Organophosphorus Pesticide Exposure Effects on Neurobehavioral Development of Zebrafish and Characterization of Developmental Switch Between Two Cation-chloride Cotransporters (zfnkcc1 and zfkcc2)
Organophosphorus pesticides (OPs) are neuroactive compounds that can affect the development of the nervous system and behavior. Using zebrafish, we compared the effectiveness of three different OPs, chlorpyrifos (CPF), diazinon (DZN) and parathion (PA) on survival, AChE inhibition and larval motility. We conclude that at equimolar concentrations CPF is the most effective OP of the three tested. 300nM CPF inhibited AChE when measured at 5 days post fertilization (dpf) by 80%, which correlated to a 35% decrease in larval motility when measured at 6 dpf.
We isolated the cDNA of <italic>zfkcc2</italic>, which encodes for an important cation-chloride cotransporter responsible for establishing a chloride gradient that allows for the neurotransmitter GABA to become inhibitory for a subset of neurons. By isolating the cDNA of <italic>zfkcc2</italic>, we were able to characterize its expression pattern up to 4 dpf. Using qRT-PCR, we showed that the expression of <italic>zfkcc2</italic> rises from 2 to 96 hours post fertilization (hpf). The expression of <italic>zfkcc2</italic> is found at 1 dpf in the forebrain, midbrain and hindbrain in a distinct pattern. By 3 dpf, zfkcc2 expression is found throughout most of the developing zebrafish brain. We were then able to characterize the expression pattern of zfnkcc1, which encodes for a cation-chloride cotransporter that works opposite to <italic>zfkcc2</italic> and establishes a chloride gradient allowing GABA to be excitatory during development. Expression of <italic>zfnkcc1</italic> is found at 1 dpf throughout most of the developing zebrafish head including in some non-neural tissue, but appears concentrated to the otic vesicle at 3 dpf. Using double in situ hybridization, we investigated the nature of the developmental switch from zfnkcc1 to zfkcc2 by looking at regions where there was an interaction between both genes' expression patterns in the developing zebrafish nervous system.
We preliminarily examined how affecting nicotinic signaling with exposures to nicotinic agonists/antagonists can affect the expression of <italic>zfnkcc2</italic> and <italic>zfkcc2</italic>. While there were no apparent changes in the expression pattern from any of the exposures at 2 dpf, we found that by 3 dpf nicotine and CPF exposures resulted in an incomplete expression pattern for <italic>zfkcc2</italic> that seemed to be delayed. However, the expression of <italic>zfnkcc1</italic> at the otic vesicle was unaffected by exposure.
Our results suggest that disrupting nicotinic signaling during development can affect the expression of an important cation-chloride cotransporter, <italic>zfkcc2</italic>, that plays a major role in establishing the necessary chloride gradient for the proper functioning of GABA during development. Future studies should focus on investigating the nature of the developmental switch between <italic>zfnkcc1</italic> and <italic>zfkcc2</italic> to see if the possibility of their regulation being coupled is a target for exposures.
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