The dose-response relationship of morphine in a zebrafish ( Danio rerio) model

Previous experiments in zebrafish (Danio rerio) have shown that a noxious ("painful") stimulus results in behavioural changes, namely a marked decrease in swimming activity. Analgesics may be effective at blocking this behavioural change but there is little evidence-based data regarding the efficacy of analgesics in fish. Before any analgesic can be used, a dose-response relationship must be demonstrated and first needs to be shown with morphine, the gold standard analgesic. I tested a model using subcutaneous acid injection as the noxious stimulus which has previously been shown to decrease swimming activity in zebrafish. Fish activity, before and after treatment, was recorded with a video camera and analyzed with Loligo ® software. To alleviate this response, morphine was administered intraperitoneally at doses of 1,3,10,30, or 100 mg/kg in an attempt to show a dose-response relationship. Fish receiving doses of 1 or 100 mg/kg came from a different source and behaved so differently that their results could not be included in the statistical analysis. Acetic acid (5%) at both 5 and 10 μL significantly reduced activity in zebrafish in comparison with saline injected controls (p<0.0001), with the difference scaling with stimulus intensity. Morphine at doses of 10 and 30 mg/kg was effective at attenuating the decrease in activity associated with the noxious stimulus. The ED 50 of morphine was 12.3 ± 1.2 mg/kg (90% C.I. 9.7-15.5). Activity of 10 mg/kg morphine/acid injected fish was not significantly different from control fish that did not receive the noxious acid injection at 60 and 90 min post injection (p=0.39). Activity of morphine-injected controls (no noxious stimulus) did not differ significantly from saline control fish at 60 and 90 min post injection (p=0,88). Effective doses of morphine (10 and 30 mg/kg) were then injected in conjunction with naloxone, a known opioid antagonist. Naloxone, at both 10 and 30 mg/kg, was effective at attenuating the analgesic effect of 10 mg/kg morphine. These results show that morphine acts dose-dependently on opioid receptors to reverse behavioural changes associated with a noxious event in zebrafish. These results are consistent with other studies on zebrafish and confirm the robustness of the acetic acid-zebrafish model in testing analgesic drugs.