Identification and Functional Expression of a Family of Nicotinic Acetylcholine Receptor Subunits in the Central Nervous System of the Mollusc Lymnaea stagnalis

Abstract

We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 subunits that display a high sequence similarity to nAChR subunits, of which 10 are of the ␣-type, 1 is of the ␤-type, and 1 was not classified because of insufficient sequence information. Heterologous expression of identified subunits confirms their capacity to form functional receptors responding to acetylcholine. The ␣-type subunits can be divided into groups that appear to underlie cation-conducting (excitatory) and anion-conducting (inhibitory) channels involved in synaptic cholinergic transmission. The expression of the Lymnaea nAChR subunits, assessed by real time quantitative PCR and in situ hybridization, indicates that it is localized to neurons and widespread in the CNS, with the number and localization of expressing neurons differing considerably between subunit types. At least 10% of the CNS neurons showed detectable nAChR subunit expression. In addition, cholinergic neurons, as indicated by the expression of the vesicular ACh transporter, comprise ϳ10% of the neurons in all ganglia. Together, our data suggested a prominent role for fast cholinergic transmission in the Lymnaea CNS by using a number of neuronal nAChR subtypes comparable with vertebrate species but with a functional complexity that may be much higher. Nicotinic acetylcholine receptors (nAChRs)3 belong to the Cys loop family of pentameric ligand-gated ion channels (LGICs) together with the 5-HT 3 , GABA type A/C, and glycine receptors. nAChRs consist of an extracellular ligand-binding domain (LBD), a transmembrane ion channel, and an intracellular domain (1). In the mammalian central nervous system (CNS), eight ␣-type (␣2-7 and ␣9 -10) and three ␤-type (␤2-4). nAChR subunits have been identified that selectively assemble into nAChR subtypes with different pharmacology, cation conductance, and cellular localization. In the mammalian CNS, nAChRs predominantly mediate presynaptic modulation of neurotransmitter release (for example see Ref.2) and are to a limited extent involved in direct, fast synaptic transmission (for example see Refs. 3-7). In contrast, in the molluscan CNS, the mode of fast synaptic cholinergic transmission seems to prevail. The CNS of the freshwater snail Lymnaea stagnalis consists of ϳ20,000 large and identifiable neurons, of which many were shown to express functional nicotinic acetylcholine receptors (8, 9). In particular, in well described neuronal networks, various nAChR subtypes were shown to mediate synaptic transmission (10, 11). Uniquely, molluscs possess excitatory and inhibitory nAChR subtypes conducting cations and anions, respectively (9, 12-15). Also, different excitatory and inhibitory nAChR subtypes mediate cholinergic transmission at single identified synapses, the differential contribution of which is regulated by soluble extracellular factors (11, 16). Therefore, functio...

Neonicotinoid insecticides inhibit cholinergic neurotransmission in a molluscan (Lymnaea stagnalis) nervous system

“…The acetylcholine receptors (AChRs) are particularly often involved in toxic effects of bioactive substances, like animal venoms, plant toxins, and also by neonicotinoid type insecticides (Daly 2005;Dutertre and Lewis 2006;Tomizawa and Casida 2003). The basic structural and pharmacological information on AChRs initially arose from vertebrates (Dani, 2001), but further studies suggest their much higher diversity in invertebrates (van Nierop et al, 2006;Wu and Lukas, 2011;Holden-Dye et al, 2013). In insects, moreover, molecular studies have also revealed taxonomical differences among the subclasses of nACh receptors (Dupuis et al, 2012;Liu et al, 2013).…”

“…the pond snail, Lymnaea stagnalis or the edible snail, Helix pomatia) the identifiable giant neurons (with diameters up to 100 µm) allow potential toxic effects to be examined using intracellular electrophysiology techniques. Acetylcholine is a neurotransmitter and modulatory substance both in the CNS and the periphery of these organisms; moreover, cholinergic receptor subtypes including nAChRs have also been established (Walker et al, 1996;Vulfius et al, 2005;van Nierop et al, 2006;Krajcs et al, 2014). Therefore the identifiable snail neurons provide a suitable tool to characterize the interactions between the ACh receptors (AChRs) and potentially toxic substances including heavy metals, insecticides or mycotoxins (Arvanov et al, 1993;Gyori et al, 1994;Gyori et al, 2007).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…Frustratingly, the heterologous expression of invertebrate nAChRs has proved to be extremely difficult [59,95] and, in several instances, this has often been achieved only by co-expression with vertebrate nAChR subunits [65,91,96,97]. More recently, expression studies in Xenopus oocytes have identified a family of anion-selective nAChRs, cloned from both the nematode Caenorhabditis elegans [98] and the mollusc Lymnaea stagnalis [99,100].…”

Anatomical and physiological background permitting spatial odor sensation in stylommatophoran molluscs

“…Pharmacological and immune data therefore were further supported by the presence of transcript in the FM coding for the LnAChR A subunit. The LnAChR A subunit is related to the homo-oligomeric human α7 nAChR and when expressed in oocytes it Acta Biologica Hungarica 67, 2016 is activated by cytosine and nicotine and blocked by αBgTX, αCTX-IMI [94]. However the nAChR in the snail FMs desensitizes slowly and also conducts Na + along with Ca 2+ , suggesting that the receptor is not a homopentamer [49].…”