Quantitative autoradiographic mapping of the ORL1, μ-, δ- and κ-receptors in the brains of knockout mice lacking the ORL1 receptor gene

doi:10.1016/S0006-8993(01)02531-8

Brain Research

Volume 906, Issues 1–2, 6 July 2001, Pages 13-24

https://doi.org/10.1016/S0006-8993(01)02531-8 Get rights and content

Abstract

Until recently the opioid receptor family was thought to consist of only the μ-, δ- and κ-receptors. The cloning of opioid receptor like receptor (ORL1) and its endogenous ligand nociceptin/orphanin FQ, which displayed anti-opioid properties, has raised the issue of functional co-operativity of this system with the classical opioid system. ORL1 receptor knockout mice have been successfully developed by homologous recombination to allow the issue of potential heterogeneity of this receptor and also of compensatory changes in μ-, δ- or κ-receptors in the absence of ORL1 to be addressed. We have carried out quantitative autoradiographic mapping of these receptors in the brains of mice that are wild-type, heterozygous and homozygous for the deletion of the ORL1 receptor. ORL1, μ-, δ- and κ-receptors were labelled with [³H] leucyl-nociceptin (0.4 nM), [³H] DAMGO (4 nM), [³H] deltorphin-I (7 nM), and [³H] CI-977 (2.5 nM) respectively. An approximately 50% decrease in [³H] leucyl-nociceptin binding was seen in heterozygous ORL1 mutant mice and there was a complete absence of binding in homozygous brains indicating the single gene encodes for the ORL1 receptor and any putative subtypes. No significant gross changes in the binding to other opioid receptors were seen across genotypes in the ORL1 mutant mice demonstrating a lack of major compensation of classical opioid receptors in the absence of ORL1. There were a small number of region specific changes in the expression of classical opioid receptors that may relate to interdependent function with ORL1.

Introduction

The opioid receptor family has until recently consisted of three major classes of receptor that are each derived from a single gene μ- [5] δ- [8], [18] and κ- [51]. The opioid receptors are widely yet distinctly distributed throughout the rat [26] and mouse [19], [42] central nervous system. The receptor distribution largely accords with the probable functional roles of opioid peptides particularly in the regulation of nociception but also in wider physiological roles in stress [23], cardiovascular control [22], immunomodulation [39] and memory [16].

An additional member of the opioid receptor family, the opioid receptor like one (ORL1) receptor, was isolated and cloned in mouse (MOR-C) [37], rat (LC132, oprl, ROR-C, C3, XOR1S, Hyp-8-1) [3], [4], [13], [21], [47], [48] and human (ORL1) [30] species during the screening of cDNA libraries for further genes encoding opioid receptor subtypes. This receptor is also a G-protein coupled receptor [7], [13] and is highly homologous to the classical opioid receptors with around 80% homology in the 2nd, 3rd and 7th membrane spanning domains [30]. Despite this structural homology the ORL1 receptor does not bind opioid ligands with any appreciable affinity [3], [4], [13], [21], [30], [47], [48]. The ORL1 receptor distribution pattern is quite distinct from that of any of the opioid receptors, is diffusely distributed throughout the rat CNS [33] and the pattern of receptor distribution is also closely correlated with its mRNA expression [34]. Scatchard plots for the ORL1 receptor in transfected CHO cells indicated the presence of a homogenous receptor site [40] however saturation and competition studies in mouse brain membranes have suggested the possible presence of both high and low affinity sites [27]. More recently an alternatively spliced transcript of the ORL1 receptor has been reported and is differentially distributed in tissues [49]. The truncated receptor displays very poor affinity and non-selective binding for nociceptin and other opioid peptides and does not mediate any inhibitory effects on cAMP production [49]. However the possibility of the existence of ORL1 receptor subtypes is still an open debate.

The endogenous ligand for the ORL1 receptor is a hepatadecapeptide isolated by two groups and termed nociceptin and orphanin FQ respectively [28], [40]. Nociceptin/orphanin FQ binds the ORL1 receptor in the nanomolar range [28], [40] and displays high sequence homology to dynorphin A but has an N-terminal phenylalanine residue in place of a tyrosine required for the activation of classical opioid receptors. The nociceptin/orphanin FQ peptide and its messenger RNA are widely distributed in the central nervous system and are largely correlated with the distribution of ORL1 receptors [2], [34].

Soon after its discovery nociceptin/orphanin FQ was described as an anti-opioid peptide [29] able to reverse or prevent morphine antinociception. This anti-opioid property appears to be restricted to the supraspinal level with spinal administration of nociceptin/orphanin FQ leading to antinociception [50]. In addition, i.c.v. administration of nociceptin/orphanin FQ has been shown to attenuate the development of morphine place preference [31] while also differentially reducing dopamine release in the nucleus accumbens following administration of morphine [14]. These behavioural observations have raised the question of functional interaction between nociceptin/orphanin FQ and endogenous opioid systems and an association between the ORL and μ-, δ- and κ-receptors.

