A choice behavior for morphine reveals experience-dependent drug preference and underlying neural substrates in developing larval zebrafish.

Transparent larval zebrafish offer the opportunity to unravel genetic and neuronal networks underlying behavior in a developing system. In this study, we developed a choice chamber paradigm to measure reward-associated behavior in larval zebrafish. In the chamber where larval zebrafish have a choice of spending their time in either a water- or morphine-containing compartment, larvae that have previously experienced morphine spend significantly more time in the compartment containing morphine. This behavior can be attentuated by pre-treatment with antagonists of the opioid receptor or the dopamine receptor, and furthermore, is impaired in the too few mutant, which has a genetic deficiency in the production of specific groups of dopaminergic and serotonergic neurons in the ventral forebrain. These results uncover a choice behavior for an addictive substance in larval zebrafish that is mediated through central opioid and monoaminergic neurotransmitter systems.

Dissociation of food and opiate preference by a genetic mutation in zebrafish.

Both natural rewards and addictive substances have the ability to reinforce behaviors. It has been unclear whether identical neural pathways mediate the actions of both. In addition, little is known about these behaviors and the underlying neural mechanisms in a genetically tractable vertebrate, the zebrafish Danio rerio. Using a conditioned place preference paradigm, we demonstrate that wildtype zebrafish exhibit a robust preference for food as well as the opiate drug morphine that can be blocked by the opioid receptor antagonist naloxone. Moreover, we show that the too few mutant, which disrupts a conserved zinc finger-containing gene and exhibits a reduction of selective groups of dopaminergic and serotonergic neurons in the basal diencephalon, displays normal food preference but shows no preference for morphine. Pretreatment with dopamine receptor antagonists abolishes morphine preference in the wildtype. These studies demonstrate that zebrafish display measurable preference behavior for reward and show that the preference for natural reward and addictive drug is dissociable by a single-gene mutation that alters subregions of brain monoamine neurotransmitter systems. Future genetic analysis in zebrafish shall uncover further molecular and cellular mechanisms underlying the formation and function of neural circuitry that regulate opiate and food preference behavior.

Chemobehavioral changes induced by short-term exposures to prochloraz, nicosulfuron, and carbofuran in goldfish.

The behavioral effects of short periods (2, 4, 6, 8 h) of static exposure to prochloraz (imidazole fungicide) and nicosulfuron (sulfonylurea herbicide) were recorded in goldfish (Carassius auratus). Observations were also made in an olfactometer to assess the effects of 8-h exposures to these two pesticides and to carbofuran (carbamate insecticide) on the behavioral responses to the flow of a solution of four L-amino acids (glycine, alanine, valine, taurine), mixed in the same relative proportions as in the urine of conspecifics. Each pesticide was tested at three sublethal concentrations (25, 50, 100 microg/L), and the behaviors recorded were related to swimming pattern, social interactions, and comfort movements. Static exposures to prochloraz affected horizontal displacements, burst swimming, grouping, and buccal movements. Static exposures to nicosulfuron affected burst swimming and grouping. In pesticide-unexposed fish (control), the flow of the amino acid solution induced attraction, decreased sheltering, and increased horizontal displacements, burst swimming, buccal movements, and antagonistic interactions. Compared to the controls, some of the behavioral responses to the solution of amino acids were significantly different after 8 h of subacute exposure to prochloraz and carbofuran. Both pesticides decreased attraction and increased sheltering. In addition, carbofuran decreased buccal movements and antagonistic interactions. Contrastingly, exposure to nicosulfuron showed no significant effect. This study further confirms the great vulnerability of fish behavior and chemocommunication processes to exposure to waterborne pesticides.

Behavioral and olfactory responses to prochloraz, bentazone, and nicosulfuron-contaminated flows in goldfish.

The immediate behavioral responses of goldfish (Carassius auratus) to pesticide-contaminated flows were recorded in a countercurrent olfactometer. In addition, electro-olfactograms were recorded from the epithelial surface of the olfactory rosette as a preliminary check for the olfactory sensitivity of the fish to the pesticides tested. All tests were run on prochloraz (imidazole fungicide), bentazone (diazine herbicide), and nicosulfuron (sulfonylurea herbicide). Behavioral effects were assessed, at four concentrations (10 microg/L, 100 microg/L, 1 mg/L, 10 mg/L), on endpoints related to swimming pattern (preference-avoidance responses, burst swimming reactions), comfort activities (buccal movements, feeding attempts), and social relations (antagonistic acts, grouping). The behavior of the fish appeared particularly sensitive to prochloraz exposure. As a whole, prochloraz-contaminated flows showed significant effects on the six behaviors studied; bentazone and nicosulfuron affected three and five, respectively. At the lowest concentration, prochloraz also showed more effects than the two other pesticides. Some of the behavioral endpoints were found particularly sensitive to a given chemical. Pesticide-contaminated flows also induced significant changes in swimming orientation of the fish. Attraction was observed in response to flowing solutions of prochloraz (1 mg/L, 10 mg/L), bentazone (10 microg/L, 10 mg/L), and nicosulfuron (1 mg/L, 10 mg/L). At a concentration of 1 mg/L, none of the pesticides induced a noticeable depolarization of the olfactory epithelium, suggesting that these chemicals are not detected by the olfactory sense of the fish. These results are discussed in the light of the data concerning effects of pesticides on behavior and chemical communication in fish.

Effects of carbofuran, diuron, and nicosulfuron on acetylcholinesterase activity in goldfish (Carassius auratus).

Juvenile goldfish (Carassius auratus) were exposed to three widely used pesticides; carbofuran, diuron, and nicosulfuron. Acetylcholinesterase (AChE) activity and molecular forms of AChE were first characterized in brain and skeletal muscle of unexposed fish. Skeletal muscle had higher AChE activity than brain (306 and 215 nmol/min/mg protein, respectively). In brain, four molecular forms of AChE were found: A12, G4, G2, and G1. In the muscle, three molecular forms were found A12, A8, and G2. AChE activity was then evaluated in both tissues of fish exposed to different concentration of pesticides (5, 50, and 500 microg/L) for 6, 12, 24, and 48 h. In brain, AChE activity was significantly inhibited during all the periods of exposure in response to 50 microg/L (19-28%) and 500 microg/L (85-87%) carbofuran. Such effect was observed in the muscle only at 500 microg/L (86-92%). Carbofuran had no effect on the distribution of molecular forms. Significant inhibitions (9-12%) of brain AChE activity were also observed in response to diuron and nicosulfuron at 500 microg/L during all periods of exposure and for 50 microg/L nicosulfuron after 24 and 48 h. This study pointed out short-term effects of exposure to sublethal concentrations of the three pesticides, ranging among different chemical families, on brain and muscle AChE in goldfish.