Research reportEffects of opioid receptor antagonists on the effects of i.v. morphine on carrageenin evoked c-Fos expression in the superficial dorsal horn of the rat spinal cord
Introduction
Many experimental and clinical studies have been performed to study the site and modality of action of morphine in the control of pain processes. These experimental studies were mainly based on electrophysiological and behavioural techniques, using various sites of administration (intravenous—i.v., subcutaneous—s.c., intraperitoneal—i.p., intracerebroventricular—i.c.v., intrathecal—i.t.). From these studies and from binding studies, it is well-established that morphine acts mainly through μ-opioid receptors to produce analgesia. This was recently confirmed in mice lacking the μ-opioid receptor gene [30]. However, when the population of functional μ-receptors is drastically reduced by alkylation with β-FNA, Takemori and Portoghese [43]have observed that morphine interacts with both δ- and κ-opioid receptors to produce analgesia in acetic acid writhing test. Moreover, a recent investigation in mice lacking for the μ-opioid receptor gene described that functional interaction may take place between μ- and δ-opioid receptors, in certain specific neuronal pathway including those involved in nociception [31].
The above-mentioned studies are based on behavioural investigations. Thus, the aim of the present study was to consider more precisely, under pathological conditions, the possible interaction of systemic morphine with μ-, δ- and κ-opioid receptors at the superficial level of the dorsal horn, which is considered as one of the main site of action of this drug to explain its antinociceptive effects [see Ref. [13]]. In this purpose, we evaluated the effects of high doses of μ-, δ-, and κ-specific antagonists on the well-known depressive effect of morphine on the expression of c-Fos protein evoked in the superficial dorsal horn laminae by intraplantar injection of carrageenin 12, 19, 33, 34, 46. Interestingly, the superficial dorsal horn which corresponds to the main termination site of nociceptive Aδ and C primary afferent fibers, contains numerous neurones driven specifically or not by noxious stimulation [see Refs. 3, 45] and is extremely rich in opioid receptors 2, 8.
To perform this study, we used the immunodetection of c-Fos protein, which has been used widely to study the pharmacology of nociceptive transmission at the spinal cord level [see Ref. [6]]. Indeed, several studies using c-Fos expression as an indirect marker of nociceptive processes have shown that pre-administered morphine significantly reduces c-Fos labelling in the spinal cord induced by various types of peripheral nociceptive stimulation [see Ref. [6]]. These depressive effects were reversed by naloxone, a non-specific opioid receptor antagonist.
To investigate the respective role of the three receptors subtypes in the morphine induced depression of c-Fos expression induced by i.pl. carrageenin at the level of the spinal cord, we used the following opioid receptor antagonists: CTOP, a somatostatin analogue that is highly selective for μ-receptors, naltrindole hydrochloride (NTI) and nor-binaltorphimine (nor-BNI), which are non-peptide naltrexone-derived antagonists for δ- and κ-opioid receptors, respectively.
Section snippets
Experimental animals
Adult male albino Sprague–Dawley rats (Charles River, France), weighing 175–200 g on arrival, were housed six per cage in a room with controlled temperature (22±1°C) and a 12-h alternating light–dark cycle for a week before experiments. Food and water were made available continuously. Guidelines on ethical standards for investigations of experimental pain in conscious animals were followed [49].
Sixty-three rats were divided into eight groups. A control group of rats received i.v. saline 10 min
Carrageenin evoked spinal c-Fos expression
Two hours after i.pl. carrageenin injection, Fos-LI neurones, which were stained to a variable degree, were located in the ipsilateral dorsal horn of the spinal cord. The number of Fos-LI neurones in the contralateral dorsal horn was not significantly different from the now well-established, extremely low number of spinal Fos-LI neurones in non-stimulated rats (<10 neurones/40 μm section [7]). The Fos labelling was mainly located in the superficial dorsal horn. In the absence of drug
Discussion
In this study, we evaluated the respective roles of μ-, δ- and κ-opioid receptors in the depressive effects of systemic morphine on carrageenin-induced c-Fos expression at the spinal cord level. These studies using c-Fos expression have a number of advantages over other approaches: (1) c-Fos technique allows a cellular resolution in freely moving rats, (2) we monitored neurones in the superficial laminae of the dorsal horn; most of them receive either exclusively or not noxious inputs, (3)
Acknowledgements
The authors wish to thank Mr. R. Rambur for the photographs and Pr A.H. Dickenson for English revisions. S. Le Guen was supported by a fellowship from the Ministère de l'Enseignement Supérieur, de la Recherche et de la Technologie. G. Catheline was supported by European BIOMED 2 Program no. BMH4-CT95-0172.
References (49)
- et al.
Autoradiographic distribution of mu, delta and kappa opioid binding sites in the superficial dorsal horn, over the rostrocaudal axis of the rat spinal cord
Brain Res.
(1991) - et al.
Subcutaneous formalin induces a segmental release of met-enkephalin-like material from the rat spinal cord
Pain
(1990) - et al.
