From the Cover: Zebrafish Larvae Are Insensitive to Stimulation by Cocaine: Importance of Exposure Route and Toxicokinetics

Abstract

Zebrafish (Danio rerio) larvae have been suggested as vertebrate model to complement or even replace mammals for rapidly assessing behavioral effects of psychoactive drugs. Yet, divergent responses have been reported in mammals and fish despite the conservation of many drug targets. Cocaine, eg, acts as stimulant in mammals but no such response has been documented for zebrafish larvae. We hypothesized that differences in exposure routes (inhalation or injection in mammals vs waterborne in fish) may be a reason for differences in behavioral responses. We characterized cocaine toxicokinetics by liquid chromatography-mass spectrometry and found its rapid uptake into larvae. We used Matrix-assisted laser desorption ionization-mass spectrometry imaging for the first time to characterize internal distribution of cocaine in zebrafish larvae. Surprisingly, eyes accumulated the highest amount of cocaine and retained most of it even after 48 h depuration. We attribute this to trapping by pigment melanin, a thus far little explored mechanism that may also be relevant for other basic drugs. Cocaine also reached the brain but with levels similar to those in trunk indicating simple passive diffusion as means of distribution which was supported by toxicokinetic models. Although brain levels covered those known to cause hyperactivity in mammals, only hypoactivity (decreased locomotion) was recorded in zebrafish larvae. Our results therefore point to cocaine's anesthetic properties as the dominant mechanism of interaction in the fish: upon entry through the fish skin and gills, it first acts on peripheral nerves rapidly overriding any potential stimulatory response in the brain.

Behavioral screening of the LOPAC1280 library in zebrafish embryos

“…Similar to our findings within embryonic zebrafish, a recently published study demonstrates that, even within larval zebrafish harboring a more complex nervous system, the observed behavioral response is not always consistent with the expected behavioral response based on the known mechanism and mode of action within mammals (Kirla et al, 2016). Within this study, the authors relied on cocaine as a model compound, as cocaine acts on the monoaminergic neurotransmitter systems and is a stimulant within mammals.…”

“…Within this study, the authors relied on cocaine as a model compound, as cocaine acts on the monoaminergic neurotransmitter systems and is a stimulant within mammals. However, contrary to the expected outcome (hyperactivity), acute exposure to non-teratogenic concentrations of cocaine resulted in a concentration-dependent decrease in locomotion (hypoactivity) within both dark and light conditions following a waterborne exposure of cocaine (Kirla et al, 2016). Importantly, the authors also quantified cocaine uptake and distribution using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI), and observed significant accumulation within the brain, eyes, and trunk of larval zebrafish.…”

“…Importantly, the authors also quantified cocaine uptake and distribution using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI), and observed significant accumulation within the brain, eyes, and trunk of larval zebrafish. Therefore, the authors concluded that cocaine-induced hypoactivity was as a result of systemic (rather than targeted) cocaine uptake across the skin of larval zebrafish, leading to an anesthetic effect on the peripheral nervous system that suppressed stimulatory targets present in the central nervous system (Kirla et al, 2016). Likewise, within our assay, there is a strong possibility that, for compounds expected to act as stimulants, waterborne exposure may have led to non-targeted, systemic uptake and distribution that overwhelmed the intended target and significantly biased behavior (spontaneous activity) toward an unidirectional hypoactive response.…”

Cocaine Accumulation in Zebrafish Eyes Leads to Augmented Amplitudes in the Electroretinogram

“…Cocaine acts on monoaminergic neurotransmission and is a central nervous system stimulant in mammals, while in zebrafish it resulted in a decrease of locomotor activity [3]. As suggested by [4], this may be due to the local anesthetic effect of the drug acting on the peripheral nervous system before it reaches its targets in the brain. This work highlighted the importance of considering the application route when comparing different experimental species.…”

“…This work highlighted the importance of considering the application route when comparing different experimental species. Another intriguing aspect discovered in cocaine exposed zebrafish larvae was a strong and sustained accumulation of cocaine in the eye, likely due to a high affinity of cocaine to melanin [4]. Cocaine is known to inhibit monoaminergic neurotransmitter reuptake, leading to an increased synaptic concentration of these neurotransmitters [5].…”