Nicotinic Acetylcholine Receptors Containing the α7-Like Subunit Mediate Contractions of Muscles Responsible for Space Positioning of the Snail, Helix pomatia L. Tentacle

Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh) responses in muscles to determine the properties of the ACh receptor (AChR), the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium), methyllycaconitine, α-Bungarotoxin (αBgTx) and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na+ and Ca2+ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.


Introduction
Acetylcholine (ACh), the first neurotransmitter to be discovered, is commonly distributed throughout the animal kingdom [1,2]. Biochemical and histochemical studies in gastropod and cephalopod molluscs demonstrated the presence of ACh, the synthesizing enzyme choline acetyltransferase, and the degrading enzyme acetyl cholinesterase, in the central nervous system (CNS) [3,4]. The effects of ACh on molluscan neurons have been shown to involve three separate receptors, one excitatory and two inhibitory, each corresponding to a specific permeability change of the surface membrane [5][6][7]. Kehoe [8] analyzed the ACh-receptor types in the pleural ganglion of Aplysia, concluding that the receptors mediating Cl-dependent inhibition and Na-dependent excitation do resemble nicotinic acetylcholine receptors (nAChR). Thereafter both muscarinic and nicotinic receptors have been found in the CNS of molluscs, located mainly in the neuropil, axons, and glial cells and rarely observed on the soma [9][10][11]. The AChRs cloned so far from invertebrate and vertebrate animals have revealed substantial homologies and also differences between amino acid sequences [2,12]. At the periphery of molluscs, application of ACh has been found to produce depolarization and contraction of the anterior byssus retractor, the pharynx levator, the radula protractor, the gill, and the buccal and swim muscles [10,[13][14][15][16][17][18][19][20][21]. ACh is also the excitatory neurotransmitter at the salivary and mucus gland cells, and has either an inhibitory or a biphasic effect in the heart [15,[22][23][24][25][26][27]. In Aplysia, ACh has been shown to exert a biphasic effect on the radula closer and the parapodial muscle, acting simultaneously at hyperpolarizing and depolarizing ACh receptors [28,29].
Taken together, the results listed above underpin the view that ACh is an important and widely distributed neurotransmitter in molluscs. However, pharmacological data from various muscles in molluscs give a confusing picture regarding the nature of the receptor. At the periphery, the ACh receptor is mostly classified as nicotinic or of mixed subtype; however the receptor classification is far from complete, because the pharmacology of the neuromuscular AChRs appears to vary considerably between different molluscan species and presumably between different muscles [19,30,31]. In vertebrates there are many nAChR subtypes, each consisting of a combination of 17 subunits and mediating diverse physiological functions. Vertebrate nAChRs are widely expressed in the CNS, while in the periphery they mediate transmission at the neuromuscular junction. nAChRs are also found in nonneuronal/muscle cells such as keratinocytes, epithelia, macrophages, etc. [32]. Previously, we have described that externally applied ACh at physiologically relevant concentrations is able to evoke contraction in the flexor muscles (FM) of snail tentacles. The tentacle muscles execute specific tentacle movements during olfaction thereby they are an important part of feeding, avoidance and mating behaviors in the snail [33]. Scanning of the environment by protracted tentacles, and twitching and quivering movements performed during olfactory orientation, are primarily due to the contraction of three FMs: M1, M2 and M3. The ACh effect is comparable to that elicited by electrical nerve stimulation and both responses are effectively attenuated by cholinergic antagonists [34]. It is suggested therefore that contraction evoked by the excitatory neurotransmitter ACh is due to agonist binding at specific membrane receptors, resulting in the opening of cationic (mainly Na + ) channels, which in turn stimulates the Na-Ca exchange mechanism, thereby increasing the concentration of cytosolic Ca 2+ entering from the extracellular space [35]. Here we examine the pharmacology of the neuromuscular contacts of FMs as a model for cholinergic transmission, since the pharmacological properties of the cholinergic response have not been investigated in detail. To address this issue, we characterized the pharmacological profile of the muscle AChR, identified the receptor subtype by Western blot and PCR, and assessed the cellular distribution by immunohistochemistry.

