Zebrafish Models to Understand Respiratory Depression and Analgesia by Opioids and to Identify Safe Opioid Pain Therapies

Introduction Opioid drugs are the mainstay of pain management, but their use is limited by their severe side‐effects that can be lethal with overdose. Indeed, opioid drugs induce respiratory depression, that can lead to severe hypoxemia and respiratory arrest when opioids are abused. The current antidote naloxone (Narcan) is a life‐saving therapy, but its use is limited because it can only be given after the overdose occurs, so it is not a preventive treatment. The main challenge in opioid drug discovery is therefore to develop new opioid therapies with potent analgesia but reduced respiratory depression, so opioids can be safely prescribed. Objectives To accelerate drug discovery, we established phenotype‐based approaches using in vivo zebrafish models of respiratory depression and analgesia. Zebrafish is an amenable model to study respiratory depression because its respiratory circuits are similar to mammalian circuits. Also, zebrafish μ‐opioid receptors have 70% homology of amino acids with their mammalian counterparts. Our aim was to developed a high‐throughput screening platform that combines drug screening and behavioural profiling so new preventive therapies can be identified. Methods To determine respiratory depression, we assessed buccal movements, as an index of respiratory activity, in zebrafish larvae (day post‐fertilization 14), and its response to the μ‐opioid receptor analgesic fentanyl. We used a video‐recording system to assess zebrafishes in multi‐well plate. To assess opioid analgesia, we induced mild pain in zebrafish larvae by submerging it in a solution of formalin, or formalin/fentanyl, and measuring its subsequent locomotor or swimming response. Results Fentanyl (0.02 μM) significantly decreased the rate of buccal movements by 84% (baseline 41.7 breath/min, fentanyl 6.71 breath/min, n=11, p=0.038), a depression reversed by naloxone (5 μM, 44.3 breath/min, p=0.01). Similarly, respiratory depression by fentanyl was reversed by the AMPA receptor modulator ampakine CX‐614 (50 μM, p=0.04, n=10) and the 5‐HT4 agonist BIMU‐8 (10 μM, n=6, p=0.035). Formalin (0.05%) increased increased locomotion, and this response was significantly reduced by fentanyl (1 μM), an analgesic effect blocked by naloxone (5 μM). Discussion Our novel and unique zebrafish models mimicked well the effects of opioid drugs on respiratory activity and nociception observed in mammals. This proof‐of‐principle study suggests that zebrafish can be used for phenotype‐based high‐throughput drug and gene screening. Using these assays, we will knock‐down key‐genes involved in opioid inhibition using morpholino oligonucleotides, and test chemical screens to identify preventive therapies to minimize respiratory depression by opioids while preserving their analgesic properties. Support or Funding Information St. Michael's Hospital Foundation This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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