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


CONCLUSIONS
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.
Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida, 34987.
Akihiko
A
Ozawa
Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida, 34987.
Gloria
G
Brunori
Department Of Pharmacology, Alma Mater Studiorum, University of Bologna, Via Irnerio 48 40126, Bologna, Italy.
Daniela
D
Mercatelli
Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida, 34987.
Jinhua
J
Wu
Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida, 34987.
Andrea
A
Cippitelli
AfaSci Research Laboratories, AfaSci, Redwood City, California 94063.
Bende
B
Zou
AfaSci Research Laboratories, AfaSci, Redwood City, California 94063.
Xinmin Simon
XS
Xie
Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida, 34987.
Melissa
M
Williams
Astraea Therapeutics, Mountain View, California 94043.
Nurulain T
NT
Zaveri
Department of Anesthesiology, Perioperative and Pain Medicine, Department of Molecular and Cellular Physiology, Department of Neurosurgery, Stanford Neurosciences Institute, Stanford University, Palo Alto, California 94304.
Sarah
S
Low
Department of Anesthesiology, Perioperative and Pain Medicine, Department of Molecular and Cellular Physiology, Department of Neurosurgery, Stanford Neurosciences Institute, Stanford University, Palo Alto, California 94304.
Grégory
G
Scherrer
Douglas Research Center, Department of Psychiatry, Faculty of Medicine, McGill University, Montreal, Quebec H4H 1R3, Canada, and Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U-964, CNRS UMR-7104, Université de Strasbourg, Illkirch, F-67404 France.
Brigitte L
BL
Kieffer
Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida, 34987, ltoll@tpims.org.
Lawrence
http://orcid.org/0000-0002-4501-7573
L
Toll
D000818:Animals; D002478:Cells, Cultured; D055879:Gene Knock-In Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D011957:Receptors, Opioid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism* / Q000648:ultrastructure; D014018:Tissue Distribution; D000094942:Nociceptin Receptor
D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000094942:Nociceptin Receptor; C000722287:Oprl1 protein, mouse
GPCR; N/OFQ; NOP receptor; eGFP; histochemistry; knock-in