br Results br Discussion Dopamine regulates

Results
Discussion Dopamine regulates behavioral responses to nicotine in Drosophila (Bainton et al., 2000), but neurochemical measurements of dopamine after nicotine stimulation had not been made. Here, we demonstrate for the first time that the stimulation of nAChRs causes dopamine release in Drosophila melanogaster larval VNC. These results are similar to findings in rodents that Sarpogrelate hydrochloride receptor evokes dopamine 2010). Acetylcholine, nicotine, and neonicotinoid insecticides evoked dopamine release in the Drosophila CNS and this response was mediated by nAChRs, and not mAChRs. All previous work in Drosophila larvae had used optogenetics or P2X2 channels to stimulate dopamine release, which requires a channel to be expressed in specific neurons using genetics (Privman and Venton, 2015, Vickrey et al., 2009). Acetylcholine or nicotine stimulation evokes endogenous dopamine release, without needing to genetically modify the organism. Mutations in the subunits of the nAChRs affect neonicotinoid evoked dopamine release without changing the response to nicotine. Thus, dopamine measurements in Drosophila are an easy way to study the effects of nicotinic subunit mutations in causing excitatory responses.
Conclusions We demonstrated for the first time that the nAChR agonists acetylcholine, nicotine, and neonicotinoids stimulate dopamine release in the Drosophila larval VNC. The release is mediated by nAChRs, and not mAChRs, and is sensitive to tetrodotoxin, indicating release is exocytotic. Nicotine and neonicotinoids stimulate dopamine release that lasts longer than acetylcholine-stimulated release, likely due to higher affinities and fewer mechanisms for their metabolism and clearance. Neonicotinoid-stimulated response is significantly lower in Drosophila strains that are resistant to the neonicotinoids and have mutations in α1 or β2 nAChR subunits but these flies maintain their response to nicotine. Thus, Drosophila is an important model organism to study the effects of nAChR mutations or agonists on dopamine release and may yield important information about the pathways of acetylcholine regulation of dopamine release.
Experimental section Chemicals. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), and solutions were prepared in Milli Q water (Millipore, Billerica, MA) unless noted otherwise. Electrode calibrations were performed in phosphate buffer solution (PBS; 131.25 mM NaCl, 3.0 mM KCl, 10.0 mM NaH2PO4, 1.2 mM MgCl2, 2.0 mM Na2SO4, and 1.2 mM CaCl2) with pH adjusted to 7.4, which was made once a month and stored at 4 °C. To make the larval dissection buffer, 11.1 mM glucose and 5.3 mM trehalose were added to the PBS buffer on the day of the experiment. A 10 mM stock solution of dopamine was prepared in 0.1 M HClO4 once a month, stored at 4 °C, and diluted to 1 μM in PBS for calibrations the day of the experiment.
Acknowledgments We would like to thank Dr. Trent Perry at the University of Melbourne, Australia for donating Drosophila stocks. We would also like to thank Geoffrey Norris of Kipnis Lab at the University of Virginia for providing tetrodotoxin. This work is funded by the NIH (R01MH085159) and a Camille Dreyfus Teacher-Scholar Award.
Introduction Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels expressed in many kinds of both excitable and non-excitable cells. They mediate fast synaptic transmission in muscles and autonomic ganglia (Kalamida et al., 2007, Skok, 2002), regulate neurotransmitter release in the brain (Gotti et al., 2009) and control cell proliferation, survival and cytokine production in non-excitable tissues (Kawashima and Fujii, 2008). The nAChRs of α7 subtype (α7 nAChRs) are involved in cholinergic anti-inflammatory pathway regulating the production of anti-inflammatory cytokines by monocyte-derived cells (De Jonge and Ulloa, 2007). The nAChR expression was found in both T and B lymphocytes (Kawashima et al., 2015). Previously we showed that α7 nAChRs are required for B lymphocyte survival during differentiation in the bone marrow (Skok et al., 2006), but negatively regulate mature B lymphocyte anti-CD40-stimulated proliferation (Koval et al., 2011). Correspondingly, it was reported that mice lacking α7 nAChRs produce more IgG1 Abs (Fujii et al., 2007) and spleen cells from α7 knockout mice produce more TNF-α and IL-6 than wild-type cells (Fujii et al., 2017). Mice injected with methyllicaconitine (MLA) responded to cytochrome c with IgG antibodies faster than non-injected ones (Koval et al., 2009). We hypothesized that such effects may be also due to the deficiency of regulatory T and/or B lymphocytes. Regulatory T cells (Tregs) were shown to express α7 nAChRs and to be subjected to cholinergic regulation (Galitovskiy et al., 2011). Here we put an aim to investigate whether α7 nAChRs are important for generation, induction and functioning of regulatory B lymphocytes (Bregs) and to study in more details the mechanisms of cholinergic regulation of the antibody immune response.