The mu-delta opioid receptor heteromer activates the pertussis toxin-resistant Got, GTP-binding protein following stimulation by the delta-agonist deltorphin-II whereas and delta-receptors activate the pertussis toxin-sensitive Ga-i3 protein following stimulation by and delta-agonists, respectively. Although the regulation of the mu-delta heteromer is being investigated extensively in vitro, its physiological relevance remains elusive owing to a lack of available molecular tools. We investigated mu-delta heteromer signaling under basal conditions and following prolonged morphine treatment in rodent brain regions highly co-expressing Rand delta-receptors and Got,. Deltorphin-II induced Got, activation in the striatum and hippocampus, demonstrating the presence of mu-delta heteromer signaling in these brain regions. Prolonged morphine treatment, which desensitizes mu- and delta-receptor function, had no effect on mu-delta heteromer signaling in the brain. Our data demonstrate that 11-delta heteromer signaling does not desensitize and is regulated differently from mu- land delta-receptor signaling following prolonged morphine treatment. (C) 2014 Elsevier Inc. All rights reserved.