Background

There is increasing evidence that small molecules can be used for the treatment of neuronal disorders (Rissman and Mobley, 2011). As such, knowledge of the neurobiology of small molecule-induced behavioral changes and elucidation of their underlying mechanisms will further our understanding of the functional mechanisms of neural circuits. However, most previous behavioral studies used mice or rats as a model, and a significant amount of resources is required to complete such studies. Drosophila have been used as a tractable genetic model to investigate many genetic disorders (Mackay and Anholt, 2006), and in recent years their use as a model for behavioral studies has become increasingly common (Zhukovskaya et al., 2013; Chen et al., 2014; Hampel et al., 2015; Kaur et al., 2015; Pitmon et al., 2016; Hidalgo et al., 2017).

Muscimol is a selective agonist of GABA_A receptors, which are responsible for the induction of rapid inhibitory neurotransmission in mammals. Thus, the administration of muscimol causes sedative hypnotic-like effects (Lancel, 1999; Yoshimoto et al., 1999; Kumar and Kalonia, 2008; Zhang et al., 2016). In rats, intravenous injection of muscimol causes a dose-dependent decrease in motor activity, and high doses lead to cataleptic-like effects (Biggio et al., 1977).

We developed a novel approach for testing and quantifying the effects of the potent GABA_A agonist muscimol on fly behaviors, including “grooming”, “walking”, and “resting” phenotypes. “Grooming” and “walking” together were considered to represent the active state of the fly, and “resting” implied no movement or a cataleptic phenotype. Direct observation of these active and non-active states of fly behavior can be achieved easily by visual observation and manual recording of the duration of individual behavioral events.

We used a 5-ml Eppendorf tube to design a basic feeding chamber, into which a glass capillary filled with a target solution was inserted. Thus, the flies were able to drink easily while traveling down the capillary drain. For the behavioral assay, a 96-well plate half-filled with 1% agar solution enabled multiple parallel samples to be run simultaneously. A standard period of 9–10 hours of starvation was utilized to induce thirst and rapid consumption of the target solution. We observed in our laboratory that the flies were generally less thirsty on rainy days, so we generally avoided performing these experiments on such days. This method enables screening of the effects of neuro-modulating peptides and natural compounds, which may act in flies through different subsets of neurons; therefore, the underlying functions of neurons may be determined. Karim MR et al. (2018) recently conducted an experiment using this method to explore the role of soy β-conglycinin-derived peptide bCGα(323-333), which regulates fly grooming behavior through effects on dopamine receptors. The significance of this approach is that it allows the study of a broad range of bioactive peptides and their neuro-modular functions with a simple and cost-effective method.