Implication of KCC2 in the sensitization to morphine by chronic ethanol treatment in mice

Chronic consumption of ethanol has been reported to modify several molecular events in the central nervous system (CNS). Ethanol directly or indirectly stimulates mesolimbic dopaminergic neurons and increases the release of dopamine in the nucleus accumbens (NAc; Xiao et al., 2007). Several studies have investigated the roles of the mesolimbic dopaminergic system, which leads from the ventral tegmental area (VTA) to the NAc, in drug dependence (Shibasaki et al., 2011). It has been suggested that there is a relationship between endogenous opioid systems and alcohol addiction (Herz, 1997); ethanol increases the release of b-endorphin in the VTA, which influences the reinforcing effects of ethanol (Jarjour et al., 2009). Furthermore, the opioid receptor antagonist naltrexone reverses the ethanolinduced release of dopamine in the NAc, and the suppression of ethanol-reinforced behavior by naltrexone is associated with attenuation of the ethanol-induced increase in dopamine levels from the NAc (Gonzales and Weiss, 1998). Thus, endogenous opioid systems play a crucial role in alcohol addiction. Microdialysis studies have demonstrated that intraVTA injection of a m-opioid receptor agonist increases dopamine overflow in the NAc (Devine et al., 1993). Morphine indirectly activates dopaminergic neurons localized in the VTA as a consequence of its inhibitory effects on nondopaminergic neurons, presumably gaminobutyric acid (GABA)ergic neurons, leading to an increase in dopamine release in the NAc (Di Chiara and Imperato, 1988). Recent studies have demonstrated that the infusion of brain-derived neurotrophic factor into the VTA promotes a change in GABAergic neurons from an inhibitory to an excitatory signaling state, which depends on the mechanisms of the effects of ethanol and morphine (Ting et al., 2013; VargasPerez et al., 2009). Thus, a change in GABAergic tone may be responsible for the change in the response to ethanol and morphine. A previous study showed that a neuron-specific K–Cl cotransporter isoform, potassium-chloride cotransporter 2 (KCC2), is responsible for the generation of an inward-directed Cl electrochemical gradient in a large variety of central neurons, particularly GABAergic neurons (Gulacsi et al., 2003). Generally, Cl homeostasis is regulated by two methods in neurons; the first involves KCC2, which can regulate Cl efflux to equilibrate the membrane potential in mature neurons, and the other involves sodium-potassium-chloride cotransporter 1 (NKCC1), which can regulate Cl influx (Ben-Ari, 2002). As changes in the expression and/or function of those cotransporters are important in the pathophysiological response, chronic treatment with ethanol may influence the response induced by morphine for Cl homeostasis by regulating NKCC1 and KCC2 in GABAergic neurons. Therefore, this study was designed to investigate the effect of chronic treatment with ethanol on the hyperlocomotion induced by morphine. Furthermore, to clarify the possible changes in GABAergic neurons by ethanol, we also examined the roles of NKCC1 and KCC2 in the regulation of GABAergic neurons.