ArticleBasal levels and alcohol-induced changes in nociceptin/orphanin FQ, dynorphin, and enkephalin levels in C57BL/6J mice
Introduction
Numerous studies have demonstrated that genetic factors are important for the development of alcoholism in humans. Early twin and adoption studies have, for instance, shown that children of alcoholic parents run a greater risk of developing alcoholism than children of individuals who do not abuse alcohol (see reviews 18, 19). Several experimental animal strains show a high preference for drugs of abuse and are therefore often used in experiments trying to identify neurochemical substrates associated with excessive drug intake. For example, Lewis rats show a preference for cocaine, alcohol, and opiates 45, 47 and C57BL/6J mice show a preference for alcohol 2, 18. In addition, several rat lines that self-administer alcohol voluntarily, such as alcohol-preferring AA rats 15, 42, have been generated by selective outbreeding.
Human and animal studies of the neurobiological mechanisms underlying drug addiction have provided evidence for the involvement of multiple neurotransmitter systems in the addiction process. The mesolimbic dopamine pathway is often referred to as the brain reward system and its activation, causing dopamine release in the nucleus accumbens, is strongly associated with the reinforcing effects of various drugs of abuse, including ethanol 13, 51. Opposing endogenous opioid systems tonically controls the mesolimbic dopamine neurons; κ-receptors mediate an inhibition of dopamine release whereas μ-opioid and/or δ-opioid receptors mediate the activation of dopaminergic neurons [43]. An imbalance between the κ-receptor system and the μ/δ-opioid systems may change the sensitivity of the reward system which, in turn, may lead to a change in drug preference. Such differences have been described in drug-preferring animals. Genetically enhanced sensitivity of the μ-opioid and/or δ-opioid system receptors to ethanol has been suggested to be associated with an increased motivation to consume alcohol 11, 12, 18, 19. Moreover, an enhanced ethanol-induced release of β-endorphin (β-END) has been shown to occur in alcohol-preferring mice as well as in individuals in families with a history of alcoholism (see reviews 18, 19). An increased sensitivity in the drug reward pathway may also originate from reduced inhibition of dopaminergic neurons due to a low activity of the κ-opioid system [44]. For example, alcohol-preferring AA rats have low immunoreactive (ir) levels of the endogenous κ-agonist dynorphin (DYN) in the nucleus accumbens [34]. However, the mesolimbic dopamine neurons are not critical for ethanol reinforcement and it has been suggested that other transmitter systems contribute to the reinforcing effects 14, 39. Ethanol enhances the activity of endogenous opioid systems, which could contribute to the ethanol-induced reinforcing effects 3, 19, 22. In agreement with this theory, it has also been shown that naltrexone, an opiate receptor antagonist, reverses ethanol-induced dopamine release in animals [3] and also decreases alcohol consumption in alcoholic patients 37, 50. In animal experiments, both unselective opiate antagonists 3, 5, 23 and a selective δ-receptor antagonist reduce alcohol drinking whereas an enkephalinase inhibitor, which increases endogenous enkephalin (ENK) levels, increases alcohol intake [17]. Taken together these results suggest that the endogenous opioid peptide system is involved in, and maybe is essential for, initiation and maintenance of excessive alcohol consumption.
Nociceptin/orphanin FQ (N/OFQ) is a newly identified neuropeptide with a structure similar to DYNA (1–17) 31, 40. N/OFQ is the endogenous ligand for the ORL-1 (opioid receptor like) receptor, which has sequence similarities with opioid receptors. Thus far, N/OFQ has been implicated mostly in nociceptive transmission (see review [9]). Less information is available regarding the effects of drugs on the N/OFQ system and the involvement of N/OFQ in drug dependence mechanisms. N/OFQ shows no conditioned place preference [10]. Like DYN, N/OFQ inhibits dopamine release within the nucleus accumbens; however, unlike DYN, this effect is mediated by an enhanced GABAergic activity in the ventral tegmental area (VTA) [32]. Inherent differences in the N/OFQ system between alcohol-preferring and alcohol-avoiding animals have so far not been elucidated.
Chronic drug exposure may cause a dysfunction in brain transmitter systems that are capable of altering drug sensitivity. Studies of drug-induced adaptation are essential in order to increase the understanding of the mechanisms underlying drug dependence and to identify possible target systems that can reduce the expression of withdrawal symptoms and drug craving. The main purposes of this study are (1) to study possible adaptations in the N/OFQ and opioid peptide systems after chronic alcohol self-administration in alcohol-preferring C57BL/6J mice, and (2) to examine strain differences in the C57BL/6J mice and alcohol-avoiding DBA/2J mice. Basal levels of N/OFQ, Met-enkephalin-Arg6 Phe7 (MEAP), as a biomarker of the proenkephalin system, and DYNB, derived from proDYN, were compared in several areas of the brain and in the pituitary gland in C57BL/6J and DBA/2J mice using specific and sensitive radioimmunoassays. The short-term and long-term effects of 4 weeks of alcohol self-administration on peptide levels were studied in the C57BL/6J mice. In order to evaluate adaptive changes in peptide systems after chronic alcohol intake, peptides were measured the last day of the self-administration period and also 5 and 21 days after alcohol was withdrawn.
Section snippets
Chemicals
Peptides were purchased at Bachem AG (Bubendorf, Switzerland) and all chemicals were obtained from regular commercial sources.
Animal experiments
Male C57BL/6J and male DBA/2J mice (M & B A/S, Denmark) were housed in individual cages for 3 weeks before the start of the experiment. They were kept in a temperature and humidity controlled environment on a 12-h light/dark cycle and they were allowed to feed and drink ad libitum. The experimental procedure was approved by the Swedish Animal Protection Legislation.
In
Basal peptide levels in C57BL/6J and DBA/2J mice
The basal ir-N/OFQ, DYNB, and MEAP levels in C57BL/6J and DBA/2J mice are shown in Table 1. The ir-N/OFQ levels in the frontal cortex and in the hippocampus were higher in the C57BL/6J mice than the DBA/2J mice. Strain differences were not detected in other brain areas. In the pituitary gland ir-N/OFQ levels were not detectable.
The ir-DYNB levels in the pituitary gland and in the PAG were higher in the C57BL/6J mice than in the DBA/2J mice. Lower ir-DYNB levels were detected in the nucleus
Discussion
Studies of drug-preferring animals, such as Lewis rats, AA rats, and C57BL/6J mice, are currently being used to examine possible neurochemical substrates for individual differences in vulnerability to develop drug dependence. We have previously reported differences in endogenous opioid peptide systems in the drug-preferring AA rats [34] and Lewis rats [35] and in the present study we demonstrate differences in opioid peptide levels and differences in N/OFQ levels in C57BL/6J mice as compared to
Acknowledgements
The authors thank Jonas Venge for skilful assistance in the nociceptin/orphanin FQ assays. This study was supported by the Swedish Alcohol Research Foundation and the Swedish Medical Research Council.
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