Abamectin induces rapid and reversible hypoactivity within early zebrafish embryos.

During early zebrafish embryogenesis, spontaneous tail contractions represent the first sign of locomotion and result from innervation of primary motoneuron axons to target axial muscles. Based on a high-content screen, we previously demonstrated that exposure of zebrafish embryos to abamectin--an avermectin insecticide--from 5-25 hours post-fertilization (hpf) abolished spontaneous activity in the absence of effects on survival and gross morphology. Therefore, the objective of this study was to begin investigating the mechanism of abamectin-induced hypoactivity in zebrafish. Similar to 384-well plates, static exposure of embryos to abamectin from 5-25 hpf in glass beakers resulted in elimination of activity at low micromolar concentrations. However, abamectin did not affect neurite outgrowth from spinal motoneurons and, compared with exposure from 5-25 hpf, embryos were equally susceptible to abamectin-induced hypoactivity when exposures were initiated at 10 and 23 hpf. Moreover, immersion of abamectin-exposed embryos in clean water resulted in complete recovery of spontaneous activity relative to vehicle controls, suggesting that abamectin reversibly activated ligand-gated chloride channels and inhibited neurotransmission. To test this hypothesis, we pretreated embryos to vehicle or non-toxic concentrations of fipronil or endosulfan--two insecticides that antagonize the γ-aminobutyric acid (GABA) receptor--from 5-23 hpf, and then exposed embryos to vehicle or abamectin from 23-25 hpf. Interestingly, activity levels within abamectin-exposed embryos pretreated with either antagonist were similar to embryos exposed to vehicle alone. Using quantitative PCR and phylogenetic analyses, we then confirmed the presence of GABA receptor α1 and β2 subunits at 5, 10, and 23 hpf, and demonstrated that zebrafish GABA receptor subunits are homologous to mammalian GABA receptor subunits. Overall, our data collectively suggest that abamectin induces rapid and reversible hypoactivity within early zebrafish embryos, an effect that may be mediated through the GABA receptor.

[1]  J. Bloomquist Ion channels as targets for insecticides. , 1996, Annual review of entomology.

[2]  C. Kimmel,et al.  Stages of embryonic development of the zebrafish , 1995, Developmental dynamics : an official publication of the American Association of Anatomists.

[3]  K. Miotto,et al.  Substance abuse and violence: A review of the literature , 2003 .

[4]  D. Volz,et al.  Aberrant ligand-induced activation of G protein-coupled estrogen receptor 1 (GPER) results in developmental malformations during vertebrate embryogenesis. , 2012, Toxicological sciences : an official journal of the Society of Toxicology.

[5]  E. Facco,et al.  Conscious sedation with diazepam and midazolam for dental patient: priority to diazepam. , 2013, Minerva stomatologica.

[6]  P. Wislocki,et al.  Environmental Aspects of Abamectin Use in Crop Protection , 1989 .

[7]  D. Rugg 5.2 – The Insecticidal Macrocyclic Lactones , 2005 .

[8]  R. Mckernan,et al.  Anticonvulsant and adverse effects of avermectin analogs in mice are mediated through the gamma-aminobutyric acid(A) receptor. , 2000, The Journal of pharmacology and experimental therapeutics.

[9]  P. Whiting GABA-A receptor subtypes in the brain: a paradigm for CNS drug discovery? , 2003, Drug discovery today.

[10]  G. Johnston GABAA receptor pharmacology. , 1996, Pharmacology & therapeutics.

[11]  R. Oswald,et al.  Analysis of cyclic and acyclic nicotinic cholinergic agonists using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy. , 1993, Biophysical journal.

[12]  D. Volz,et al.  High-content screening assay for identification of chemicals impacting spontaneous activity in zebrafish embryos. , 2014, Environmental science & technology.

[13]  Pierre Drapeau,et al.  Motoneuron Activity Patterns Related to the Earliest Behavior of the Zebrafish Embryo , 2000, The Journal of Neuroscience.

[14]  G. Callard,et al.  Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment , 2008, BMC Molecular Biology.

[15]  E. M. Vieira,et al.  Lethal effects of abamectin on the aquatic organisms Daphnia similis, Chironomus xanthus and Danio rerio. , 2012, Chemosphere.

[16]  J. Lynch,et al.  Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels , 2012, British journal of pharmacology.

[17]  R. A. Dybas Abamectin Use in Crop Protection , 1989 .

[18]  J. Clark,et al.  Resistance to avermectins: extent, mechanisms, and management implications. , 1995, Annual review of entomology.

[19]  John E. Casida,et al.  Action of phenylpyrazole insecticides at the GABA-gated chloride channel , 1993 .

[20]  Francisco Sánchez Bayo,et al.  Insecticides Mode of Action in Relation to Their Toxicity to Non-Target Organisms , 2012 .

[21]  E. Brustein,et al.  Steps during the development of the zebrafish locomotor network , 2003, Journal of Physiology-Paris.

[22]  W. Campbell,et al.  Ivermectin: a potent new antiparasitic agent. , 1983, Science.

[23]  R. Mckernan,et al.  Which GABAA-receptor subtypes really occur in the brain? , 1996, Trends in Neurosciences.

[24]  J. Eisen,et al.  The Met receptor tyrosine kinase prevents zebrafish primary motoneurons from expressing an incorrect neurotransmitter , 2008, Neural Development.

[25]  A T Eldefrawi,et al.  Receptors for γ‐aminobutyric acid and voltage‐dependent chloride channels as targets for drugs and toxicants 1 , 1987, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[26]  J. Eisen,et al.  Pathfinding by zebrafish motoneurons in the absence of normal pioneer axons. , 1992, Development.

[27]  S. Ōmura,et al.  The life and times of ivermectin — a success story , 2004, Nature Reviews Microbiology.

[28]  C. Kimmel,et al.  Organization of hindbrain segments in the zebrafish embryo , 1990, Neuron.

[29]  L. Costa,et al.  Interaction of the neurotoxic pesticides ivermectin and lindane with the enteric GABAA receptor-ionophore complex in the guinea-pig. , 1993, European journal of pharmacology.

[30]  P. Drapeau,et al.  Time course of the development of motor behaviors in the zebrafish embryo. , 1998, Journal of neurobiology.

[31]  W. Campbell History of avermectin and ivermectin, with notes on the history of other macrocyclic lactone antiparasitic agents. , 2012, Current pharmaceutical biotechnology.