spinal cord, and DRG neurons. They appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function.

DOI10.1523/JNEUROSCI.5122-14.2015
Alternate JournalJ. Neurosci.
PubMed ID26290245
PubMed Central IDPMC4540802
Grant ListK99 DA031777 / DA / NIDA NIH HHS / United States
R01 DA023281 / DA / NIDA NIH HHS / United States
DA031777 / DA / NIDA NIH HHS / United States
R00 DA031777 / DA / NIDA NIH HHS / United States
DA027811 / DA / NIDA NIH HHS / United States
R01 DA027811 / DA / NIDA NIH HHS / United States
DA023281 / DA / NIDA NIH HHS / United States

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