Embryonic ethanol exposure alters synaptic properties at zebrafish neuromuscular junctions Nicole J. Sylvain a , Daniel L. Brewster a,b , Declan W. Ali a,b, ⁎ a Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 b Centre for Neuroscience, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 abstract article info Article history: Received 29 March 2010 Received in revised form 1 December 2010 Accepted 9 December 2010 Available online 16 December 2010 Keywords: Ethanol Synapse NMJ Fetal alcohol syndrome AChR mEPC Pre-natal alcohol exposure induces delays in fine and gross motor skills, and deficiencies in reflex development via mechanisms that remain to be elucidated. The purpose of the present study was to investigate the effect of embryonic ethanol exposure (16-hour exposure window with1.5%, 2% or 2.5% EtOH) on synaptic properties at the neuromuscular junction (NMJ) in 3 day post fertilization (dpf) zebrafish larvae. Immunohistochemical studies show that exposure of embryos to 2.5% ethanol for 16 h results in motor neuron axons that display abnormal branching patterns. Co-labelling embryos with pre-synaptic markers such as SV-2 or 3A10, and the post-synaptic marker, α-bungarotoxin, which irreversibly binds to nicotinic acetylcholine receptors (nAChRs), indicates that pre- and post-synaptic sites are properly aligned even when motor neuron axons display abnormal morphology. Miniature endplate currents (mEPCs) recorded from muscle fibers revealed the presence of two types of mEPCs that we dubbed fast and slow. Ethanol treated fish experienced significant changes in the frequencies of fast and slow mEPCs, and an increase in the rise time of slow mEPCs recorded from red muscle fibers. Additionally, embryonic exposure to ethanol resulted in a significant increase in the decay time of fast mEPCs recorded from white fibers. Mean mEPC amplitude was unaffected by ethanol treatment. Together, these results indicate that zebrafish embryos exposed to ethanol may experience altered synaptic properties at the NMJ. © 2010 Elsevier Inc. All rights reserved. 1. Introduction Embryonic organisms exposed to ethanol exhibit physical and mental deficits such as joint and cardiac abnormalities, motor and reflex developmental delays and mental retardation (Jones and Smith, 1973b; Jones et al., 1973; Kalberg et al., 2006; Staisey and Fried, 1983). The underlying mechanisms for many of these deficits remain unresolved. In this study we set out to determine if the motor deficits that occur as a result of embryonic exposure to ethanol, are due to abnormalities in synaptic activity at the neuromuscular junction (NMJ). This study follows from a recent one in which we found that zebrafish embryos exposed to ethanol in the first day of development, exhibited significant abnormalities in gross morphology, swimming behavior, and motor neuron and muscle fiber morphology (Sylvain et al.). These findings prompted us to ask whether synaptic activity at the NMJ was altered by exposure to ethanol. A number of recent studies have used fish embryos as a model for embryonic ethanol toxicity (Arenzana et al., 2006; Bilotta et al., 2004, 2002; Bradfield et al., 2006; Carvan et al., 2004; Dlugos and Rabin, 2003; Matsui et al., 2006; Oxendine et al., 2006; Tanguay and Reimers, 2008). Zebrafish embryos offer certain advantages over mammalian models when studying the effects of embryonic ethanol exposure. For instance, the concentration and the time course of alcohol exposure can be accurately controlled since the embryos develop in a chorion outside the female. Embryos can be accurately aged and immunohis- tochemistry performed without the need for tissue dissection. However, drawbacks of using zebrafish as a model for embryonic ethanol exposure include the lack of maternal–embryo interaction during gestation, and relatively high (brief) doses of EtOH are required to induce defects that are similar to those observed in mammalian models (Arenzana et al., 2006; Bilotta et al., 2004; Cudd, 2005; Dlugos and Rabin, 2003; Matsui et al., 2006; Tanguay and Reimers, 2008). Nevertheless, zebrafish allow for a range of studies that may be difficult to perform in other preparations. Importantly, zebrafish embryos exposed to ethanol exhibit defects that are similar to children with fetal alcohol syndrome disorders (FASD), including defects in eye development, heart rate, apoptosis in the CNS, skeletal morphogenesis and locomotion (Bilotta et al., 2004; Carvan et al., 2004). In young zebrafish, two types of muscle fibers are present: slow tonic (red) fibers, which are found in only one layer and are located right underneath the skin, and fast twitch (white) fibers, which are Neurotoxicology and Teratology 33 (2011) 313–321 ⁎ Corresponding author. Department of Biological Sciences, CW-405, Biological Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. Tel.: +1 780 492 6094; fax: +1 780 492 9234. E-mail address: declan.ali@ualberta.ca (D.W. Ali). 0892-0362/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2010.12.001 Contents lists available at ScienceDirect Neurotoxicology and Teratology journal homepage: www.elsevier.com/locate/neutera