Zebrafish provide a sensitive model of persisting neurobehavioral effects of developmental chlorpyrifos exposure: comparison with nicotine and pilocarpine effects and relationship to dopamine deficits

Abstract

Chlorpyrifos (CPF) an organophosphate pesticide causes persisting behavioral dysfunction in rat models when exposure is during early development. In earlier work zebrafish were used as a complementary model to study mechanisms of CPF-induced neurotoxicity induced during early development. We found that developmental (first five days after fertilization) chlorpyrifos exposure significantly impaired learning in zebrafish. However, this testing was time and labor intensive. In the current study we tested the hypothesis that persisting effects of developmental chlorpyrifos could be detected with a brief automated assessment of startle response and that this behavioral index could be used to help determine the neurobehavioral mechanisms for persisting CPF effects. The swimming activity of adult zebrafish was assessed by a computerized video-tracking device after a sudden tap to the test arena. Ten consecutive trials (1/min) were run to determine startle response and its habituation. Additionally, habituation recovery trials were run at 8, 32 and 128 min after the end of the initial trial set. CPF-exposed fish showed a significantly (p<0.025) greater overall startle response during the 10-trial session compared to controls (group sizes: Control N=40, CPF N=24). During the initial recovery period (8 min) CPF-exposed fish showed a significantly (p<0.01) greater startle response compared to controls. To elucidate the contributions of nicotinic and muscarinic acetylcholine receptors to developmental CPF-mediated effects, the effects of developmental nicotine and pilocarpine exposure throughout the first five days after fertilization were determined. Developmental nicotine and pilocarpine exposure significantly increased startle response, though nicotine (group sizes: Control N=32, 15 mM N=12, 25 mM N=20) was much more potent than pilocarpine (group sizes: Control N=20, 100 microM N=16, 1000 microM N=12). Neither was as potent as CPF for developmental exposure increasing startle response in adulthood. Lastly, developmental CPF exposure decreased dopamine and serotonin levels and increased transmitter turnover in developing zebrafish larvae (N=4 batches of 50 embryos/treatment). Only the decline in dopamine concentrations persisted into adulthood (group sizes: Control N=14, CPF N=13). This study shows that a quick automated test of startle can detect persisting neurobehavioral impairments caused by developmental exposure to CPF. This may be helpful in screening for persisting neurobehavioral defects from a variety of toxicants.

Fig. 1

Diagram of the zebrafish startle testing method. A) Top view showing the orientation of the vibrational startle apparatus. This fish are tested in eight cylindrical chambers arranged between computer monitors (monitors are use to elicit visual startle). B) Schematic diagram illustrating the testing chambers above that tap solenoids which are computer driven to deliver a vibrational startle at 1 min intervals (ten total trials) and at 8, 32, and 128 min after the initial session trial. C) View of the computer screen showing the video tracking of eight zebrafish during the startle task.

Fig. 2

Developmental chlorpyrifos effects on A) startle response and habituation over ten trials (mean±sem). B) Comparison of developmental nicotine exposure with developmental chlorpyrifos exposure on average startle reaction over 10 trials (mean±sem). C) Developmental chlorpyrifos exposure effects on startle response 8, 32 and 128 min after the 10-trial habituation sequence (mean±sem) (group sizes: Control N=40, CPF N=24).

Fig. 3

Developmental nicotine effects on A) startle response and habituation over ten trials (mean±sem). B) Comparison of developmental nicotine exposure on average startle reaction over 10 trials (mean±sem). C) Developmental nicotine exposure effects on startle response 8, 32 and 128 min after the 10-trial habituation sequence (mean±sem) (group sizes: Control N=32, 15 μM N=12, 25 μM N=20).

Fig. 4

Developmental pilocarpine effects on A) startle response and habituation over ten trials (mean±sem). B) Comparison of developmental pilocarpine exposure on average startle reaction over 10 trials. (mean±sem). C) Developmental pilocarpine exposure effects on startle response 8, 32 and 128 min after the 10-trial habituation sequence (mean± sem) (group sizes: Control N=20, 100 μM N=16, 1000 μM N=12).

Fig. 5

Effects of developmental CPF exposure on neurochemical measures in the adult zebrafish brain. A) Effect of CPF on DA levels *p<0.025 CPF vs. Control, B) DOPAC/DA ratios, C) 5HT levels, D) 5HIAA/5HT ratios, and E) Norepinephrine levels. (mean±sem), (group sizes: Control N=14, CPF N=13).

Fig. 6

Effects of developmental CPF exposure on neurochemical measures in the larvae (6 days post-fertilization). A) Effect of CPF on DA levels **p<0.01 CPF vs. Control, B) DOPAC/DA ratios *p<0.025 CPF vs. Control, C) 5HT levels **p<0.01 CPF vs. Control, D) 5HIAA/5HT ratios, E) Norepinephrine levels and E/NE ratio (mean±sem), N=4 batches of 50 embryos/exposure group.