Ethics Statement
There are no ethical approvals required for the research using invertebrate animals, such as the gastropod snail Helix in Hungary; however every effort was made to decrease the number and the suffering of animals. Totally ,250 snails were used. No specific permits were required for the described field collections. The animal collection site (GPS coordinates N:46 o 57,2489 and E:17 o 53,6639 and surrounding) is public and is not situated on private or protected land. Nevertheless the study was conducted in strict accordance with the recommendations in the guidelines for the treatment of animals of the Institutional Biosafety Regulations (VE-I-001/01890-10/2013).

Preparation and contraction recording
Adult specimens of the pulmonate snail, Helix pomatia, were collected from the surrounding area. FMs of the tentacles were isolated and their isotonic contraction recorded as described previously [35]. Briefly, one end of a single muscle was fixed in a slot cut out of Plexiglas, leaving the other end free, and changes in muscle length were measured in control and test solutions using the ocular micrometer on a stereomicroscope. Agonists and antagonists were dissolved in physiological solution containing (in mM) 80 NaCl, 4 KCl, 10 CaCl 2 , 5 MgCl 2 and 10 Tris-HCl (pH = 7.4), and were perfused onto the muscle via a rapid application device. The geometry of the perfusion chamber enabled a fast exchange of solutions (2-3 sec). Flow of solution through linearly arranged tubes was initiated by a gravity perfusion system. For the electrical stimulation of innervated muscles a pair of silver hooked electrodes was placed under the nerve. The ganglion was placed in a pit of the recording chamber and separated from the muscle by a Vaseline gap. 10 ms electrical pulses of 5-10 V were applied at 1-1.5 Hz.

Immunohistochemistry
The CNS and FMs were dissected and fixed in 4% paraformaldehyde diluted in 0.1 M PB, pH 7.4) for 6 h at 4uC. Immunohistochemical procedures were carried out on 40 mm thick cryostat sections made from the cerebral ganglion (CG), as well as on whole mount preparations of the tentacle FMs. Both the sections and the whole mount preparations were incubated for 24 h at room temperature with polyclonal antibodies raised against the a4 and the a7 subunit of the nAChR (human) in rabbit. The antibodies were diluted 1:1000 in PBS-TX containing 0.25% bovine serum albumin. After a short wash in PBS-TX the immunoreaction was visualized using the polymer HRP conjugated donkey anti-rabbit secondary antibody (One-step Polymer-HRP IHC Detection System, BioGenex, USA). Specificity of the antibodies was tested by pre-absorption with the control peptide of the antibody (Abcam). Method controls were performed by omitting the primary antibodies from the incubation solution.

RNA Analysis
Total RNA was extracted using TRI Reagent (Sigma) from freshly homogenated CNS, CM and tentacular FM. The total RNA content was determined by calculating the ratio of the absorbance measured at 260 nm and 280 nm in spectrophotometric analysis. The RNA content of the samples were as follows: 1,2 mg/ml for CNS, 0,55 mg/ml for CM and 0,3 mg/ml for FM.
The quality and quantity of total RNA from CNS and muscles were assessed using formaldehyde denaturation gel. Reverse transcription of the isolated total RNA was performed, and the resulting cDNA was subjected to PCR, using degenerate or nondegenerate primer-pairs designed to detect nAChR subunits in Lymnaea. Primers used to generate amplicons were as follows: non-degenerate primer LnAChR (all primers were purchased from Csertex, Budapest, Hungary and synthetized by Microsynth, Balgach, Switzerland). PCR reaction was performed in 41 cycles (95uC for 3 min, 95uC for 30 sec, 45uC for 1 min, 72uC for 1 min, 72uC for 10 min, store at 4uC) using a T1 thermocycler (Biometra, Goettingen, Germany). Amplified products were run on 2% agarose gel, using ethidium bromide UV detection. For DNA sizing GeneRuler 100 bp DNA Ladder Plus ready-to-use (0.1 mg/ml) was applied (Fermentas, Biocenter, Szeged, Hungary).

