Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play essential roles in purinergic signaling in the nervous system. Several studies suggest a link between Panx1-based channels activity and neurodegenerative disorders including Parkinson’s disease (PD), but experimental evidence is limited. Here, we applied behavioral and molecular screening of zebrafish larvae to examine the role of Panx1 in both pathological and normal conditions, using electrical stimulation in a microfluidic chip and RT-qPCR. A zebrafish model of PD was produced by exposing wildtype (panx1a+/+) and Panx1a knock-out (panx1a-/-) zebrafish larvae to 250µM 6-hydroxydopamine (6-OHDA). After 72hrs treatment with 6-OHDA a reduced electric-induced locomotor activity was observed in 5 days post fertilization (dpf) panx1a+/+ larvae. The 5dpf panx1a-/- larvae were not different from affected. The RT-qPCR data showed a reduction in tyrosine hydroxylase (TH) expression level in both panx1a+/+ and panx1a-/- groups. However, TH expression of 6-OHDA exposed panx1a-/- larvae was not decreased when compared to untreated mutants. Extending 6-OHDA treatment duration to 120hrs caused a significant reduction in the locomotor response of 7dpf panx1a-/- larvae when compared to the untreated panx1a-/- group. The RT-qPCR data also confirmed a significant decrease in TH expression levels after 120hrs treatments with 6-OHDA for both genotypes. Our results suggest that the absence of Panx1a channels compromised dopaminergic signaling in 6-OHDA-treated zebrafish larvae. We here propose that zebrafish Panx1a models offer great opportunities to shed light on the physiological and molecular basis of PD. Panx1a might play a preventive role on PD progression, and therefore deserves further investigation

Multi-phenotypic screening of multiple zebrafish larvae plays an important role in enhancing the quality and speed of biological assays. Many microfluidic devices have been presented for zebrafish phenotypic assays, but multi-organ screening of multiple larvae, from different needed orientations, in a single device that can enable rapid and large-sample testing is yet to be achieved. Here, we propose a multi-phenotypic quadruple-fish microfluidic chip for simultaneous monitoring of fin movement and heart activity of 5–7-day postfertilization zebrafish larvae trapped in the chip. In each experiment, fin movements of four larvae were quantified in the dorsal view in terms of fin beat frequency (FBF). Positioning of four optical prisms next to the traps provided the lateral views of the four larvae and enabled heart rate (HR) monitoring. The device’s functionality in chemical testing was validated by assessing the impacts of ethanol on heart and fin activities. Larvae treated with 3% ethanol displayed a significant drop of 13.2% and 35.8% in HR and FBF, respectively. Subsequent tests with cadmium chloride highlighted the novel application of our device for screening the effect of heavy metals on cardiac and respiratory function at the same time. Exposure to 5 μg/L cadmium chloride revealed a significant increase of 8.2% and 39.2% in HR and FBF, respectively. The device can be employed to improve quantitative multi-phenotypic screening of zebrafish larvae in response to chemical stimuli in various chemical screening assays, in applications such as ecotoxicology and drug discovery.

The signaling molecular mechanisms in zebrafish response to electricity are unknown, so here we asked if changes to dopaminergic signaling pathways can affect their electrically-evoked locomotion. To answer this question, the effects of multiple selective and non-selective dopamine compounds on the electric response of zebrafish larvae is investigated. A microfluidic device with enhanced control of experimentation with multiple larvae is used, which features a novel design to immobilize four zebrafish larvae in parallel and expose them to electric current that induces tail locomotion. In 6 days post-fertilization zebrafish larvae, the electric induced locomotor response is quantified in terms of the tail movement duration and beating frequency to discern the effect of non-lethal concentrations of dopaminergic agonists (apomorphine, SKF-81297, and quinpirole), and antagonists (butaclamol, SCH-23390, and haloperidol). All dopamine antagonists decrease locomotor activity, while dopamine agonists do not induce similar behaviours in larvae. The D2- like selective dopamine agonist quinpirole enhances movement. However, exposure to non-selective and D1-selective dopamine agonists apomorphine and SKF-81297 cause no significant change in the electric response. Exposing larvae that were pre-treated with butaclamol and haloperidol to apomorphine and quinpirole, respectively, restores electric locomotion. The results demonstrate a correlation between electric response and the dopamine signalling pathway. We propose that the electrofluidic assay has profound application potential as a chemical screening method when investigating biological pathways, behaviors, and brain disorders.

We previously showed that electric current can cause zebrafish larvae to move towards the anode pole along a microchannel. For a semi-mobile larva, we observed that zebrafish response to electricity depended on the current magnitude. The effects of electric signal direction, voltage magnitude and habituation to repeated exposures to electric pulses were not characterized. Here, this knowledge gap was addressed by exploiting these parameters in a microfluidic device with a head-trap to immobilize a zebrafish larva and a downstream chamber for tail movement and phenotypic characterization of response duration (RD) and tail beat frequency (TBF). We first assessed larvae’s response to electric current direction (at 3µA) and voltage magnitude. Changing the current direction significantly altered the RD and TBF with long and low-frequency responses seen when the anode was positioned at larvae’s tail. The electric voltage drop across the fish body had a significant effect on larvae’s locomotion with long RD and low TBF observed at 5.6V in the range of 1.3-9V. We also demonstrated that the zebrafish locomotor response to repeated 3µA current pulses diminished with dependency on the interstimulus interval. However, the diminished response was fully recovered after a 5-min resting period or introduction of a novel light stimulus (i.e. habituation-dishabituation strategy). Therefore, electric response suppression in zebrafish was attributed to the habituation as a form of non-associative learning. Our microfluidic platform has broad application potential in behavioral neuroscience to study cognitive phenotypes, fundamental studies on the biological roots of electric response, and pharmacological screening.

Microfluidic devices have been introduced for phenotypic screening of zebrafish larvae in both fundamental and pre-clinical research. One of the remaining challenges for the broad use of microfluidic devices is their limited throughput, especially in behavioural assays. Previously, we introduced the tail locomotion of a semi-mobile zebrafish larva evoked on-demand with electric signal in a microfluidic device. Here, we report the lessons learned for increasing the number of specimens from one to four larvae in this device. Multiple parameters including loading and testing time per fish and loading and orientation efficiencies were refined to optimize the performance of modified designs. Simulations of the flow and electric field within the final device provided insight into the flow behavior and functionality of traps when compared to previous single-larva devices. Outcomes led to a new design which decreased the testing time per larva by approximately 60%. Further, loading and orientation efficiencies increased by more than 80%. Critical behavioural parameters such as response duration and tail beat frequency were similar in both single and quadruple-fish devices. The optimized microfluidic device has significant advantages for greater throughput and efficiency when behavioral phenotyping is required in various applications, including chemical testing in toxicology and gene screening.