Adult Zebrafish (Danio rerio): An Alternative Behavioral Model of Formalin-Induced Nociception

Abstract

The zebrafish (Danio rerio) has been proposed as a low-cost and simple alternative to the use of higher vertebrates in laboratory research on novel compounds with antinociceptive potential. In this study, we tested adult zebrafish (Danio rerio) as an alternative behavioral model of formalin-induced nociception. We evaluated the nociceptive effect of 0.1% formalin (3 or 5 μL; intramuscularly [i.m.]), applied into the tail or lips, on locomotor activity, using as parameter the number of times the fish crossed the lines between the quadrants of a glass Petri dish during the neurogenic stage (0-5 min) and the inflammatory stage (15-30 min). The behavioral model was validated by testing the antinociceptive effect of morphine and indomethacin (standard analgesic drugs used in the formalin test of rodents). We also tested whether the effect of morphine could be modulated by naloxone, an opioid antagonist. The effect of morphine and indomethacin on zebrafish locomotor behavior was evaluated with the open field test. The white/black test was used to rule out the anxiolytic effect of 0.1% formalin injected into the tail on adult zebrafish. Formalin (0.1%; 3 and 5 μL injected into the tail) increased significantly the nociceptive behavior of the adult zebrafish in both stages (p < 0.001 vs. control). Morphine and indomethacin (both 0.2 mg/mL; 20 μL; intraperitoneally [i.p.]) significantly inhibited nociception induced with formalin (5 μL injected i.m. into the tail) in both stages (p < 0.001). Naloxone blocked the antinociceptive effect of morphine. No influence on locomotion was observed. Locally administered formalin (injected into the tail) induced nociception, but not anxiety. The results suggest that the adult zebrafish behavioral model is a feasible alternative to more conventional laboratory models used in research on novel compounds with antinociceptive potential.

Respiratory depression and analgesia by opioid drugs in freely-behaving larval zebrafish

“…Although pain circuit activity and its response to opioids cannot be directly assessed, the swimming escape response to nociceptive stimuli can be easily assessed in larval zebrafish. Swimming response to chemicals, such as formalin (Magalhaes et al, 2017) or acetic acid (Lopez-Luna, Al-Jubouri, Al-Nuaimy, & Sneddon, 2017) can be measured, as well as response to thermal stimuli (Malafoglia et al, 2014). Here, we propose to determine whether respiratory depression and analgesia can be measured in larval zebrafish.…”

Ethical considerations in fish research

“…Opioids inhibit neurotransmitter release pre‐synaptically thereby blocking nociceptors but these drugs also block central transmission. Several studies have shown morphine is an effective analgesic in O. mykiss (Baker et al ., ; Jones et al ., ; Sneddon, ) and other species ( e.g ., goldfish Carassius auratus (L. 1758); Nordgreen et al ., ; D. rerio , Lopez‐Luna et al ., ,b,c,d; Magalhaes et al ., ; Schroeder & Sneddon, ; Taylor et al ., ). Morphine has a pharmocokinetically similar action in fishes compared with mammals, however, morphine persists for a prolonged period after administration due to slower excretion rates (half‐life 37 h; Newby et al ., , ).…”

“…() tested the effect of flunixin, ketorolac and ketoprofen in O. mykiss , none of which appeared to be effective at ameliorating an increase in opercular beat rate 2 h after surgery at the doses employed in the study. Indomethacin injected intraperitoneally (20 μl of 0.2 mg ml −1 ) did reduce pain related responses in adult D. rerio treated with formalin (Magalhaes et al ., ). Aspirin administered via immersion prevented the reduced activity seen in adult and larval D. rerio when subjected to noxious events (Lopez‐Luna et al ., ,b,c,d; Schroeder & Sneddon, ).…”

“…Drugs with analgesic properties could be employed during any invasive procedures such as invasive tagging, surgery, cannulation, exposure to noxious chemicals or after damage to the fish occurs via factors such as net abrasion or injuries from aggressive encounters or diseases. The major classes of analgesic drugs are opioids, nonsteroi-Several studies have shown morphine is an effective analgesic in O. mykiss(Baker et al, 2013;Jones et al, 2012;Sneddon, 2003b) and other species (e.g., goldfish Carassius auratus (L. 1758);Nordgreen et al, 2009; D. rerio, Lopez-Luna et al, 2017a,b,c, d;Magalhaes et al, 2017;Schroeder & Sneddon, 2017;Taylor et al, 2017). Morphine has a pharmocokinetically similar action in fishes compared with mammals, however, morphine persists for a prolonged period after administration due to slower excretion rates (half-life 37 h;Newby et al, 2006Newby et al, , 2009.…”

Mapping of Morphine-Induced OPRM1 Gene Expression Pattern in the Adult Zebrafish Brain

“…The morphine treatment procedure was chosen based on a protocol previously reported (Stewart et al, 2011). As morphine is an analgesic, it does not cause any form of pain or suffering to the fish, and the doses used are those utilized in previous studies that do not cause toxicity (Magalhães et al, 2017;Chatigny et al, 2018). Therefore, no major effects from the treatments on fish were expected and only normal healthy fish were used in this study.…”