Statistics
Data are presented as the mean 6 S.E.M. Statistical significance was assessed in all experiments using one-sample ttest. Statistical significance was accepted when P,0.05 or P,0.01.

Results
The effect of ACh on FMs of the superior tentacles was a slow and consistent tonic, concentration-dependent contracture with no phasic activity. The threshold for the depolarizing effect of ACh was between 10 28 and 10 27 M while the maximum response was observed between 10 24 -10 23 M. The effective concentration of the ACh (EC 50 = 7610 26 M see [34] to induce contraction is sufficiently low to provide compelling evidence that ACh is an excitatory neurotransmitter at the neuromuscular contacts of FMs. At concentrations producing maximum contraction (10 24 M), repeated ACh application did not lead to a reduction in size of the response, demonstrating slow desensitization properties of the muscle AChRs (Fig. 1).

Cholinergic agonists
A variety of cholinergic agonists were tested in an attempt to establish the pharmacological nature of the muscle AChR (Fig. 2.). The action of ACh on muscle cells was mimicked by the nonspecific nAChR agonist nicotine, ChCl and TMA, and closely matched by carbachol and suxamethonium or Succ ( Fig. 2A). PNU-282987, which is a drug acting as potent and selective agonist at the vertebrate homomeric a7 subtype of neural nAChR, elicited a contraction which was 25% of that elicited by ACh. 3-BrCy a potent agonist at vertebrate a7 nAChR at 31.6 nM elicited contraction which was 32% of that elicited by ACh (Fig. 2 B). The RJR 2403 had no potency to evoke contraction (Fig. 2B). The rank order of effectiveness in inducing contraction at a 10 24 M agonist concentration was as follows: ACh.ChCl.TMA.nicotine.carbachol.Succ.PNU-282987. The 3-BrCy at 10 26 M elicited contraction which was 32% of the contraction induced by external ACh. Succ is a depolarizing nAChR agonist used to induce muscle relaxation, and here elicited contraction which was 60% of that induced by ACh. Nicotine and carbachol are known to be muscle type nAChR agonists, while ChCl is a selective and full agonist at the a-BgTx sensitive a7 subunit containing nAChR. The non-selective muscarinic agonists Oxo (M2 agonist) and Arec (partial agonist on nAChR and mAChR) applied at 10 24 M had no potency to induce contraction ( Fig. 2A). These data confirmed that ACh responses of the FM were nAChR-ionophore mediated.

