Anxiolytic effects of nicotine in zebrafish
Introduction
Nicotine has a wide variety of effects on the central nervous system which impact behavioral function (reviews in [11], [13], [14], [20], [21], [24]). Three main effects which seem to prominently underlie cigarette smoking are reward, cognitive enhancement, and anxiolysis [3]. Rodent models of have classically been used to assess nicotinic receptor roles in reinforcement, cognitive function and anxiety. Recently, zebrafish, Danio rerio, have emerged as a useful complementary model for the study of a variety of neurobehavioral functions including reward and cognitive function [6], [10], [15], [16], [17], [18]. Anxiolysis was studied in the present experiment using the zebrafish model of nicotine effects, because it is thought to play a role in nicotine addiction and remission, and a demonstration that nicotine affects anxiety in the zebrafish would open new avenues of investigation in nicotinic receptor function. With a rapid and inexpensive model system many novel compounds could be efficiently screened for further study in mammalian models. Molecular mechanisms for the critical neuropharmacological effects could be readily studied in the great array of mutant zebrafish strains.
Zebrafish behavioral assays are becoming increasingly versatile and utilized in studies of genetic and neural underpinnings of cognitive function [1], [7], [22], [27]. A dose–effect nicotine study on zebrafish in a delayed alternation task [16] found results consistent with rats, monkeys, and humans [19]: low doses improve memory, while high doses are detrimental.
Nicotine has varied effects on anxiety in humans and animals [25]; these effects are frequently correlated with tobacco addiction [8]. In humans, stress increases the rate of smoking [12]; this relation between stress and nicotine seeking is also found in rodents [5]. In rodents, nicotine has been found by some investigators to have anxiolytic effects as measured by choices on an elevated plus maze [4]. However, others have found nicotine-induced anxiogenic or mixed effects in rodents [9], [29]. Nicotine dose seems to be quite important in the nature of its effects on anxiety. In mice, low dose nicotine (0.05 mg/kg) caused a significant anxiolytic effect as measured by open arm choice in the elevated plus maze [2]. This effect was abolished by co-administration of the endocannabinoid (CB1) antagonist rimonabant. In the same study a higher nicotine dose (0.8 mg/kg) caused an anxiogenic effect on the plus maze. This anxiogenic effect of high dose nicotine appeared to involve a different neural substrate than the nicotine low-dose anxiolysis inasmuch as this effect was not altered by rimonabant.
The current study was directed at determining whether nicotine effects on anxiety could be modeled in the zebrafish. The testing procedure developed took advantage of the natural tendency for zebrafish to spend the majority of time at the bottom when introduced into a novel environment and then gradually over a period of minutes expand their position of swimming to include the higher portions of the test tank. This appears analogous to rats' choices of walled arms in an elevated plus maze and positions near the walls of an open field thigmotaxic under stressful circumstances. Assessment of anxiety in zebrafish is relatively new. Peitsaro et al. [23] found that zebrafish swim along the edges of a novel test environment and that previous experience in the environment reduced the proportion of time that the fish spent near the walls of the environment relative to the center. They did not assess the vertical location choices of the fish. The new paradigm we have developed shows that assessing position choice along the vertical dimension provides a rapid metric of the adaptation of zebrafish to a novel environment and the effect of drug treatments on adaptation to a novel environment.
Section snippets
Subjects
Zebrafish (Danio rerio) were kept at approximately 28.5 °C on a 12:12-h light/dark cycle. Behavioral testing of drug effects took place during the light phase between 8:00 a.m. and 5:00 p.m. The tank water used de-ionized H2O and sea salts (Instant Ocean, 1.2 g/20 l of water). The tanks with the groups of adult fish were maintained with constant filtration and aeration. Fish were fed daily with brine shrimp and flake fish food.
Drug administration
Nicotine ditartrate and mecamylamine HCl were administered by
Experiment 1
The swimming activity of the fish as indexed by the distance traveled each minute of the 5-min session (Fig. 1). The linear trend of change over the 5-min session differed (p < 0.0005) between the different nicotine treatment conditions. The placebo treated fish showed a relatively low activity during the first minute, which quickly increased during the next couple of minutes to more than double the distance traveled in the first minute. This rate of increase in swimming behavior over the 5-min
Discussion
In zebrafish, as in rodents, nicotine caused significant effects on locomotor activity. The effects were consistent with the anxiolytic actions seen in rodents and humans. The diving response is a common reaction in prey fish in response to stress. This would serve to help them avoid predators. In vehicle-treated fish, substantial time is spent in the bottom third of the tank when first introduced into the tank and then rapidly diminished their time in the bottom of the tank as the session
Acknowledgement
Research Supported in part by Duke University Superfund Basic Research Center ES010356 and in part by Philip Morris USA Inc. and Philip Morris International.
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