NEUROSCIENCE RESEARCH ARTICLE Cole A. Malloy et al. / Neuroscience 411 (2019) 47–64 Pharmacological identification of cholinergic receptor subtypes: modulation of locomotion and neural circuit excitability in Drosophila larvae Cole A. Malloy, a,b, * Eashwar Somasundaram, a Aya Omar, a Umair Bhutto, a Meagan Medley, a Nicole Dzubuk a and Robin L. Cooper a a Department of Biology and Center for Muscle Biology, University of Kentucky, Lexington, KY 40506, USA b Molecular Neurophysiology and Biophysics Section, Eunice Kennedy Shriver NICHD, NIH, Bethesda, MD 20892-3715, USA Abstract—Acetylcholine (ACh) is an abundant neurotransmitter and neuromodulator in many species. In Drosophila melanogaster ACh is the neurotransmitter used in peripheral sensory neurons and is a primary excitatory neurotrans- mitter and neuromodulator within the central nervous system (CNS). The receptors that facilitate cholinergic transmis- sion are divided into two broad subtypes: the ionotropic nicotinic acetylcholine receptors (nAChRs) and the metabotropic muscarinic acetylcholine receptors (mAChRs). This receptor classification is shared in both mammals and insects; however, both the pharmacological and functional characterization of these receptors within the Droso- phila nervous system has lagged behind its mammalian model counterparts. In order to identify the impact of ACh receptor subtypes in regulating the performance of neural circuits within the larval CNS, we used a behavioral and elec- trophysiological approach to assess cholinergic modulation of locomotion and sensory-CNS-motor circuit excitability. We exposed intact and semi-intact 3 rd instar larvae to ACh receptor agonists and antagonists to observe their roles in behavior and regulation of neural circuit excitability and to investigate AChR pharmacological properties in vivo. We combined this with targeted AChR RNAi-mediated knockdown to identify specific receptor subtypes facilitating ACh modulation of circuit efficacy. We identify a contribution by both mAChRs and nAChRs in regulation of locomotor behavior and reveal they play a role in modulation of the excitability of a sensory-CNS-motor circuit. We further reveal a conspicuous role for mAChR-A and mAChR-C in motor neurons in modulation of their input-output efficacy. © 2019 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: acetylcholine, Drosophila melanogaster, pharmacology, electrophysiology, behavior. INTRODUCTION Acetylcholine (ACh) has long been recognized as a primary neurotransmitter and neuromodulator in the nervous system of Drosophila melanogaster. It is the principal neurotrans- mitter used in sensory neurons and is a prominent excitatory neurotransmitter and neuromodulator within the CNS (Lee and O'Dowd, 1999; Yasuyama and Salvaterra, 1999; Su and O'Dowd, 2003). The primary enzymes involved in ACh metabolism, choline acetyltransferase and the degradative enzyme acetylcholinesterase (AChE), are highly expressed in sensory neurons and within the CNS (Buchner, 1991). Null mutations in these genes impart embryonic lethality, signaling the importance of ACh in Dro- sophila development and nervous system function (Buchner, 1991). Despite this documented significance, lit- tle is known regarding the modulatory role of ACh in modify- ing neural circuit and network activity in this species. While elegant work has enhanced our understanding of ACh sig- naling and the specific receptor subtypes that regulate a number of behaviors in adult flies, including in olfactory information processing (Gu and O'Dowd, 2006; Silva et al., 2015), motion detection (Takemura et al., 2011) in mediat- ing giant fiber escape response (Fayyazuddin et al., 2006) *Corresponding author at: Molecular Neurophysiology and Biophysics Section, Eunice Kennedy Shriver NICHD, NIH, 35 Convent Dr., Bldg 35, Room 3C908, Bethesda, MD 20892-3715. E-mail address: cole.malloy@nih.gov (Cole A. Malloy). Abbreviations: ACh, Acetylcholine; AChE, Acetylcholinesterase; AChRs, Acetylcholine receptors; m6, Abdominal muscle 6; BD, Benzo- quinonium dibromide; BDSC, Bloomington Drosophila Stock Center; CS, Canton S; CNS, Central nervous system; CPG, Central pattern generator; EJP, Excitatory junction potential; GluR, Glutamate receptor; HL-3, Hemolymph-like 3 (Saline); MLA, Methyllycaconitine citrate salt; mEJPs, Mini Excitatory junction potentials; mAChRs, Muscarinic acetyl- choline receptors; nAChRs, Nicotinic acetylcholine receptors; Pestanal, Piperonyl butoxide; SEM, Standard error of the mean; curare, Tubocurarine. https://doi.org/10.1016/j.neuroscience.2019.05.016 0306-4522/© 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 47 Downloaded for Anonymous User (n/a) at University of Kentucky from ClinicalKey.com by Elsevier on August 11, 2019. For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.