ORL1 receptor knockout mice have been generated and their phenotype characterised in a number of studies [24], [25], [36], [38], [44], [45]. These mice display normal nociceptive thresholds and nociceptin/orphanin FQ-induced hyperalgesia is lost in homozygous animals [36]. Although antinocicetive responses to morphine are normal in ORL1 knockout mice these animals do not develop tolerance to this opioid to the extent that occurs in wild-type mice [45]. This has led to the suggestion that there are important interrelationships between the ORL1 receptor and the μ-opioid receptor in the development of tolerance to morphine [45]. More recently it has been shown that both tolerance and dependence, but not analgesic potency of morphine is attenuated in ORL1 knockout mice [44].

To further investigate the possible relationship between the ORL1 receptor and the classical opioid system we have carried our quantitative autoradiographic mapping in a novel line of ORL1 knockout mice. These mice deficient in the ORL1 receptor gene have been generated by deletion of exons 2 and 3 of the ORL1 receptor gene. In these animals we have determined if there are any receptors derived from different genes to which nociceptin/orphanin FQ binds and addressed if any compensatory changes in μ-, δ- and κ-receptors occur in the brains of these mice, which might explain the behavioural phenotype of these animals.

Section snippets

Generation of knockout mice

Mice containing a deletion of the ORL1 gene were produced by standard procedures. The mice are homozygous for a mutant allele in which the neomycin gene was substituted for a Hind III-EcoRV fragment that encodes exons 2 and 3 of mouse ORL1. Mice were maintained according to AALAC standards as approved by the Animal Care and Use Committee at UMDNJ.

Autoradiographic procedures

Intact whole brains from six to eight week old male wild-type, heterozygous and homozygous mice (n=5 for each genotype) were removed from the

ORL1 receptors

Quantitative analysis of [³H] leucyl-nociceptin (0.4 nM) binding revealed a diffuse distribution throughout the wild-type mouse brain. Moderate to high levels of binding (between 50 and 90 fmol/mg tissue) were observed throughout all parts of the cortex with no marked rostral to caudal pattern. In non-cortical structures high levels of binding (greater than 70 fmol/mg tissue) were observed in the accessory olfactory bulb, suprachiasmatic nucleus and the amygdala. Moderate levels of expression

Discussion

The diffuse distribution pattern of ORL1 receptors observed here reflects the wide physiological roles of this receptor system reported to date. The distribution and levels of binding of the ORL1 receptor in the hippocampus is consistent with the enhancement of long term potentiation memory and learning observed in ORL1 knockout mice [24], [25] and the impairment of spatial learning reported following intracerebroventricular administration of nociceptin/orphanin FQ [41]. The ORL1 receptor is

Acknowledgements

This work was supported by a MRC Merck, Sharp and Dohme CASE studentship to S.C., by NIH grant DA-09040 (JP) and by a PMA post-doctoral fellowship (ZC).

