Expression of Cys‐loop receptor subunits and acetylcholine binding protein in the mechanosensory neurons, glial cells, and muscle tissue of the spider Cupiennius salei

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Pentameric Cys‐loop receptors play key roles in chemical synapses throughout central and peripheral nervous systems. Mammalian Cys‐loop receptors include anion‐selective γ‐aminobutyric acid type A (GABAA) and glycine receptors, and cation‐selective nicotinic acetylcholine (nACh) and 5‐HT3 receptors (Miller and Smart, 2010). Invertebrates have several types of GABAA and nACh receptors as well as glutamate‐ (GluCl), histamine‐ (HisCl), and pH‐ (pHCl) gated Cl channels (Hosie et al., 1997; Ozoe et al., 2013; Lees et al., 2014). Invertebrate Cys‐loop receptors are prime targets for widely used control agents against agricultural pests and disease vectors (Tomizawa and Casida, 2009; Wolstenholme, 2010). However, these toxins also bind to homologous receptors in beneficial species, such as spiders (Lynagh and Lynch, 2012; Zemkova et al., 2014).
Functional Cys‐loop receptors are composed of five subunits that assemble around a central ion‐conducting pore. Some well‐known mammalian receptors consist of five identical subunits, but most are heteropentamers assembled from two or more different subunits, and this arrangement determines the pharmacological and physiological properties of these receptors (Smart and Paoletti, 2012). Native invertebrate Cys‐loop receptor subunit compositions are unknown, but genes that have been expressed in cultured cells or oocytes form homo‐ and/or heteropentameric channels with varying resemblance to native receptors (Eguchi et al., 2006; Hosie et al., 2007; Hibbs and Gouaux, 2011; Kita et al., 2014). To understand the physiological roles of these receptors in vivo and to determine how they respond to toxins, it is essential to decode the subunit compositions of native receptors.
The Central American wandering spider, Cupiennius salei (Keys), is a model species for neurophysiological and developmental studies (Barth, 2002; McGregor et al., 2008). Some of its highly developed mechanosensory organs are accessible to electrophysiological and imaging experiments that are possible in only a small number of animal models (Seyfarth 1985; Barth 2002; French et al., 2002). In addition, all mechanosensory neurons and muscle fibers in the spider legs receive extensive innervation via centrally originating efferents that contain GABA and glutamate, and synaptic contacts also occur between neighboring efferents (Fabian‐Fine et al., 2002), making these preparations accessible models for synaptic modulation. Electrophysiological and immunocytochemical investigations have suggested that the mechanosensory neurons and muscle fibers have a variety of Cys‐loop receptors (Maier et al., 1987; Panek et al., 2002; Panek and Torkkeli, 2005; Widmer et al., 2005; Torkkeli et al., 2011), but until now the lack of molecular information in spiders has made their identification impossible.
Recently, we assembled and characterized 16 Cys‐loop subunits from the C. salei central and peripheral nervous system transcriptomes (Torkkeli et al., 2015). These sequences include 12 subunits that are predicted to form anion‐permeable channels, plus three cation‐conducting nACh receptor subunits and a putative acetylcholine‐binding protein (AChBP) that lacks the transmembrane region. This molecular information makes it possible to explore the expression patterns of Cys‐loop subunits in the spider mechanosensory and proprioceptive organs. The transmitter responses of neurons innervating mechanosensory VS‐3 slit sensilla in the spider patella have been extensively investigated (Panek et al., 2002; Panek and Torkkeli, 2005; Pfeiffer et al. 2009; Torkkeli et al., 2012), and the major aim here was to use in situ hybridization to discover which combinations of Cys‐loop subunits form the receptors that mediate these responses. Spider muscle fibers are innervated by excitatory motor nerves and inhibitory efferent fibers (Maier et al., 1987), but it is not known on which types of receptors they act. Therefore, we also explored the gene expression in muscle tissue by using quantitative real‐time PCR.
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