Buspirone, chlordiazepoxide and diazepam effects in a zebrafish model of anxiety

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Abstract

Zebrafish are becoming more widely used to study neurobehavioral pharmacology. We have developed a method to assess novel environment diving behavior of zebrafish as a model of stress response and anxiolytic drug effects. In a novel tank, zebrafish dwell in the bottom of the tank initially and then increase their swimming exploration to higher levels over time. We previously found that nicotine, which has anxiolytic effects in rodents and humans, significantly lessens the novel tank diving response in zebrafish. The specificity of the diving effect was validated with a novel vs. non-novel test tank. The novel tank diving response of zebrafish was tested when given three anxiolytic drugs from two different chemical and pharmacological classes: buspirone, chlordiazepoxide and diazepam. When the test tank was novel the diving response was clearly seen whereas it was significantly reduced when the test tank was not novel. Buspirone, a serotonergic (5HT1A receptor agonist) anxiolytic drug with some D2 dopaminergic effect, had a pronounced anxiolytic-like effect in the zebrafish diving model at doses that did not have sedative effects. In contrast, chlordiazepoxide, a benzodiazepine anxiolytic drug, which is an effective agonist at GABA-A receptors, did not produce signs of anxiolysis in zebrafish over a broad dose range up to those that caused sedation. Diazepam another benzodiazepine anxiolytic drug did produce an anxiolytic effect at doses that did not cause sedation. The zebrafish novel tank diving task can be useful in discriminating anxiolytic drugs of several classes (serotonergic, benzodiazepines and nicotinic).

Introduction

Zebrafish (Danio rerio) are becoming widely used for the study of the molecular bases of neurobiology with applications in neuropharmacology and neurotoxicology (Linney et al., 2004, Teraoka et al., 2003). As a model organism, the zebrafish provides an inexpensive, potentially high-throughput test subject that could prove useful in gaining a greater understanding of neuropharmacological mechanisms in mammals and facilitate drug discovery. Zebrafish studies have recently begun use assessments of complex behavioral functions such as, learning, memory and anxiety response (Arthur and Levin, 2001, Bass and Gerlai, 2008, Bencan and Levin, 2008, Colwill et al., 2005, Dahm and Geisler, 2006, Eddins et al., 2009, Hicks et al., 2006, Levin and Chen, 2004, Levin et al., 2007, Levin et al., 2003, Levin et al., 2006, López-Patiño et al., 2008, Ninkovic and Bally-Cuif, 2006, Pietsaro et al., 2003, Pradel et al., 1999, Pradel et al., 2000, Rawashdeh et al., 2007, Williams et al., 2002, Xu et al., 2007). Development of validated behavioral tests for complex behavioral function in zebrafish will facilitate the analysis of the neural and molecular mechanisms of behavioral function as well as economical screening for promising new drugs for treating behavioral dysfunction.

Anxiety and stress responses are beginning to be investigated in zebrafish. Anxiety-like behavior in zebrafish has been shown through patterns of swimming along the edge (Pietsaro et al., 2003) and towards the bottom of novel environments (Levin et al., 2007). Nicotine at a dose of 50 mg/l given by immersion for 3 min caused a significant attenuation of the diving response (time spent dwelling in the bottom of the tank) during the initial part of the 5-minute test session. The higher 100 mg/l nicotine caused a significant reduction in the bottom dwelling throughout the session. There is typically increased swimming activity over the course of the test session. This was significantly attenuated by both 50 and 100 mg/l of nicotine. These types of behaviors are not seen as frequently when the fish had previously been exposed to its surroundings. We are using this paradigm in zebrafish to determine the efficacy of different classes of anxiolytics. Nicotine has been shown in our previous study to reduce anxiety in zebrafish placed in a novel environment and could be reversed by the nicotinic acetylcholine receptor antagonist, mecamylamine (Levin et al., 2007). The 100 mg/l nicotine dose has previously been seen in our studies to significantly improve learning and memory in zebrafish (Levin et al., 2007, Levin et al., 2006).

