Pain is a significant health problem. Mu opioid receptor agonists are used clinically as analgesics, but their use is constrained by high abuse liability. Intracranial self-stimulation (ICSS) is a preclinical behavioral procedure that has been used to assess abuse potential of opioids, and drug-induced facilitation of ICSS is interpreted as an abuse-related effect. ICSS can also be used as a behavioral baseline to detect affective dimensions of pain. Specifically, pain-related depression of ICSS can model pain-related depression of behavior and mood, and drug-induced blockade of pain-related ICSS depression can serve as a measure of affective analgesia. This dissertation used mu agonists that vary in efficacy at the mu receptor (methadone> fentanyl> morphine> hydrocodone> buprenorphine> nalbuphine) and compared their effects on ICSS in the absence (phase one) or presence (phase 2) of pain. Adult male Sprague-Dawley rats were equipped with intracranial electrodes targeting the medial forebrain bundle and trained to lever press for brain stimulation. Different frequencies of stimulation maintained a frequency-dependent increase in ICSS rates, and permitted detection of both rate-increasing and rate-decreasing treatment effects. During phase 1, medium- and high-efficacy mu agonists produced initial rate-decreasing effects, followed by abuse-related rate-increasing effects at later time points. Repeated morphine administration produced tolerance to its own rate-decreasing effects, cross-tolerance to rate-decreasing effects of other mu agonists, and enhanced expression of rate-increasing effects. Low efficacy mu agonists only produced rate-increasing effects, which were enhanced after repeated morphine. These results suggest that previous opioid exposure increases expression of abuse-related facilitation of ICSS by mu agonists regardless of efficacy. During phase 2, intraperitoneal administration of lactic acid (1.8%) served as a noxious stimulus to depress ICSS. All mu agonists blocked acid-induced depression of ICSS at doses similar to those that facilitated ICSS in the absence of pain. A higher intensity noxious stimulus (5.6 % acid) produced further depression of ICSS and reduced the antinociceptive potency of both methadone and nalbuphine. Morphine antinociception was resistant to tolerance in the assay of acid-depressed ICSS. Overall, these results provide a basis for comparing determinants of abuse-related opioid effects in the absence of pain with their affective analgesic effects in the presence of pain.

Opioid Receptors (ORs) are involved in the pathophysiology of several neuropsychiatric conditions yet remain an untapped therapeutic resource. Although only mu-, delta-, and kappa-OR types have been cloned, additional subtypes result from complexes generated by direct receptor-receptor interactions. Mu- and delta-ORs form a heteromeric receptor complex with unique pharmacological and signalling properties distinct from those of mu- and delta-OR homomers. In these studies, we sought to characterize the ligand binding pocket and agonist-induced internalization profile of the mu-delta heteromer, to investigate mu-delta heteromer-specific signalling in brain, and to interrogate the contribution of this receptor complex to opioid-mediated behavioural effects. In competition radioligand binding studies, delta-agonists displaced high affinity mu-agonist binding from the mu-delta heteromer but not the muOR homomer, suggestive of delta-agonists occupying or allosterically modulating the muOR ligand binding pocket within the heteromer. Delta-agonists induced internalization of the mu-delta heteromer in a dose-dependent, pertussis toxin resistant, and muOR- and deltaOR-dependent manner from the cell surface via the clathrin and dynamin endocytic machinery. Agonist-induced internalization of the mu-delta heteromer persisted following chronic morphine treatment conditions which desensitized the muOR homomer. Using Galpha-specific GTPgammaS binding assays, we demonstrated that mu-delta heteromer signalling previously characterized in cell lines was present in the striatum and hippocampus, and did not desensitize following prolonged morphine treatment conditions which desensitized muOR homomer-mediated signalling. Since delta-agonists which also target the mu-delta heteromer possess antidepressant-like and anxiolytic-like properties, we investigated the role of this receptor complex in mood regulation. We devised a strategy to selectively analyze the effects of the mu-delta heteromer by dissociating it using a specific interfering peptide aimed at a sequence implicated in mu-delta heteromerization. The interfering peptide abolished the unique pharmacological and trafficking properties of delta-agonists at the mu-delta heteromer and dissociated this receptor complex in vitro. Intra-accumbens administration of the interfering peptide disrupted the mu-delta interaction in vivo and allowed for isolation of the mu-delta heteromer contribution to the mood-regulatory effects of a delta-agonist with activity at the heteromer. Activation of the mu-delta heteromer in the nucleus accumbens produced antidepressant-like and anxiolytic-like actions in animal models of depression and anxiety.

Opioid Receptors (ORs) are involved in the pathophysiology of several neuropsychiatric conditions yet remain an untapped therapeutic resource. Although only mu-, delta-, and kappa-OR types have been cloned, additional subtypes result from complexes generated by direct receptor-receptor interactions. Mu- and delta-ORs form a heteromeric receptor complex with unique pharmacological and signalling properties distinct from those of mu- and delta-OR homomers. In these studies, we sought to characterize the ligand binding pocket and agonist-induced internalization profile of the mu-delta heteromer, to investigate mu-delta heteromer-specific signalling in brain, and to interrogate the contribution of this receptor complex to opioid-mediated behavioural effects. In competition radioligand binding studies, delta-agonists displaced high affinity mu-agonist binding from the mu-delta heteromer but not the muOR homomer, suggestive of delta-agonists occupying or allosterically modulating the muOR ligand binding pocket within the heteromer. Delta-agonists induced internalization of the mu-delta heteromer in a dose-dependent, pertussis toxin resistant, and muOR- and deltaOR-dependent manner from the cell surface via the clathrin and dynamin endocytic machinery. Agonist-induced internalization of the mu-delta heteromer persisted following chronic morphine treatment conditions which desensitized the muOR homomer. Using Galpha-specific GTPgammaS binding assays, we demonstrated that mu-delta heteromer signalling previously characterized in cell lines was present in the striatum and hippocampus, and did not desensitize following prolonged morphine treatment conditions which desensitized muOR homomer-mediated signalling. Since delta-agonists which also target the mu-delta heteromer possess antidepressant-like and anxiolytic-like properties, we investigated the role of this receptor complex in mood regulation. We devised a strategy to selectively analyze the effects of the mu-delta heteromer by dissociating it using a specific interfering peptide aimed at a sequence implicated in mu-delta heteromerization. The interfering peptide abolished the unique pharmacological and trafficking properties of delta-agonists at the mu-delta heteromer and dissociated this receptor complex in vitro. Intra-accumbens administration of the interfering peptide disrupted the mu-delta interaction in vivo and allowed for isolation of the mu-delta heteromer contribution to the mood-regulatory effects of a delta-agonist with activity at the heteromer. Activation of the mu-delta heteromer in the nucleus accumbens produced antidepressant-like and anxiolytic-like actions in animal models of depression and anxiety.