Phenotypic chemical and mutant screening of zebrafish larvae using an on-demand response to electric stimulation

Abstract

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.

A vertebrate model to reveal neural substrates underlying the transitions between conscious and unconscious states

“…Electrical stimulation can be applied to the larvae in a defined, constant burst 38 , 39 that can be varied to explore dose–response effects. ELEC allows a higher throughput with less operator bias than other noxious stimuli, and will allow simultaneous imaging and mapping of neural pathways, so that comparisons to the TAP reflex pathways 40 can occur.…”

Visuomotor deficiency in panx1a knockout zebrafish is linked to dopaminergic signaling

“…Fish Husbandry and embryo collection. Zebrafish (Danio rerio) of strain Tupfel long fin (TL) were used to generate the panx1a −/− mutant line 62 . The fish were maintained in a recirculation system (Aquaneering Inc., San Diego, CA) at 28 °C on a 14 hours light/ 10 hours dark cycle.…”

Modeling Lysosomal Storage Diseases in the Zebrafish

“…Given the frequent use of transgenic zebrafish in imaging-based screens, it can be crucial that larvae remain stationary and consistently oriented across all wells. Technologies for optimizing larval positioning include the SideView plate that facilitates lateral imaging (Rovira et al, 2011), 3D printing of customizable molds for agarose-filled multiwells (Wittbrodt et al, 2014), microfluidic device for head immobilization that leaves the tail region free for behavioral monitoring (Khalili et al, 2019), and the VAST BioImager Platform (Union Biometrica) that positions individual larva in a capillary for imaging (Pardo-Martin et al, 2010;Chang et al, 2012;Pardo-Martin et al, 2013). Coupling of the VAST platform with confocal microscopy enabled high throughput imaging of myelinating oligodendrocytes in transgenic zebrafish larvae, which was expanded into a chemical screen that identified several enhancers of myelinating oligodendrocyte formation (Early et al, 2018).…”