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High levels of binding were observed in the cortex and striatum, as has been reported previously (Clarke et al., 2001). Finally, also [3H]CI-977 binding to the κ-opioid receptor was not affected by genotype (Fig. 4C, Table 4;F(2,116)=1.77, N.S.), and there was no significant genotype×brain region interaction (F(12,116)=1.18, N.S.) High levels of binding were found in the striatum and hypothalamus, which corresponds to previous studies (Clarke et al., 2001). Using recently established gene knockout technology (Smits et al., 2006, 2008; van Boxtel et al., 2008b) we obtained a rat knockout for NOPr.
The nociceptin/orphanin FQ (N/OFQ) opioid peptide receptor (NOPr) is a new member of the opioid receptor family consisting of μ, δ and κ opioid receptors. The anti-opioid properties of its endogenous ligand, N/OFQ provide the receptor interesting potentials in symptoms and processes related to drug addiction, learning and memory, anxiety and depression, and nociception. Using target-selected N-ethyl-N-nitrosourea (ENU)-driven mutagenesis we recently generated a rat model bearing a premature stop codon in the opioid-like receptor (oprl1) gene, and here we describe the primary characterization of this novel model. Data revealed that [³H]N/OFQ binding to brain slices was completely absent in rats homozygous for the premature stop codon (oprl1^−/−). Heterozygous rats displayed an intermediate level of NOPr binding. Oprl1 receptor transcript levels, as determined by Northern blot analysis, were reduced by approximately 50% in oprl1^−/− rats compared to wild-type controls (oprl1^+/+), and no alternative spliced transcripts were observed. Quantitative autoradiographic mapping of μ, δ and κ opioid receptors using [³H]DAMGO, [³H]deltorphin and [³H]CI-977, respectively, did not show any changes in opioid receptor binding. In conclusion, we present a novel mutant rat lacking NOPr without compensatory changes in μ, δ and κ opioid receptors. We anticipate that this mutant rat will have heuristic value to further understand the function of NOPr.
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The effects with pretreatment with nociceptin (0.03–30 nmol, i.c.v.) were evaluated on the threshold for eliciting afterdischarge (ADT), generation and spread of seizure activity and postictal depression in rats with kindling stimulation. Nociceptin produced a decrease in ADT (32–45%) in rats with partial seizures (PS, stage II–III), and an increase (61–92%) in rats with generalized seizures (GS, kindled state). Nociceptin did not modify the behavioral changes, spike frequency and duration of afterdischarge elicited at ADT in both experimental groups. In rats with GS, nociceptin enhanced postictal depression (34–44%) evaluated with a recycling paradigm. Autoradiography experiments revealed enhanced nociceptin opioid receptor (NOP) binding in medial amygdala (22–26%), frontal (21–23%) and entorhinal (27–32%) cortices, and reduced binding in the substantia nigra pars compacta (28%) and medial central gray (29%) of rats with PS. The GS group displayed significant decreased NOP binding (40–70%) in most of the brain areas evaluated. These results suggest that nociceptin facilitates ictal activity in rats with PS, whereas in animals with GS, it induces inhibitory effects on ADT and enhances the postictal period. These effects correlate with significant changes in NOP binding.
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The average cell density per unit volume was determined by counting them under a light microscope equipped with a digitalized image analysis system, and according with the procedure described previously.15,16 The autoradiography experiments were carried out on adjacent sections thaw-mounted on gelatin-coated slides according to the procedures described previously.17–19 Experimental conditions for mu, delta and nociceptin autoradiography are summarized in Table 2.
Opioid receptor binding was evaluated in parahippocampal cortex (PHC) obtained from patients with intractable mesial temporal lobe epilepsy (MTLE) with and without subacute high frequency electrical stimulation (HFS) in this brain area. Mu, delta and nociceptin receptor binding was determined by autoradiography in PHC of five patients (ESAE group) with MTLE history of 14.8 ± 2.5 years and seizure frequency of 11 ± 2.9 per month, two of them (40%) with mesial sclerosis. This group demonstrated antiepileptic effects following subacute HFS (130 Hz, 450 μs, 200–400 μA), applied continuously during 16–20 days in PHC. Values were compared with those obtained from patients with severe MTLE (history of 21.7 ± 2.8 years and seizure frequency of 28.2 ± 14 per month) in whom electrical stimulation did not induce antiepileptic effects (ESWAE group, n = 4), patients with MTLE in whom no electrical stimulation was applied (MTLE group, n = 4) and autopsy material acquired from subjects without epilepsy (n = 4 obtained from three subjects). Enhanced 3H-DAMGO (MTLE, 755%; ESAE, 375%; ESWAE, 693%), 3H-DPDPE (MTLE, 242%; ESAE, 80%; ESWAE, 346%) and 3H-nociceptin (MTLE, 424%; ESAE, 217%; ESWAE, 451%) binding was detected in the PHC of all epileptic groups. However, tissue obtained from ESAE group demonstrated lower opioid receptor binding (3H-DAMGO, 44.5%, p < 0.05; 3H-DPDPE, 47%, p < 0.05; 3H-nociceptin, 39.3%, p < 0.5) when compared with MTLE group. The present results indicate that a high effectiveness to the antiepileptic effects induced by HFS is associated with reduced opioid peptide binding.

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Research reportQuantitative autoradiographic mapping of the ORL1, μ-, δ- and κ-receptors in the brains of knockout mice lacking the ORL1 receptor gene

Abstract

Introduction

Section snippets

Generation of knockout mice

Autoradiographic procedures

ORL1 receptors

Discussion

Acknowledgements

Life Sci.

Neuroscience

FEBS Lett.

FEBS Lett.

Brain Res.

Neurosci. Lett.

Brain Res.

Neurosci. Lett.

FEBS Lett.

Brain Res.

Pharmacol. Biochem. Behav.

Brain Res.

Trends Pharmacol. Sci.

Eur. J. Pharmacol.

Brain Res.

Biochem. Biophys. Res. Comm.

Neuroscience

FEBS Lett.

Brain Res.

Biochem. Biophys. Res. Comm.

J. Biol. Chem.

Int. J. Immunopharmacol.

Brain Res.

Neuroscience

FEBS Lett.

Research report
Quantitative autoradiographic mapping of the ORL1, μ-, δ- and κ-receptors in the brains of knockout mice lacking the ORL1 receptor gene