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.