Benzodiazepines are commonly used as sedatives, sleeping aids, and anti-anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K + -Cl À cotransporter 2 (KCC2) in the sensitization to morphine-induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of c-aminobutyric acid A-type receptor (GABA A R) a1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine-induced hyperlocomotion, which is accompanied by the up-regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down-regulation of protein phosphatase-1 (PP-1) as well as the up-regulation of phosphorylated protein kinase C c (pPKCc). Furthermore, PP-1 directly associated with KCC2 and pPKCc, whereas pPKCc did not associate with KCC2. On the other hand, pre-treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine-induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKCc-PP-1-KCC2 pathway by chronic treatment with zolpidem. Keywords: benzodiazepine, drug dependence, KCC2, morphine, nucleus accumbens, zolpidem. Benzodiazepines are used as sedatives, sleeping aids, and anti-anxiety drugs. However, the long-term use of benzodiazepines induces psychological and physiological dependence. Psychological dependence is considered to accompany the neuroplastic changes in the dopaminergic system which projects from the ventral tegmental area to the nucleus accumbens, and activation of the mesolimbic dopaminergic system induces the rewarding effects and sensitization to locomotor activity induced by opioids and psychostimulants Narita et al. 2003Narita et al. , 2004Narita et al. , 2006Fukakusa et al. 2008). On the other hand, even though benzodiazepines induce psychological dependence without increasing the release of dopamine (Invernizzi et al. 1991), the mechanisms by which benzodiazepines induce psychological dependence are not fully understood. c-aminobutyric acid A-type receptor (GABA A R) consists of a five-subunit complex (2 a, 2 b, and 1 c subunit), containing Cl À channels, and the activation of GABA A R by GABA results in an increase in Cl À influx. Benzodiazepines bind to the benzodiazepine receptor that is located on GABA A R between a and c subunits. The activation of GABA A R containing a1 subunit is associated with sleeping, sedation, abuse potential, and anti-epilepsia.