Zebrafish can be used to study the neural mechanisms of a variety of types of behavior. Key to this effort is the development of sensitive, efficient and reliable tests of the behavioral functions of interest. We have found that the novel tank diving response of zebrafish can provide insight into stress reactivity and anxiety (Levin et al., 2007). The diving response to dwell in the bottom of a novel tank and subsequently explore higher levels of the tank resembles thigmotaxis (position choice along the wall) seen in rodents upon initial presentation into a novel open field that is commonly interpreted as anxiety response to stress (Simon et al., 1994, Treit and Fundytus, 1988). Previously, we found that nicotine administration at a dose level, which significantly improves learning and memory (Levin and Chen, 2004, Levin et al., 2006) significantly attenuates this diving response (Levin and Chen, 2004). This is congruent with the findings of anxiolytic effects of nicotine in rodents and humans. To determine the similarities and differences in the neural bases of anxiety and stress response in zebrafish with mammals we examined broad dose effects of three different drugs with anxiolytic effects in humans and rat models: chlordiazepoxide, diazepam and buspirone.

Benzodiazapine receptors have been found in a wide variety of species including boney fish but not lower vertebrates and invertebrates (Nielsen et al., 1978). A variety of studies have identified benzodiazepine-GABA-A receptors in fish and shown that they have similar binding characteristics as those in rodents and humans (Anzelius et al., 1995a, Anzelius et al., 1995b, Carr et al., 1999, Friedl et al., 1988, Wilkinson et al., 1983), though functionally they may have some differences from mammals (Betti et al., 2001). There is evidence that benzodiazepine-GABA receptors are less pharmacologically responsive in fish, at least with regard to controlling convulsant activity (Corda et al., 1989). With regard to potential anxiolytic effects, Rehnberg et al. found in fathead minnows that exposure to the fish alarm pheromone reduced swimming activity in control fish but that the reduction in swimming effect was not seen in fish after exposure to 20 mg/l of chlordiazepoxide for an hour (Rehnberg et al., 1989). However, in their experiment, that dose of chlordiazepoxide had decreased activity relative to controls before the alarm pheromone was given.

The current study was conducted to test the effect of two different types of anxiolytic drugs on the novel environment diving response in zebrafish to determine the involvement of benzodiazepine and serotonergic systems in this zebrafish model of anxiety. Specifically, we studied extensive dose-effect functions with chlordiazepoxide, and diazepam a benzodiazepine anxiolytics, and buspirone, a serotonergic anxiolytic, on the novel environment diving response as well as swimming activity. This study together with our previous work with nicotine will help determine the applicability of this zebrafish model across anxiolytic drug classes and the participation of different transmitter receptor systems in anxiolytic response in zebrafish.

Section snippets

Subjects

Zebrafish (D. rerio) obtained from Triangle Tropical Fish (Durham, NC) were kept at approximately 28.5 °C on a 12:12-h light/dark cycle in an automated flow-through continuously filtered water system by Aquatic Habitats (Apopka, FL, USA). Behavioral testing of drug effects took place during the light phase between 8:00 a.m. and 5:00 p.m. In each study the fish were randomly sorted into treatment groups and vehicle-treated controls so that there was no confounding of breeding or holding

Experiment 1: importance of novelty of the test tank

To document the importance of novelty of the test tank we conducted a study testing zebrafish, which were housed in the same-sized 1.5-liter tanks in which they were tested (N = 50). Thus, the tanks were not novel in the sense of size and dimensions. Their responses to being placed in the test tank were compared with fish, which were housed in larger 3-liter tanks, which were twice as wide as the 1.5-liter tanks (N = 20). All fish were tested in identical 1.5-liter, narrow tanks. All the fish had

Discussion

The current study demonstrated in a zebrafish model of stress response anxiolytic effects of two drugs: buspirone and diazepam which have been shown to be effective anxiolytics in humans and rodent models. A third drug, chlordiazepoxide was not found to be effective in this zebrafish model.

Chlordiazepoxide, a classic benzodiazepine anxiolytic, which acts as an effective agonist at GABA-A receptors did not show any signs of anxiolytic effects in the current zebrafish study over a wide dose

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