Int. J. Devl Neuroscience 58 (2017) 9–16 Contents lists available at ScienceDirect International Journal of Developmental Neuroscience j ourna l ho me page: www.elsevier.com/locate/ijdevneu Cellular protein and mRNA expression of 1 nicotinic acetylcholine receptor (nAChR) subunit in brain, skeletal muscle and placenta Atqiya Aishah a,b , Tina Hinton a,b , Rita Machaalani b,c,∗ a Discipline of Pharmacology, The University of Sydney, NSW, 2006, Australia b The Bosch Institute, The University of Sydney, NSW, 2006, Australia c Department of Medicine, The University of Sydney, NSW, 2006, Australia a r t i c l e i n f o Article history: Received 6 October 2016 Received in revised form 17 January 2017 Accepted 23 January 2017 Available online 30 January 2017 Keywords: CHRNB1 Human Brainstem Formalin fixed in situ hybridization Immunohistochemistry a b s t r a c t The 1 nicotinic acetylcholine receptor (nAChR) subunit is a muscle type subunit of this family and as such, is found predominantly in muscle. Recent reports document its expression in other tissues and cell lines including adrenal glands, carcinomas, lung and brain. However, the majority of studies were of tissue lysates, thus the cellular distribution was not determined. This study aimed to determine the cellular distribution of the 1 nAChR subunit in the brain, at both the mRNA and protein levels, using non- radioactive in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, and to compare it to two muscle tissue types, skeletal and placenta. Tissue was formalin fixed and paraffin embedded (all tissue types) and frozen (placenta) from humans. Additional control tissue from the piglet and mouse brain were also studied, as was mRNA for the 3 nAChR and N-methyl-d-aspartate receptor 1 (NR1) subunit. We found no 1 nAChR subunit mRNA expression in the human and piglet brain despite strong protein expression. Some signal was seen in the mouse brain but considered inconclusive given the probes designed were not of 100% homology to the mouse. In the skeletal muscle and placenta tissues, 1 nAChR subunit mRNA expression was prominent and mirrored protein expression. No 3 nAChR or NR1 mRNA was seen in the skeletal muscle, as expected, although both subunit mRNAs were present in the placenta. This study concludes that further experiments are required to conclusively state that the 1 nAChR subunit is expressed in the human, piglet and mouse brain. © 2017 ISDN. Published by Elsevier Ltd. All rights reserved. 1. Introduction Nicotinic acetylcholine receptors (nAChRs) are part of the Cys- loop family of ligand gated ion channels. The receptor is formed as a pentamer, with five subunits arranged around a central pore that is permeable to cations upon binding of an agonist, such as acetyl- choline (ACh) or nicotine. A total of seventeen nAChR subunits have been characterized and two types of nAChRs are known; muscle type and neuronal type. The 1 nAChR subunit is traditionally char- acterized as a muscle type subunit, expressed in either (1) 2 1d or (1) 2 1d receptor complexes (Millar, 2003; Mishina et al., 1985). Receptors containing 1 subunits have been found pre- dominantly in the neuromuscular junction (NMJ), and have been attributed to the efficient clustering of nAChRs and anchoring of the receptors to the cytoskeleton (Wheeler et al., 1994). This is impor- ∗ Corresponding author at: Department of Medicine, Room 206, Blackburn Build- ing, DO6, University of Sydney, NSW, 2006, Australia. E-mail address: ritam@med.usyd.edu.au (R. Machaalani). tant for formation of synapses in the NMJ, evidenced by data where the exchange of the 1 with 2 subunits in nAChRs expressed in Xenopus oocytes resulted in decreased cluster formation of the receptor proteins (Kalamida et al., 2007; Wheeler et al., 1994). Furthermore, lack of the 1 subunit in 1 complexes resulted in weaker channel activity upon agonist binding (Kalamida et al., 2007). The characterization of nAChRs in skeletal muscle of rat has been extensively reviewed (Schuetze and Role, 1987), with more recent data reporting expression of the 1 nAChR subunit mRNA (GTEx; www.gtexportal.org/home/gene/CHRNB1) and pro- tein within muscle tissue as well as other rat and human peripheral tissue types including liver, adrenal medulla, bronchial cells, and the placenta, summarized in Table 1. However, most of these studies applied polymerase chain reaction methods for mRNA expression and immunoblotting for protein expression; thus, the cellular distribution of the subunit has not been delineated. Expres- sion at the cellular level provides novel data in determining cell type-specific functionality. In situ hybridization (ISH) and immuno- histochemistry (IHC) are two common techniques used in cellular http://dx.doi.org/10.1016/j.ijdevneu.2017.01.011 0736-5748/© 2017 ISDN. Published by Elsevier Ltd. All rights reserved.