4. Discussion
Previous reports have established that zebrafish larvae display changes in their natural behavior (i.e., behaviour without external stimulation) following exposure to drugs that are known to act on DA receptors in mammals[29,30,32,33,37,38]. In our earlier works, we reported that the acute administration of neuroactive drugs that alter DAergic signaling (e.g., apomorphine, SKF-38393, quinpirole, 6-OHDA and levodopa) caused similar effects on locomotor activity in zebrafish larvae as in mammals[12,16]. The aim of this study was to advance the knowledge about zebrafish larvae’s response to electric field. Using our quadruple fish microfluidic device, the sensitivity of zebrafish larvae’s electric-induced response to different chemicals that target DAergic receptors in mammals was evaluated for the first time, based on two quantitative behavioral readouts of RD and TBF.
Based on the data presented for general locomotion assay, both apomorphine and butaclamol (non-selective DAergic agents) produce their maximum effect after 20 minutes exposure[33]. These nonselective compounds, acting on both D1- and D2- receptors, are commonly described as rapid-acting as the other agonists and antagonists used in our study required longer exposure times. The selective agonists had shorter exposure times than the selective antagonists, with the D2-like drugs taking effect faster than those targeting the D1 receptors.
We benefited from our novel quadruple fish device to examine a possible link between zebrafish’s electric induced response and the DA receptors activities. Exposure to apomorphine, a non-selective DA agonist, had no impact on the electric-induced response (Fig. 3). This may be either due to the drug affecting multiple different receptors[39] or possible saturation of the DA receptors after treatment with this specific dose[40,41]. In contrast, exposure to the non-selective antagonist butaclamol, resulted in a decrease in electric-induced movement (Fig. 3), possibly because of suppression of the DAergic signalling[30], matching observations from the literature for 6 dpf zebrafish larvae[33]and mammals[42–44], suggesting the induction of analogous actions in zebrafish and mammals caused by DA receptor agents. For the first time, we showed that the hypoactivity caused by butaclamol was reversed through subsequent treatment with apomorphine, restoring the locomotor response to match that observed in the control group (Fig. 3).
Exposure to D1 and D2-like selective DA antagonists of SCH-23390 and haloperidol caused lower general locomotor activity in 6 dpf zebrafish larvae[33] and rodents[44–47]. King et al also reported haloperidol having sedative effects on humans[48]. These results suggest a similar drug action mechanism for mammals and zebrafish triggered by the mentioned selective antagonists, possibly due to blockage of either D1- or D2-like receptors. SCH-23390 and haloperidol, reduced electric induced movement of zebrafish larvae in our microfluidic device (Fig. 4 and Fig. 5), consistent with previous findings reported in the literature[33,44–48].
SKF-81297 (a D1-like selective DA agonist) induced no significant effects on general locomotor response when freely swimming 6 dpf zebrafish larvae were treated with the drug[49]. However, the resulting phenotypes for the D1 receptor-deficient mice were not uniform for all studies and did not align with expected observations of D1 receptor ligand treatment[50]. Some researchers reported an increase in general locomotor activity in rodents[51,52]. The effect was less prevalent in younger organisms such as 5-10 days old compared to weaned rats[53]. In contrast other researchers observed no significant difference in general movement of WT weaned mice[54] or Swiss Webster mice[55] when exposed to D1-like receptor agonists. Treatment with SKF-81297 resulted in no significant change in electric-induced locomotor response of zebrafish larvae (Fig. 4), suggesting that D1- DA receptors might not be heavily involved in modulating zebrafish larvae’s electric induced response. The results obtained in our experiments match the previously reported results, showing no significant change in electric induced locomotor response upon exposure to SKF-81297[49,54,55].
Researchers also reported that treatment with quinpirole (a D2-like selective DA agonist), not only stimulated general locomotor activity in zebrafish larvae[31,38], but in rodents as well[44,56]. Similarly, exposure to quinpirole significantly increased the electric induced movement of larvae (Fig. 5), revealing a dominant involvement of D2-like DA receptors in electric induced response.
As another novel aspect of our project, SKF-81297 and quinpirole (DA-selective agonists) were employed to ascertain whether they would restore the disrupted electric induced RD and TBF of larvae pretreated with SCH-23390 and haloperidol (DA-selective antagonists), respectively (Fig. 4 and Fig. 5).
To the best of our knowledge, we demonstrate for the first time that posttreatment with quinpirole restores the electric induced locomotor response, while posttreatment with SKF-81297 did not fully improve activity. We propose a mechanism of electric-induced behavioral response which requires D2-DA receptors and the advanced microfluidic platform as a powerful investigative tool.