Cholinergic antagonists
In order to characterize the receptor involved, a broad spectrum of nicotinic antagonists were tested at a 10 24 M concentration. The muscle cell response to 10 25 M ACh was rapidly (within 2 min) and reversibly reduced or blocked when the circulating bathing medium contained competitive or non-competitive antagonists such as Myt (a potent nAChR antagonist), DMDA (a peripheral ACh synthesis inhibitor that antagonizes nAChR), Succ, a depolarizing neuromuscular blocker that non-competitively binds to muscle -type nicotinic receptors, Hex, a nondepolarizing, non-specific nAChR blocker, Hem, which blocks the reuptake of choline by the high-affinity choline transporter and is classified as an indirect ACh antagonist or dTC, a competitive antagonist of several ligand gated channels (Fig. 3A). The order of effectiveness in blocking ACh elicited contraction was as follows: DMDA.Myt.dTC.Succ.Hex.Hem. The ACh-elicited contraction-blocking effects of two additional specific antagonists were tested, applied at increasing concentrations (Fig. 3B). MLA, which is a potent antagonist for a7 containing neuronal nAChRs, attenuated the contractions by 35% at 1 nM. Higher concentrations (100 nM and 100 mM) further inhibited the contractions, although at concentrations.40 nM it also interacts with a4b2 and a6b2 receptors. DHbE has moderate selectivity for the neuronal a4 receptor subunit (IC 50 values are 0.19 and 0.37 mM for a4b4 and a4b2 receptors, respectively). At low concentration (1 nM) it attenuated the ACh-evoked contraction of FMs by 13%. At To determine the properties of the AChR and compare with those expected for a7-nAChRs, the response of tentacle muscles was next investigated using ligands that specifically bind to the a7subunit of the receptor. It is known that ACh, ChCl and PNU -282987 are specific activators of the a7-subunit containing nAChR. The a7-nAChR is a cation-selective, rapidly desensitizing receptor which binds a-BgTx with high affinity in mammals. The valuable property of aBgTx is that it binds to muscle nAChRs with high specificity. When aBgTx was applied at 50 and 100 nM to FMs, a concentration dependent decrease in the ACh elicited response was observed (Fig. 4A). The toxin alone showed no effect on the FMs; however, it blocked the ACh elicited muscle contraction by 34% at 100 nM. This effect was partly reversible with 80% of the ACh-elicited contraction recovering after washing the preparation for 15 min. We also examined the effect of aA-CTx PIVA and a-CTx ImI. aA-CTx PIVA is a member of nAChR-targeted Conus peptides. This peptide reversibly blocks postsynaptic muscle fetal a1/b1/c/d and muscle adult a1/b1/e/d nAChRs (Hopkins et al, 1995, Teichert et al, 2006); here, the aA-CTx PIVA attenuated the ACh elicited contraction by 17%. On the other hand, a-CTx ImI, a 12 amino acid peptide isolated from Conus imperialis venom that effectively blocks postsynaptic a7-containing nAChRs in mammal, almost completely blocked the ACh-elicited contraction (Fig. 4B). Muscarinic agonists were unable to elicit contraction of the muscle, but more surprisingly, classic muscarinic antagonists such as Atr, Lev, Scop and Str effectively antagonized contractions evoked by ACh (Fig. 5). These blocking effects were comparable to those of DMDA and Myt. However, the specific muscarinic receptor antagonist Orph proved to be inefficient in antagonizing the ACh-elicited contraction. Overall, the results of the pharmacological experiments presented here suggested that ACh-elicited contractions in the M1, M2 and M3 tentacle muscles were attributable to nAChRs containing the a7 or a7-like receptor subunit.
