Allyl Isothiocyanate (Mustard Oil) Mediates Short- and Long-Term Sensitization of Anxiety-Like Behavior in Both Restrained and Free-Swimming Larval Zebrafish (Danio rerio)

Zebrafish (Danio rerio) holds a promise as a model vertebrate system for neurobiological investigations of learning and memory. Here, we sought to demonstrate two forms of nonassociative learning: first, short-term sensitization of tail movements and second, long-term sensitization of thigmotaxis, induced by application of allyl isothiocyanate, a chemical irritant, in both restrained and free-swimming larval zebrafish. We found that allyl isothiocyanate increased both tail movements and tail movements’ duration for several minutes after the chemical irritant was removed from the bath. Moreover, pharmacological manipulations were observed to alter the behavior. Experimental manipulations included exposure to methiothepin, an antagonist of serotonin (5-HT) receptors. Methiothepin application was observed to block MO-induced sensitization of thigmotaxis in larval zebrafish. Our results demonstrate that a noxious stimulus, allyl isothiocyanate, can reliably induced tail movements and thigmotaxis sensitization in both restrained and free-swimming larval zebrafish, and thus facilitate future investigations of in vivo neural circuits

Induction of Short-Term Sensitization by an Aversive Chemical Stimulus in Zebrafish Larvae

Larval zebrafish possess a number of molecular and genetic advantages for rigorous biological analyses of learning and memory. These advantages have motivated the search for novel forms of memory in these animals that can be exploited for understanding the cellular and molecular bases of vertebrate memory formation and consolidation. Here, we report a new form of behavioral sensitization in zebrafish larvae that is elicited by an aversive chemical stimulus [allyl isothiocyanate (AITC)] and that persists for ≥30 min. This form of sensitization is expressed as enhanced locomotion and thigmotaxis, as well as elevated heart rate. To characterize the neural basis of this nonassociative memory, we used transgenic zebrafish expressing the fluorescent calcium indicator GCaMP6 (Chen et al., 2013); because of the transparency of larval zebrafish, we could optically monitor neural activity in the brain of intact transgenic zebrafish before and after the induction of sensitization. We found a distinct brain area, previously linked to locomotion, that exhibited persistently enhanced neural activity following washout of AITC; this enhanced neural activity correlated with the behavioral sensitization. These results establish a novel form of memory in larval zebrafish and begin to unravel the neural basis of this memory