Investigating Opioid‐induced Respiratory Depression and Analgesia Using Larval Zebrafish

Introduction Opioid drugs present the severe side‐effect of respiratory depression which can be lethal. In fact, every year, in North America, over 70,000 people die of opioid overdose. Our current understanding is limited due to a lack of simple and amenable animal models to study the effects of opioid drugs on the nervous system. We aim to understand respiratory depression and analgesia by opioid drugs using novel zebrafish models, allowing for high throughput drug screening and gene editing. Using these models, we will demonstrate whether respiratory depression can be reversed by serotonergic agonists, glutamatergic modulators and calcium channel activators. Methods We have established phenotype‐based approaches using in‐vivo zebrafish models of respiratory depression and analgesia by fentanyl, a clinically relevant opioid analgesic. To quantify respiratory depression, we measured respiratory activity by recording mandible movements in 12–14 day post‐fertilization larvae. To quantify analgesia, we measured the escape swimming response to nociceptive stimuli such as formalin and administered fentanyl to induce analgesia. To show that μ‐opioid receptors (MORs) are expressed in respiratory circuits in the brainstem, we used in‐situ hybridization for Oprm1 (mRNA of the MOR). Results In comparing respiratory sensitivity to opioids between zebrafish strains, we showed that Tübingen (TU) fish did not respond to fentanyl (1μM, P=0.4240 , n=9), whereas AB fish showed pronounced respiratory rate depression (54±7.7% decrease, P<0.001 , n=11). Zebrafish crossed between AB and TU did not show respiratory depression ( P=0.104 , n=7). In AB zebrafish, a dose‐dependent decrease in respiratory rate was observed, with 1μM fentanyl showing the most potent respiratory depression ( P=0.01 , n=11). The opioid receptor antagonist naloxone (5μM) or the selective MOR antagonist CTAP (4μM) reversed respiratory depression to baseline levels ( P=0.002 , n=8). Similarly, respiratory depression was reversed by the positive allosteric modulator of AMPA receptors CX614 (50μM, P=0.187 , n=10) or the serotonergic 5‐HT 4 agonist BIMU8 (10μM, P=0.024 , n=6). To test the role of N‐type calcium channels, we showed that nefiracetam, a calcium channel activator, reversed respiratory depression by fentanyl (57±19%, P=0.002 , n=9). Formalin (0.05%) significantly increased swimming velocity (320±313% of baseline, P=0.003 , n=24–30), which was reduced by fentanyl (3μM, 117±151%, n=10). The effect of fentanyl was blocked by naloxone (5μM, P=0.001 , n=16) and CTAP (4μM, P=0.029 , n=16). MOR mRNA was expressed in the medulla where respiratory circuits are located. Discussion Our models show that respiratory depression and analgesia by opioids can be mimicked in zebrafish larvae. The strain differences suggest a potential genetic protection against respiratory depression by opioids which can be uncovered using gene screening and knockout animals. Our novel zebrafish models are powerful models to investigate opioid‐induced respiratory depression and analgesia. Using them, we identified potential molecular targets to develop safe opioid pain therapies, such as calcium channels. Support or Funding Information St. Michael’s Hospital Foundation and the J.P. Bicknell Foundation Biomedical Grant

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