Zebrafish movement before and after TAP and ELEC. (A) The zebrafish were behaviorally equilibrated within the chambers used to induce stimuli. Prior to a stimulus (1) the distance moved over 2 s was stable in both chambers, which the circular tap wells having a higher average distance moved when compared to the rectangular ELEC chamber. At the 0 time point, the zebrafish larvae were stimulated with either tap or electrical shock. The following 36–40 s (block box magnification) the zebrafish with the TAP (green) moved normally whereas the ELEC demonstrated a decrease in movement (purple). (B) When averaging the 2 s of distance moved for the 120 s prior to the stimulus (1), the 40 s immediately following the stimulus (2) and 40–90 s after the stimulus, there is no significant difference in movement for the TAP (green). However, for the ELEC (purple), there is a statistically significant decrease in movement following the stimulus but the larvae recover back to baseline movement after approximately 40 s. Five replicates where assessed with 8–12 larvae per replicate.

Hill plots of the inhaled and IV anesthetics. Zebrafish respond to inhaled anesthetics, halothane (A), isoflurane (B) and sevoflurane (C), and IV anesthetics, propofol (D) and etomidate (E), at physiological concentrations. For all anesthetics, the SPONT (blue) is lost at a lower concentration as compared to the TAP (green). For the IV anesthetics, ELEC (purple) is also lower than SPONT. Interestingly, although propofol TAP (green) and ELEC (purple), are similar, etomidate requires a sevenfold higher dose of drug for TAP versus ELEC. All graphs were created with 3–8 replicates per point and 15–18 zebrafish larvae used per replicate.

Ketamine hill plots and absorption. (A, B) Change in behavior for SPONT (blue), TAP (green) and ELEC (purple) required very high doses of ketamine at pH 7.2 (A) versus pH 8 (B). For the TAP (A, green), complete loss of movement was unable to be attained. Graph was created with a minimum of three replicates per point and 8–12 zebrafish larvae used per replicate. Ketamine at pH 8 (B) requires a fourfold higher concentration for TAP versus ELCT. (C) We used HPLC to determine the ketamine concentration in the zebrafish larvae over time. We found a significantly higher concentration of ketamine at pH 8 versus pH 7.2. Additionally, this shows that the ketamine equilibrated at approximately 30 min. Each point was replicated five times and contained 20–25 larval zebrafish per replicate.

Hill plots of a sedative drug. Zebrafish respond to dexmedetomidine at physiological concentrations for loss of SPONT (A, light blue circle). This response is able to be reversed using an alpha-2 adrenergic antagonist, atipamezole (A, dark blue square) at 100 µM. There is a decrease in movement seen in the ELEC (A, purple triangle). However, even at the highest doses, we were unable to achieve complete loss of movement. (B) There is no significant difference seen with this drug using the TAP (p = 0.4). All graphs were created with 3–8 replicates per point and 8–12 zebrafish larvae used per replicate.

Electrical stimulation apparatus made to create a low amplitude, consistent noxious stimulus. (A) The box was 3D printed using free software (TinkerCad) with five chambers. The first and last chamber held a gold pin for the electrical current. The middle three chambers were for zebrafish larvae. The chambers were separated by a semi-permeable cloth membrane (dotted lines). This allowed liquid and, therefore, current, to flow but did not allow the larvae to move between chambers. (B) The apparatus (red arrowhead) attached to the Grass stimulator (black arrowhead) was placed within a Noldus DanioVision behavior box (black arrow) and a video of their movement was recorded (red arrow). (C) The zebrafish larvae moved 93.3% ± 6.1% of the time to the ELEC stimulus. n = 5 replicates with 12 zebrafish per replicate. (D, E) Two separate boxes were fabricated and there was no significant difference between the µAmps generated in the lanes of the two boxes. n = 3 replicates.