The presence of a4 and a7 subunits of the nAChR in the posterior tentacles and the CG The pharmacological studies described above strongly suggested that a7-containing, cation selective nAChRs participate in the regulation of the FM contraction. In order to provide additional evidence corroborating the presence and location of the a7 or a7like subunit of the receptor, the protein extract of the FM was analyzed by probing immunoblots with several monoclonal a1-3-5-, and polyclonal a4and a7-specific antibodies (Ab) directed against an epitope located at the extracellular N-terminal domain of human nAChR. No positive reaction was obtained with the  monoclonal Ab. When using the a7 Ab (ab10096), ,60 and 90 kDa bands were observed in the CNS homogenates which were completely eliminated by preincubating the Ab with the appropriate blocking peptide. Using the same Ab, ,62 and 110 kDa bands were labeled in FM homogenate and they were reduced to a faint appearance with the immunogen but not blocked completely. In the CM sample similarly to FM, ab10096 labeled two bands (,60 and 110 kDa) and the 110 kDa band could be almost fully eliminated with the immunogen (Fig. 6A). We tested another anti a7 Ab (ANC-007) purchased from a different producer, to see if there is difference in their specificity. ANC-007 labeled one single band (,160 kDa) only in the CNS homogenate, which could however be completely eliminated with the blocking peptide. In FM homogenate no labeled band(s) was observed (Fig. 6B). Using the a4 Ab (ANC-004), positive reactions were observed as multiple bands in the CNS (,72, 80, 93 and 97 kDa) and FM (,85 and 90 kDa) samples, and all bands were blocked with the immunogen. The ,90 kDa band observed in the FM is the mass, which is also suggested by the Ab (Alomone) producer for a4 subunit (Fig. 6C).
Immunohistochemical experiments further demonstrated that a4 Ab immunoreactivity was mainly confined to neuronal elements in both the tentacle muscles and the CG. In the tentacles, a positive immune response was found in axonal fibers innervating the FMs (Fig. 7A, B, and C), and in the CG (Fig. 7D). In the muscles, immunolabeled axons could be seen running along the muscle fibers, frequently displaying a varicose appearance (Fig. 7A-C). In the CG, medium diameter immunolabeled cell bodies were observed on both the ventral and dorsal surfaces. The majority of the medium diameter neurons were located to the procerebrum and to the pleural lobe of the ganglion. Groups of small diameter neurons were also identified in the pleural lobe. In addition, separated labeled nerve trunks were seen in the neuropil area of the ganglion (Fig. 7D). The a7 Ab revealed positive immunoreactivity in non-neuronal elements in the tentacles, but also in a few neuronal elements of the CG (Fig. 8). In wholemount preparations made from FMs the immunoreaction was located to the muscle fibers, displaying a dotted appearance along the FM and CM at higher magnification ( Fig. 8A-D). In the tentacle stem, immunoreaction could be identified in the CM (Fig. 8 E), at the base of gland cells (Fig. 8 F) and in haemolymph  vessel walls (Fig. 8 G) suggesting a wide distribution of the a7-nAChR subtype at the periphery. To further demonstrate the existence of a7 or a7-like nAChR in FM and verify the specificity of the Abs, experiments were conducted to test for the presence of a7-subunit in the CNS and in the FMs . Fig 9. shows the results of a representative PCR-experiment using non-degenerating primer pair for the amplification of LnAChRA subunit. When degenerating primers for the amplification of the LnAChR B and E (which displays homology to the vertebrate a4 subunit) subunits were used cDNA products could not be visualized. Thus PCR results confirmed the existence of LnAChR A in FM, CM and brain tissues of the snail underpinning evidences obtained by pharmacological and molecular data.

