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. 2015 Aug 19;35(33):11682-93.
doi: 10.1523/JNEUROSCI.5122-14.2015.

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

Akihiko Ozawa  1 , Gloria Brunori  1 , Daniela Mercatelli  2 , Jinhua Wu  1 , Andrea Cippitelli  1 , Bende Zou  3 , Xinmin Simon Xie  3 , Melissa Williams  1 , Nurulain T Zaveri  4 , Sarah Low  5 , Grégory Scherrer  5 , Brigitte L Kieffer  6 , Lawrence Toll  7
Affiliations

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

Akihiko Ozawa et al. J Neurosci. .

Abstract

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.

Keywords: GPCR; N/OFQ; NOP receptor; eGFP; histochemistry; knock-in.

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Figures

Figure 1.
Figure 1.
Knock-in construct, genotyping scheme and NOP-eGFP mRNA levels in NOP+/+, NOP+/eGFP, and NOPeGFP/eGFP mice. A, Targeting strategy. Oprl1 exons, eGFP cDNA, and the floxed (triangles) neomycin cassette are displayed as empty, gray, and black boxes, respectively. Homologous recombination (HR) was followed by Cre recombinase treatment (Cre) in ES cells. B, mRNA levels were determined by performing RT-PCR using whole brains, as described in Materials and Methods. N = 4 mice of each genotype. Error bars denote SD.
Figure 2.
Figure 2.
In vitro and in vivo activity of N/OFQ at NOP receptors in NOP+/+, NOP+/eGFP, and NOPeGFP/eGFP mice. [3H]N/OFQ binding and [35S]GTPγS binding to brain membranes, electrophysiological recordings, and the radiant heat tail-flick assay were performed as described in Materials and Methods. In vitro experiments shown in A and B are from single experiments conducted in quadruplicate ([3H]N/OFQ binding) or triplicate ([35S]GTPγS binding), with error bars denoting SD among the replicates. Each experiment was repeated at least 2 additional times with similar results. C, Electrophysiological response to N/OFQ in a DRG neuron with current-clamp recording. Application of 1 μm N/OFQ reversibly hyperpolarized membrane potential. D, The effect of N/OFQ on field EPSP in slices of hippocampus. Application of N/OFQ (0.3 μm, 3 μm) reversibly inhibited the field EPSP slopes. Left, Top, The representative traces of field EPSP before, during and after application of 3 μm of N/OFQ. Left, Bottom, The time course of the inhibitory effect of N/OFQ. The bar indicated the period of drug application. Right, The summary results of N/OFQ and antagonist, SR14148. Application of vehicle (0.01% of DMSO, n = 4) or SR14148 alone (n = 3) had no significant effect on field EPSP. E, Nociceptive response to thermal stimulation in the tail-flick test in NOP+/+ (left) and NOPeGFP/eGFP (right) mice following the combined administration of SR16835 (0, 10, and 30 mg/kg) and morphine (M; 3 mg/kg). Tail-flick latency was determined immediately before morphine administration (T = 0) that followed SR16835 treatment. Tail flick response was then repeated 30 (T = 30) and 60 min (T = 60) following morphine treatment. Data are mean %MPE ± SEM. ***p < 0.001 difference from T = 0; ##p < 0.01, ###p < 0.001 difference from SR 0 + morphine treatment.
Figure 3.
Figure 3.
N/OFQ activates NOP-eGFP receptors in primary neuron cells. Hippocampal primary neurons (10 d in vitro) isolated from NOP-eGFP mice were treated with 1 μm N/OFQ for the indicated time periods to examine the changes in the subcellular localization of NOP-eGFP. NOP-eGFP receptors were visualized with an anti-GFP antibody and secondary antibody conjugated to AlexaFluor488. Scale bar, 25 μm. The 5 min time point appears to have more punctate processes in some cells.
Figure 4.
Figure 4.
NOP-eGFP receptor expression in the brain. A, Evaluation of the specific NOP-eGFP receptor expression in the brain derived from the wild-type (WT) and NOP-eGFP mice. Brain sections from NOP-eGFP mice were incubated with (Ab+) or without (Ab−) an anti-GFP antibody. B, NOP-eGFP receptor distribution was observed in a wide range of brain region. The position of all sections is given relative to bregma (mm); the numbers highlighted in white. Scale bars, 100 μm.
Figure 5.
Figure 5.
NOP-eGFP receptors are highly distributed in laminae I–III and X. Tissue sections from the spinal cord were incubated with anti-GFP, and -CGRP (laminae I and IIo; A) or -PKCγ (ventral border of lamina Iii; B). Tissues were also treated with biotinylated IB4 (dorsal border of lamina IIi) and streptavidin-conjugated to AlexaFluor 555 (A, B). C, Immunostaining of lamina X. Immunoreactivity of NOP-eGFP was observed in laminae I–III, where CGRP-immunoreactive terminals, PKCγ interneurons and IB4-positive interneurons are located. Scale bars, 100 μm.
Figure 6.
Figure 6.
Various types of DRG neurons express NOP-eGFP receptors. To characterize the NOP-eGFP distribution in DRG neurons, sections were incubated with anti-GFP antibody together with anti-μ receptor antibody, DRG markers; anti-CGRP, -NF200 antibodies, or biotinylated IB4. A, NOP-eGFP expression in DRG neurons (green). Nuclei were stained with DAPI (blue). The NOP-eGFP containing DRG neurons were quantified by determining the percentage of eGFP-positive cells compared with the total number of sensory neurons (n = 1396). The total number of DRG sensory neurons was determined by counting the total number of DAPI-stained cells and excluding those from glia and connective tissues. Tissue sections were also costained with anti-CGRP and -NF200 antibodies, (B) in which the white arrowheads indicate NOP-eGFP+, CGRP-myelinated medium DRG neurons, or (C) biotinylated IB4. In each panel, white arrows indicate the cells where costaining occurs. D, Size profiling of DRG neurons that are expressing NOP-eGFP, CGRP, or NF200, or bind to IB4. E, Identity of NOP-eGFP+ DRG neurons. F, Percentage of medium NOP-eGFP+ DRG neurons that are myelinated and not coexpressing CGRP. Data are represented as mean ± SEM, G, colocalization of NOP-eGFP and μ receptors in DRG. Small unmyelinated neurons coexpress NOP-eGFP and μ receptors. White arrows depict the cells coexpressing NOP-eGFP and μ receptors. Scale bars, 100 μm.
Figure 7.
Figure 7.
Colocalization of NOP-eGFP and μ receptors was examined in brain regions that are essential for pain and reward system. A, VTA and fr; B, amygdala; C, PAG; D, IPN; E, medial habenula; F, fr in a sagittal section, G, H, High-power images of the white and yellow squares respectively in image E. In the merged representative images, yellow indicates that NOP-eGFP colocalizes with μ receptor. Scale bars: A–F, 100 μm; G, H, 15 μm.
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References

