Elsevier

Brain Research

Volume 824, Issue 1, 3 April 1999, Pages 105-111
Brain Research

Research report
Effects 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

https://doi.org/10.1016/S0006-8993(99)01207-XGet rights and content

Abstract

This study performed in freely moving rats evaluated the ability of specific opioid receptor antagonists to reverse the inhibitory effects of morphine on carrageenin-induced c-Fos expression in the spinal cord. Our study focused on the superficial dorsal horn (laminae I–II), which is the main termination site of nociceptive primary afferent fibers and is rich in opioid receptors. In order to replicate clinical routes of administration, all agents were administered intravenously (i.v.). As previously demonstrated, pre-administered i.v. morphine (3 mg/kg) produced a marked decrease (58±5%) in the number of Fos-LI neurones measured at 2 h after intraplantar (i.pl.) carrageenin (6 mg/150 μl) and yet was without influence on peripheral oedema. This decrease in c-Fos expression was completely blocked by combined administration of morphine with the μ-opioid receptor antagonist, [d-Phe-Cys-Tyr-d-Orn-Thr-Pen-Thr-NH2] (CTOP-1+1 mg/kg). Naltrindole (NTI-1+1 mg/kg), a δ-opioid receptor antagonist partially blocked the effects of systemic morphine, so that the inhibitory effects of morphine after NTI injection are now 40±4%. However, this effect of NTI was weak since the depressive effects of morphine were still highly significant (p<0.001). In contrast, nor-binaltorphimine (nor-BNI-1+1 mg/kg), a κ-opioid receptor antagonist, had no significant effect on the effects of morphine. These results indicate the major contribution of μ-opioid receptors to the antinociceptive effects of systemic morphine at the level of the superficial dorsal horn. The observed effect of NTI is not necessarily related to a direct action of morphine on δ-opioid receptors and some possible actions of this antagonist are discussed.

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)

  • P. Honoré et al.

    When is the maximal effect of pre-administered systemic morphine on carrageenin evoked spinal c-Fos expression in the rat?

    Brain Res.

    (1995)
  • D.N. Jones et al.

    Long term kappa-opioid receptor blockade following nor-binaltorphimine

    Eur. J. Pharmacol.

    (1992)
  • D. Le Bars et al.

    The spinal site of action of morphine in pain relief: from basic research to clinical applications

    Trends Pharmacol. Sci.

    (1981)
  • A. Mansour et al.

    Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications

    Trends Neurosci.

    (1995)
  • K. Noguchi et al.

    Preproenkephalin mRNA in spinal dorsal horn neurons is induced by peripheral inflammation and is co-localized with Fos and Fos-related proteins

    Neuroscience

    (1992)
  • K. Noguchi et al.

    Dynorphin expression and Fos-like immunoreactivity following inflammation induced hyperalgesia are colocalized in spinal cord neurons

    Mol. Brain Res.

    (1991)
  • K. Omote et al.

    δ-Receptor involvement in morphine suppression of noxiously evoked activity of spinal WDR neurons in cats

    Brain Res.

    (1991)
  • M.H. Ossipov et al.

    The increase in morphine antinociceptive potency produced by carrageenin-induced hindpaw inflammation is blocked by naltrindole, a selective δ-opioid antagonist

    Neurosci. Lett.

    (1995)
  • F. Porreca et al.

    Modulation of morphine antinociception by peripheral (Leu5) enkephalin: a synergistic interaction

    Eur. J. Pharmacol.

    (1990)
  • B.K. Taylor et al.

    Continuous intravenous infusion of naloxone does not change behavioral, cardiovascular, or inflammatory responses to subcutaneous formalin in the rat

    Pain

    (1997)
  • W. Wongchanapai et al.

    Differential involvement of opioid receptors in intrathecal butorphanol-induced analgesia: compared to morphine

    Pharmacol. Biochem. Behav.

    (1998)
  • M. Zimmermann

    Ethical guidelines for investigations of experimental pain in conscious animals

    Pain

    (1983)
  • J.U. Adams et al.

    Receptor selectivity of icv morphine in the rat cold water tail-flick test

    Drug Alcohol Depend.

    (1993)
  • J.-M. Besson et al.

    Peripheral and spinal mechanisms of nociception

    Physiol. Rev.

    (1987)
  • Cited by (22)

    • Delta opioid receptor agonist BW373U86 attenuates post-resuscitation brain injury in a rat model of asphyxial cardiac arrest

      2014, Resuscitation
      Citation 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 Pharmacology
      Citation 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).

    • Remifentanil preconditioning confers delayed cardioprotection in the rat

      2007, British Journal of Anaesthesia
      Citation 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).

    View all citing articles on Scopus
    View full text