Objective

Opioids play a central role in the control of pain and regulate a number of other behavioural and physiological responses including gastrointestinal mobility, mood and respiration. Whilst opioid receptors are important targets for pain management there is an unmet need to develop novel drugs for the treatment of pain and opioid dependency without the serious problems associated with the clinical use of opioids. Opioids used clinically, eg morphine (full agonist) and buprenorphine (partial agonist) exert their pharmacological actions via mu-opioid receptors, while (-)-pentazocine (full agonist) exerts its effects via kappa opioid receptors. This ex vivo autoradiographic study investigated the occupancy of mu and kappa-opioid receptors in slices of various rat brain regions following peripheral administration of morphine, buprenorphine and (-)-pentazocine. [3H]DAMGO and [3H]U-69,593 were used to label mu and kappa-opioid receptors, respectively. Male,Sprague-Dawley rats (300±50g) were administered vehicle, morphine [3, 10 or 30], buprenorphine [0.1, 0.3 or 1] or (-)-pentazocine [5, 10 or 20] and terminated 60 min (morphine, buprenorphine) or 30 min ((‑)‑pentazocine) post-administration. Doses in square brackets are [mg/kg, ip]. Brains were removed. Coronal sections(20um)containing cortex, striatum, hippocampus and periaqueductal grey (PAG) were cut and incubated in buffer containing either [3H]DAMGO (2 or 5nM) or [3H]U-69,593 (2.5nM) for 10 or 90 min, respectively. Non‑specific binding was determined by 50uM (‑)Naloxone or 10uM U-69,593 for [3H]DAMGO or [3H]U-69,593 autoradiography, respectively. Binding was terminated by aspiration and sections washed in buffer (3x5 min). Radioactivity bound to the section was determined using a Beta-Imager. Results are expressed as % occupancy, n = 3-5 rats/group. Significant differences versus the vehicle-treated group: *p<0.05,**p<0.01, ***p<0.001. Morphine [10 and 30] occupied mu-opioid receptors labelled by [3H]DAMGO in the cortex (49** and 61 %***), striatum (52** and 74 %***), hippocampus (48* and 56 %**) and PAG (33 %*, top dose only). Buprenorphine [0.3 and 1] occupied mu-opioid receptors in the cortex (92*** and 93 %***), striatum (87*** and 89 %***) and hippocampus (88 %***, both doses). The lowest dose of buprenorphine tested also occupied mu‑opioid receptors in the cortex (49 %*).(‑)‑Pentazocine [5, 10 and 20] occupied kappa-opioid receptors labelled by [3H]U‑69,593 in the striatum (54*, 44* and 55 %**). Morphine (mu-opioid agonist) and buprenorphine (partial mu-opioid agonist/kappa-opioid antagonist) occupied central mu-opioid receptors in a dose‑dependent manner. All doses of (‑)‑pentazocine (kappa-opioid agonist/mu-antagonist) occupied central kappa-opioid receptors. Using these tool compounds which have a range of opioid receptor profiles, we have validated these ex vivo autoradiography assays for opioid receptors. Ex vivo autoradiography is a powerful tool that can be used to quantify mu- and kappa-opioid receptor occupancy in vivo by novel drug-candidates for pain and opioid dependency.