The interaction of morphine and γ-aminobutyric acid (GABA)ergic systems in anxiolytic behavior: using μ-opioid receptor knockout mice

Abstract

We investigated the interaction of morphine and γ-aminobutyric acid (GABA)ergic systems in anxiolytic action using μ-opioid receptor knockout and wild-type female mice. An elevated plus-maze test was used to assess anxiolytic behavior. The anxiolytic activities were monitored after experimental animals were pretreated with morphine [15 mg/kg, subcutaneous (s.c.)] and 3 h later received a single dose of muscimol (0.5 mg/kg, s.c.). In another experiment, mice received a single dose of opioid antagonist, naloxone [1.0 mg/kg, intraperitoneal (i.p.)], or GABA_A receptor antagonist, (+)bicucullin (2.0 mg/kg, i.p.), 2.5 h after the morphine and 30 min before the muscimol injection. Control mice received vehicle only. The results show that morphine enhanced muscimol-induced staying time in open arms by 160% in wild-type mice. Moreover, the effect of morphine in the wild type was inhibited by the pretreatment of either naloxone or (+)bicucullin. Autoradiographic analysis indicated that morphine-administration raised [³H]muscimol binding by around 25% of the basal level in posterior thalamic, mediodorsal thalamic, and amygdaloid areas, but not in the hippocampal area in wild-type mice. In contrast, morphine administration did not alter the [³H]muscimol binding in μ-opioid receptor knockout mice. The present results reveal that μ-opioid receptor may play a role in the modification of anxiolytic behavior regulated by GABAergic neurotransmission.

Introduction

Previous results from several laboratories indicate that an interaction occurs between the brain γ-aminobutyric acid (GABA)ergic function and the opiate function. Morphine may modulate brain GABA/benzodiazepine receptors. It was reported that opioids affect benzodiazepine, GABA, or glycine binding in the central nervous system (CNS) of the rat 2, 15 and GABA-induced inward current in the CNS of Lymnaea stagnalis L. [19]. In addition, GABA modulators also exert their action on morphine-induced pharmacological effects, such as endorphine secretion [11], catalepsy, and analgesia in rat [14]. It seems particularly interesting that GABAergic system plays an important role in acute opiate withdrawal in rodents 20, 21 or alteration of contractility in illeums isolated from the guinea pig [1]. Despite the evidence by immuno-cytochemical studies showing that GABA neurons in the CNS express μ-opioid receptor (MOR) 5, 6, especially the hippocampal neurons of rats [4], very little direct evidence is available concerning interaction of opioid and GABA receptors in animals that are deficient in the MOR gene.

An elevated plus-maze test was used as an accurate and convenient method for assessing anxiolytic behavior [9]. This methodology is very suitable for assessing interactions between the opioid and GABA system involved in anxyolitic behaviors. Many reports have indicated that animal models of anxiety that assess unconditioned responses are more accurate to assess anxiolytic functions of GABAergic system than are traditional operant-based procedures [3]. Moreover, it has been shown that administration of estrogen induces higher levels of enkephalin mRNAs in the hypothalamus of mice [18] and estradiol induces a preproenkephalin gene expression in the hypothalamus of the rat [22]. This effect is greater in female than in male rats [8], indicating that female mice are more sensitive toward opioid-mediated functions. The recent data in which alteration in emotional responses was shown in female preproenkephalin-gene knockout mice [13] strongly support this suggestion. Therefore, female MOR knockout mice could provide a useful model to investigate the direct interaction of MOR and GABA_A receptor in anxiolytic action. Here, we investigated the effects of morphine and muscimol, a GABA_A agonist, on the anxiolytic behavior in MOR knockout mice, and on [³H]muscimol binding in the brain following behavior tests.