Cholecystokinin B receptor antagonism enhances the ability of a low dose of morphine to reduce c-Fos expression in the spinal cord of the rat
Neuroscience
(1995) - et al.
Receptor localization in the mammalian dorsal horn and primary afferent neurons
Brain Res. Rev.
(1997) - et al.
Opioid receptors on peripheral sensory axons
Brain Res.
(1997) - et al.
Temporal analysis of increases in c-fos, preprodynorphin and preproenkephalin mRNAs in rat spinal cord
Mol. Brain Res.
(1989) - et al.
The antinociceptive action of supraspinal opioids results from an increase in descending inhibitory control: correlation of nociceptive behavior and c-Fos expression
Neuroscience
(1991) - et al.
Inflammation of the rat paw enhances axonal transport of opioid receptors in the sciatic nerve and increases their density in the inflamed tissue
Neuroscience
(1993) - et al.
Carrageenin-evoked c-Fos expression in rat lumbar spinal cord: the effects of indomethacin
Eur. J. Pharmacol.
(1995) - et al.
The effects of morphine on carrageenin-induced spinal c-Fos expression are completely blocked by β-Funaltrexamine, a selective mu opioid receptor antagonist
Brain Res.
(1996)
When is the maximal effect of pre-administered systemic morphine on carrageenin evoked spinal c-Fos expression in the rat?
Brain Res.
Long term kappa-opioid receptor blockade following nor-binaltorphimine
Eur. J. Pharmacol.
The spinal site of action of morphine in pain relief: from basic research to clinical applications
Trends Pharmacol. Sci.
Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications
Trends Neurosci.
Preproenkephalin mRNA in spinal dorsal horn neurons is induced by peripheral inflammation and is co-localized with Fos and Fos-related proteins
Neuroscience
Dynorphin expression and Fos-like immunoreactivity following inflammation induced hyperalgesia are colocalized in spinal cord neurons
Mol. Brain Res.
δ-Receptor involvement in morphine suppression of noxiously evoked activity of spinal WDR neurons in cats
Brain Res.
The increase in morphine antinociceptive potency produced by carrageenin-induced hindpaw inflammation is blocked by naltrindole, a selective δ-opioid antagonist
Neurosci. Lett.
Modulation of morphine antinociception by peripheral (Leu5) enkephalin: a synergistic interaction
Eur. J. Pharmacol.
Continuous intravenous infusion of naloxone does not change behavioral, cardiovascular, or inflammatory responses to subcutaneous formalin in the rat
Pain
Differential involvement of opioid receptors in intrathecal butorphanol-induced analgesia: compared to morphine
Pharmacol. Biochem. Behav.
Ethical guidelines for investigations of experimental pain in conscious animals
Pain
Receptor selectivity of icv morphine in the rat cold water tail-flick test
Drug Alcohol Depend.
Peripheral and spinal mechanisms of nociception
Physiol. Rev.
Cited by (22)
Stimulation of μ-opioid receptors dilates retinal arterioles by neuronal nitric oxide synthase-derived nitric oxide in rats
2017, European Journal of PharmacologyDelta opioid receptor agonist BW373U86 attenuates post-resuscitation brain injury in a rat model of asphyxial cardiac arrest
2014, ResuscitationCitation Excerpt :Naltrindole hydrochloride (Sigma, USA) and BW373U86 (Tocris, UK) were dissolved in NS containing 5% DMSO. The optimal dosages of BW373U86 and Naltrindole were determined according to existing references and our preliminary experimental results.18–23 Protein levels of CREB and pCREB were analyzed 24 h after ROSC using western blot in protocol I (n = 6/group).
Intracerebroventricular administration of morphine confers remote cardioprotection-Role of opioid receptors and calmodulin
2011, European Journal of PharmacologyCitation Excerpt :All chemicals were from Sigma Chemical Co. (St. Louis, MO). The choice of doses of nor-binaltorphimine (Nielsen et al., 2007; Portoghese et al., 1987), naltrindole (Gao et al., 2010; Portoghese et al., 1988), CTOP (Briski and Sylvester, 2001; Catheline et al., 1999; Hawkins et al., 1989) and TFP (Budai and Kasa, 1987) were according to previous studies. Data are expressed as mean ± S.D. and data analysis was performed with a statistical software package (Prism v4.0; GraphPad Software, San Diego, CA).
Further analyses of mechanisms underlying the antinociceptive effect of the triterpene 3β, 6β, 16β-trihydroxylup-20(29)-ene in mice
2011, European Journal of PharmacologyEffects of transgene-mediated endomorphin-2 in inflammatory pain
2009, European Journal of PainRemifentanil preconditioning confers delayed cardioprotection in the rat
2007, British Journal of AnaesthesiaCitation Excerpt :Nor-binaltorphimine11 17 (nor-BNI, a KOP receptor selective antagonist) 5 mg kg−1 i.v. 24 h before ischaemia. CTOP11 17–19 (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2, a MOP receptor selective antagonist) 1 mg kg−1 i.v. 24 h before ischaemia. RPC (remifentanil 10 μg kg−1 min−1 infusion 24 h before ischaemia).