Deep brain stimulation (DBS) of the nucleus accumbens (NAc) has been applied to attenuate drug relapses in animal studies and clinical treatments. However, the mechanisms by which DBS produces its therapeutic effects remain largely unknown. This study is the first to combine manganese-enhanced magnetic resonance imaging (MRI) with DBS to explore the effects of accumbal DBS on the relapse pathway of rats. The images suggested that DBS produced an antidromic activation of the cortico-accumbal afferents. To verify the activating effects of DBS, the expression levels of the protein encoded by the immediate early gene c-Fos following DBS were measured using immunochemical methods. Intra-shell DBS increased c-Fos expression at the site of stimulation as well as in the infralimbic prefrontal cortex (IL), and increased c-Fos-expressing GABAergic interneurons in the IL This activity is consistent with a previous study demonstrating that DBS of the NAc shell attenuated cocaine reinstatement via local activation and/or the activation of GABAergic interneurons in the medial prefrontal cortex (mPFC). For further examination, lidocaine was microinjected into the NAc shell to block the fibers during DBS before the reinstatement test. Inactivation of the target fibers attenuated the effects of DBS on the reinstatement of morphine seeking. Moreover, GABA receptor antagonists administered into the IL weakened the effect of DBS, while GABA receptor agonists microinjected into the IL attenuated the reinstatement of morphine seeking. (C) 2016 Elsevier Inc. All rights reserved.