Behavioral responses of zebrafish larvae to environmental cues are important functional readouts that should be evoked on-demand and studied phenotypically in behavioral, genetical and developmental investigations. Very recently, it was shown that zebrafish larvae execute a voluntary and oriented movement toward the positive electrode of an electric field along a microchannel. Phenotypic characterization of this response was not feasible due to larva’s rapid movement along the channel. To overcome this challenge, a microfluidic device was introduced to partially immobilize the larva’s head while leaving its mid-body and tail unrestrained in a chamber to image motor behaviors in response to electric stimulation, hence achieving quantitative phenotyping of the electrically evoked movement in zebrafish larvae. The effect of electric current on the tail-beat frequency and response duration of 5–7 days postfertilization zebrafish larvae was studied. Investigations were also performed on zebrafish exposed to neurotoxin 6-hydroxydopamine and larvae carrying a pannexin1a (panx1a) gene knockout, as a proof of principle applications to demonstrate on-demand movement behavior screening in chemical and mutant assays. We demonstrated for the first time that 6-hydroxydopamine leads to electric response impairment, levodopa treatment rescues the response and panx1a is involved in the electrically evoked movement of zebrafish larvae. We envision that our technique is broadly applicable as a screening tool to quantitatively examine zebrafish larvae’s movements in response to physical and chemical stimulations in investigations of Parkinson’s and other neurodegenerative diseases, and as a tool to combine recent advances in genome engineering of model organisms to uncover the biology of electric response.

Zebrafish or Danio rerio is an established model organism for studying the genetic, neuronal and behavioral bases of diseases and for toxicology and drug screening. The embryonic and larval stages of zebrafish have been used extensively in fundamental and applied research due to advantages offered such as body transparency, small size, low cost of cultivation and high genetic homology with humans. However, the manual experimental methods used for handling and investigating this organism are limited due to their low throughput, labor intensiveness and inaccuracy in delivering external stimuli to the zebrafish while quantifying various neuronal and behavioral responses. Microfluidic and lab-on-a-chip devices have emerged as ideal technologies to overcome these challenges. In this review paper, the current microfluidic approaches for investigation of behavior and neurobiology of zebrafish at embryonic and larval stages will be reviewed. Our focus will be to provide an overview of the microfluidic methods used to manipulate (deliver and orient), immobilize and expose or inject zebrafish embryos or larvae, followed by quantification of their responses in terms of neuron activities and movement. We will also provide our opinion in terms of the direction that the field of zebrafish microfluidics is heading toward in the area of biomedical engineering.

Background Microfluidic devices are being used for phenotypic screening of zebrafish larvae in fundamental and pre‐clinical research. A challenge for the broad use of these microfluidic devices is their low throughput, especially in behavioral 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. Methods and Results Multiple parameters including loading and testing time per fish and loading and orientation efficiencies were refined to optimize the performance of modified designs. Flow and electric field simulations 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 ≈60%. Further, loading and orientation efficiencies increased by more than 80%. Critical behavioral parameters such as response duration and tail beat frequency were similar in both single and quadruple‐fish devices. Conclusion The developed 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.

Multi-phenotypic screening of zebrafish larvae, such as monitoring the heart and tail activities, is important in biological assays. Microfluidic devices have been developed for zebrafish phenotypic assays, but simultaneous lateral–dorsal screening of the same larva in a single chip is yet to be achieved. We present a multi-phenotypic microfluidic device for monitoring of tail movement and heart rate (HR) of 5–7-day postfertilization zebrafish larvae. Tail movements were stimulated using electric current and quantified in terms of response duration (RD) and tail beat frequency (TBF). The positioning of a right-angle prism provided a lateral view of the larvae and enabled HR monitoring. Investigations were performed on zebrafish larvae exposed to 3% ethanol, 250 μM 6-hydroxydopamine (6-OHDA) or 1 mM levodopa. Larvae exposed to ethanol showed a significant drop in HR, whereas electric stimulation increased the HR temporarily. Larvae experienced a significant drop in RD, TBF and HR when exposed to 6-OHDA. HR was not affected by levodopa post-treatment, whereas RD and TBF were restored to normal levels. The results showed potential for applications that involve monitoring of cardiac and behavioral parameters in zebrafish larvae. Tests can be done using the same chip, without changing the larvae’s orientation. This eliminates undue stress caused by reorientation, which may affect their behavior, and the use of separate devices to obtain dorsal and lateral views. The device can be implemented to improve multi-phenotypic and quantitative screening of zebrafish larvae in response to chemical and physical stimuli in different zebrafish disease models.

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