br Acknowledgments The authors would
Acknowledgments The authors would like to thank Ms. Ashley Davis for her administrative assistance in preparing this article and Mr. Dan Beck for his artistic contributions to Fig. 1.
Introduction Acetylcholine is the most abundant neurotransmitter in the central nervous system (CNS) of insects and the neuromuscular junction in vertebrates (Gupta, 1987). In insects, the cholinergic system mediates wing movements (Gauglitz and Pfluger, 2001), locomotion, learning, and memory (El Hassani et al., 2008). The cholinergic system is a target of insecticides such as organophosphates (Fukuto, 1990), that inactivate acetylcholinesterase, and neonicotinoids (Matsuda et al., 2001), that are Sarpogrelate hydrochloride receptor agonists (Brown et al., 2006). Acetylcholine receptors are either nicotine sensitive (nicotinic acetycholine receptors, nAChR), muscarine sensitive (muscarinic acetylcholine receptor, mAChR), or of mixed nicotinic/muscarinic nature (Breer and Sattelle, 1987). nAChRs are more abundant than mAChRs in the nervous system of insects, including Drosophila melanogaster (Breer, 1981, Lummis and Sattelle, 1985, Salvaterra and Foders, 1979). Acetylcholine modulates neural activity in insects, causing depolarization in cockroach giant interneurons (Harrow and Sattelle, 1983) and large excitatory currents and action potential bursts in the Drosophila larval central nervous system (Rohrbough and Broadie, 2002). In rodents, nicotine increases dopamine release during phasic activity (Rice and Cragg, 2004) and depletion of acetylcholine or nAChR antagonist administration decreases stimulated dopamine (Zhou et al., 2001). However, there are no studies of nAChR mediated dopamine release in insects. Nicotinic acetylcholine receptors are composed of α and β subunits, which assemble as pentamers to form a cation channel (Chamaon et al., 2000, Lansdell et al., 2012). α subunits contain a Cys-Cys pair and are required for acetylcholine binding (Kao and Karlin, 1986). Receptors with both α and β subunits form ligand-binding site at the interface of the different subunits (Corringer et al., 2000, Gill et al., 2011). Benke and Breer were the first to suggest the existence of nAChRs in insects with differing affinity for α-bungarotoxin and different agonist sensitivities (Benke and Breer, 1989). Since then, several different types of nAChRs subunit combinations have been identified which have different agonist affinity and activation kinetics (Exley and Cragg, 2008). While not a plant pest, Drosophila melanogaster is a popular model organism to study the insect nervous system and is particularly helpful to study structures such as insect nAChRs that are difficult to express in host cells (Thany et al., 2007). In Drosophila, ten different subunits of nAChRs have been identified, Dα1–Dα7 and Dβ1–Dβ3 (Lansdell et al., 2012). Combinations of subunits and mutations are key to nAChR function; for example the α5 subunit is involved in α-bungarotoxin sensitivity (Wu et al., 2005), and the α6 subunit is essential for the insecticidal effect of spinosad (Perry et al., 2007). In mammals, α4β2 nAChRs are located on dopamine neurons and regulate dopamine release (Chen et al., 2003, Maskos, 2010). Drosophila strains with mutations of Dα1, Dα2, or Dβ2 nAChR subunits are highly resistant to the neonicotinoids nitenpyram and imidacloprid (Perry et al., 2008). The behavioral effects of nicotine on Drosophila are regulated by dopamine (Bainton et al., 2000), and Dβ1 subunit in dopaminergic neurons play a role in acute locomotor hyperactivity caused by nicotine in male Drosophila (Zhang et al., 2016). Therefore, understanding how nAChR control dopamine release is critical for understanding the effects of neonicotinoids. In this study, we characterized acetylcholine, nicotine, and neonicotinoid-stimulated dopamine release in Drosophila larval ventral nerve cord (VNC) for the first time. Our lab has pioneered measurements of dopamine in Drosophila using fast-scan cyclic voltammetry (FSCV) at implanted carbon-fiber microelectrodes (CFMEs), but previous experiments primarily used optogenetically-stimulated release (Privman and Venton, 2015, Vickrey et al., 2009). Here, we focus on release mediated by nAChRs and establish that acetylcholine, nicotine, and neonicotinoids cause dopamine release in the larval VNC. We also show that Drosophila larvae can be used to study how agonist sensitivities are altered when nAChR subunits are mutated. Longer duration release is evoked by nicotine and neonicotinoids than with acetylcholine. Stimulated release is sensitive to α-bungarotoxin (α-BTX) and tetrodotoxin (TTX) and is exocytotic. Neonicotinoid-stimulated release is significantly lower in Drosophila nAChR subunit mutants that were previously found to have increased resistance to imidacloprid and nitenpyram. Thus, mutations that confer resistance to neonicotinoids in these strains also affect neonicotinoid-stimulated dopamine release. nAChR agonists stimulate dopamine release in Drosophila larval VNC; therefore, Drosophila can be used to study agonist sensitivities at mutated nAChRs subunits.