Differential effects of dopamine receptor agonists ropinirole and quinpirole on locomotor and anxiolytic behaviors in larval zebrafish (Danio rerio): A role for the GABAergic and glutamate system?

Zebrafish are frequently used as a vertebrate model to elucidate toxicological and pharmacological mechanisms of action in the central nervous system. Pharmacological studies demonstrate that dopamine, signaling via several receptor subtypes, regulates zebrafish larval behavior. Quinpirole is a selective dopamine receptor agonist for D2 and D3 subtypes while ropinirole exhibits selectivity toward D2, D3, and D4 receptors. The main objective of this study was to determine the short-term actions of quinpirole and ropinirole on the locomotor activity and anxiolytic/anti-anxiolytic behaviors of zebrafish. Furthermore, dopamine signaling can cross talk with other neurotransmitter systems, including the GABAergic and glutamatergic system. As such, we measured transcriptional responses in these systems to determine whether dopamine receptor activation modulated GABAergic and glutaminergic systems. Ropinirole reduced locomotor activity of larval fish at concentrations of 1 µM and greater but quinpirole did not affect locomotor activity at all concentrations tested. Anxiolytic-related behaviors were also compared between the two pharmaceuticals. Noteworthy was that both dopamine receptor agonists at 1 µM increased the activity of zebrafish in the light phase of a light-dark preference test, which may be related to the activation of D2 and/or D3 receptors. In terms of interactions with other neurotransmitter systems, ropinirole up-regulated transcripts in larvae zebrafish related to both the GABAergic and glutamatergic systems (abat, gabra1, gabrb1, gad1b, gabra5, gabrg3, and grin1b). Conversely, quinpirole did not alter the abundance of any transcript measured, suggesting that dopamine-GABA interaction may involve D4-receptors, which has been noted in mammalian models. This study demonstrates pleiotropic actions of dopamine agonism on the GABA and glutamate system in larval zebrafish. This study has relevance for characterizing toxicants that act via dopamine receptors and for elucidating mechanisms of neurological disorders that involve motor circuits and multiple neurotransmitter systems, like Parkinson's disease.

Developmental and behavioral toxicity assessment of glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) in zebrafish embryos/larvae.

The relative toxicity of glyphosate (GLY) and its metabolite aminomethylphosphonic acid (AMPA) to zebrafish were compared. Embryos/larvae were exposed to one dose of either GLY (0.1, 1, or 10 µM), AMPA (0.1, 1, or 10 µM), or a 1 µM mixture for 7-days post-fertilization. Survival, success of hatch, and deformity frequency were not different from controls. Neither chemical induced reactive oxygen species in larval fish. GLY increased superoxide dismutase 2 mRNA in larvae while AMPA increased catalase and superoxide dismutase 1 in a concentration-specific manner. GLY increased cytochrome c oxidase subunit 4 isoform 1 and citrate synthase mRNA in larvae while AMPA decreased cytochrome c oxidase I and increased 3-hydroxyacyl CoA dehydrogenase transcripts. Hyperactivity was noted in fish treated with GLY, but not AMPA nor the mixture. Anxiety-like behaviors were absent with exposure to GLY or AMPA. GLY and AMPA may exert different effects at the molecular and behavioral level.

Molecular and behavioral assessment in larval zebrafish (Danio rerio) following exposure to environmentally relevant levels of the antineoplastic cyclophosphamide.

Antineoplastics treat cancers and enter aquatic ecosystems through wastewater and hospital effluent. Risks associated with antineoplastics are not well characterized in aquatic organisms. We conducted zebrafish embryo/larvae toxicity assays to evaluate responses to cyclophosphamide (0.01-50 µM). Zebrafish survival was affected by 5 µM cyclophosphamide and deformities were noted at > 1 µM. Oxidative respiration remained unchanged in embryos with exposure up to 200 µM. Reactive oxygen species were not increased by 50 µM cyclophosphamide exposure. More than 15 oxidative stress and immune-related transcripts were measured. Superoxide dismutase 2 and heat shock protein 70 and 90a were induced in larvae by cyclophosphamide. Immune-related transcripts were assessed due to immunosuppressive properties of cyclophosphamide, and mmp9 and myd88 levels were altered in expression. Hyperactivity of larvae was noted following 5 µM cyclophosphamide exposure. There was no change in anxiety-related endpoints (light-dark preference). Risks for larval fish exposed to cyclophosphamide in the environment may be low.