    1. Abraira VE, Ginty DD. The sensory neurons of touch. Neuron. 2013;79:618–639. doi: 10.1016/j.neuron.2013.07.051. - DOI - PMC - PubMed
    1. Adapa ID, Toll L. Relationship between binding affinity and functional activity of nociceptin/orphanin FQ. Neuropeptides. 1997;31:403–408. doi: 10.1016/S0143-4179(97)90032-9. - DOI - PubMed
    1. Anton B, Fein J, To T, Li X, Silberstein L, Evans CJ. Immunohistochemical localization of ORL-1 in the central nervous system of the rat. J Comp Neurol. 1996;368:229–251. doi: 10.1002/(SICI)1096-9861(19960429)368:2<229::AID-CNE5>3.3.CO%3B2-G. - DOI - PubMed
    1. Bardoni R, Tawfik VL, Wang D, François A, Solorzano C, Shuster SA, Choudhury P, Betelli C, Cassidy C, Smith K, de Nooij JC, Mennicken F, O'Donnell D, Kieffer BL, Woodbury CJ, Basbaum AI, MacDermott AB, Scherrer G. Delta opioid receptors presynaptically regulate cutaneous mechanosensory neuron input to the spinal cord dorsal horn. Neuron. 2014;81:1312–1327. doi: 10.1016/j.neuron.2014.01.044. - DOI - PMC - PubMed
    1. Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell. 2009;139:267–284. doi: 10.1016/j.cell.2009.09.028. - DOI - PMC - PubMed

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