Assessing sub-lethal effects of the dinitroaniline herbicide pendimethalin in zebrafish embryos/larvae (Danio rerio).

Pendimethalin is a dinitroaniline herbicide used to control broadleaf weeds by inhibiting the formation of microtubules during cell division. Its use on a variety of crops leads to its potential entry into aquatic environments, but little is known about its sub-lethal toxicity to early developmental stages of aquatic vertebrates. To address this knowledge gap, we assessed the toxicity of pendimethalin to zebrafish embryos and larvae by measuring mortality, developmental abnormalities, oxidative respiration, reactive oxygen species, gene expression, and locomotor activity following exposure to the herbicide throughout early development. Embryos at ~6 h post-fertilization (hpf) were exposed to either a solvent control (0.1% DMSO, v/v), embryo rearing medium (ERM), or one dose of either 1, 2.5, 5, or 25 µM pendimethalin for up to 7-days post fertilization depending on the bioassay. Exposure to 25 µM pendimethalin resulted in high prevalence of spinal curvature, tail malformations, pericardial edema, and yolk sac edema at 4 dpf, while exposure to 5 µM pendimethalin induced pericardial edema and lordosis in the fish exposed over 7 dpf. Exposure to pendimethalin up to 5 µM did not negatively impact oxidative respiration (e.g., basal respiration, oligomycin-induced ATP production) in embryos following a 24-h exposure. Pendimethalin did not induce reactive oxygen species at concentrations of 1-5 µM. Levels of transcripts associated with oxidative respiration and damage response were altered in 7d-larvae; cox1 mRNA was increased in larvae fish exposed to 1 µM while cox5a1 and sod2 mRNA were decreased with 2.5 µM exposure. The Visual Motor Response (VMR) test for light-dark response revealed that larval activity in the dark period was reduced for zebrafish exposed to >1 µM pendimethalin compared to ERM and DMSO solvent control groups. These data inform on the sub-lethal toxicity of pendimethalin to early stages of fish embryos and larvae.

Exposure to acetochlor impairs swim bladder formation, induces heat shock protein expression, and promotes locomotor activity in zebrafish (Danio rerio) larvae.

Acetochlor is one of the most widely used herbicides in the world, however, there are few data on the sub-lethal effects of acetochlor on early developmental stages of fish. To address this, we measured survival, deformity, swim bladder formation, embryo oxygen consumption rates, reactive oxygen species (ROS) levels, transcripts (related to swim bladder formation, oxidative damage response, and apoptosis) and behavior responses following exposure to acetochlor (0.001 µM up to 125 µM). Exposure to acetochlor at concentrations 50 µM and above exerted 100% mortality after 3 dpf, and significantly reduced the size of the swim bladder (25 µM). In embryos, basal respiration, oligomycin-induced ATP production, and maximal respiration were decreased 30-60% following a 24 h exposure to 125 µM acetochlor. Acetochlor did not affect ROS levels up to 25 µM in larvae with acute exposure. Acetochlor at 25 µM increased mRNA levels of bax1, hsp70, and hsp90a by ~4-fold in larval zebrafish. In both the visual motor response and light-dark preference test, 25 µM acetochlor increased locomotor activity of larval fish. At lower exposure concentrations, 100 and 1000 nM acetochlor increased the mean time spent in the dark zone, suggesting promotion of anxiolytic behavior. This study presents a comprehensive evaluation of sublethal effects of acetochlor, spanning molecular responses to behavior, which can be used to refine risk assessment decisions for aquatic environments.