Discussion
The nature of the AChR is nicotinic Previously we have provided convincing evidence that ACh is an excitatory transmitter mediating FM contractions in superior tentacles [34]. ACh released from axons of cholinergic neurons activated receptors which proved to be mainly nicotinic. The FM receptors of the snail tentacle were accessible and sensitive to externally applied ACh and nicotinic receptor agonists. Nicotine, TMA, 3-BrCy and ChCl were as potent as ACh. PNU-282987 (at 10 24 M) the specific a7-subunit containing AChR agonists, elicited contraction which was 25% of that of elicited by ACh. The muscarinic agonist Oxo and Arec were unable to elicit contraction confirming that the AChR is nicotinic. The ACh elicited contraction was antagonized by anti-nicotinic cholinergic drugs such as Myt, Hex, DMDA, Succ, Hem and dTC. Further evidence for a nicotinic type AChR was obtained by using specific anti-nicotinic toxins; from the nicotinic antagonists tried, aA-CTx PIVA attenuated the ACh responses, while aBgTx and a-CTx ImI potently blocked the ACh-evoked contraction of FMs. The receptor characterization was confused, however, by the ability of anti-muscarinic antagonists such as Atr, Scop, Str and Lev to block the contraction evoked by ACh. The non-specific muscarinic receptor antagonists Lev and Str, and also dTC, do block other cys-loop ligand gated channels in molluscs and probably act at the level of an ionophore common to a number of receptors, questioning their usefulness in characterizing receptor type [36]. Figure 6. Demonstration of nAChR subtypes by western blotting using different nAChR subunit specific antibodies. A -Immunodetection of the a7 subunit in homogenates of central nervous system (CNS), flexor (FM) and columellar muscle (CM). The ab10096 recognized two bands (,60 and 90 kDa) in the CNS which were blocked by the control peptide of the antibody (arrows). In the FM, two labeled bands (,62 and 110 kDa) were present which could not be totally eliminated with the immunogen. In the CM, the ,110 kDa band (but not the ,60 kDa band) was blocked (arrow). B -Another a7 specific antibody, the ANC-007 labeled a single band in the CNS at ,160 kDa, which was blocked with the immunogen (arrow). C -The a4 specific antibody ANC-004 labeled several bands between ,80 and 110 kDa both in the CNS and FM, which were completely eliminated by the blocking peptide. Arrow shows the band of the size which is suggested by the producer. doi:10.1371/journal.pone.0109538.g006 Figure 7. Distribution of ACh receptors containing the a4 subunit of nicotinic ACH receptor in the flexor muscles (A-C) and the cerebral ganglion (D). The a4 immunoreactivity is confined to neuronal elements in both the flexor muscles and the ganglion. In the flexor muscle M1 (A) trunks of axons (thick arrows) run along the longitudinal axes of the muscles. In the nerve trunks (B-C) fine immunolabeled axons could be seen (thin arrows). In the cerebral ganglion (D) the majority of labeled cell bodies is located in the pleural lobe (pll) and is gathered in loose groups (empty asterisks). Compact groups of small neurons can also be observed (asterisk). mcmesocerebrum, pc-procerebrum, cplc-cerebro-pleural connective, pn-perineurium. bar: 50 mm. doi:10.1371/journal.pone.0109538.g007 At the same time, the specific muscarinic receptor antagonist Orph did not antagonize the ACh-elicited contraction. Previously it was described that neurons of the snail brain have nicotinic or muscarinic receptor properties in response to various cholinergic agonists. It was concluded that it is not possible to classify cholinergic receptors on the basis of antagonist action alone, at least in molluscs. Based on the almost equal sensitivity to both nicotinic and muscarinic antagonists, it appears that the muscle excitation is mediated by AChRs with mixed pharmacology [31,[37][38][39]. Such a conclusion is seemingly restricted only to lower molluscs, because binding experiments made recently on the cuttlefish nervous system questioned the existence of muscariniclike receptors [9].Only a few studies have examined the detailed pharmacology of ACh-induced responses and receptor subtypes in various molluscan muscles. For example, the branchial heart of the cephalopod Sepia officinalis appears to be controlled by the aBgTx sensitive nAChR. Oxo and other muscarinic agonists do not cause diastolic arrest of the heart [40]. The inhibitory effects of Hex on the cholinergic responses of buccal muscles indicate that the receptor is related to nicotinic receptors [14,41,42]. Hex and dTC are inhibitors of vertebrate nicotinic receptors; however, they are more effective at inhibiting ganglionic than neuromuscular nicotinic receptors. The cholinergic response of Aplysia buccal muscles is also inhibited by muscarinic receptor antagonists, indicating that the cholinergic receptor in buccal muscles has a remarkably different receptor site from vertebrate cholinergic receptors, or that the muscles contain both nicotinic-like and muscarinic-like receptors. For example Ajimal and Ram [43] found that cholinergic contractions of Aplysia rectum could be blocked by both Atr and Hex and also that both agents cause contraction of the esophagus. However, when Hex and Atr were applied together the ACh response was not completely blocked, and the nicotinic antagonist mecamylamine did not leave any unblocked response, as might have been expected if a second type of receptor were mediating a portion of the response. In the current study, the receptors examined were activated exclusively by nAChR agonists and are thus different from those observed previously in the snail brain and muscles. Our results indicate the expression of only one nAChRs subtype in snail FM that is aBgTx sensitive, slowly desensitizing receptor. However muscarinic as well as non-cholinergic antagonists also inhibit the receptor function, distinguishing the tentacle muscle receptor from those of vertebrates. The rank order of agonist potency obtained in this study differs from that found on a7-nAChRs of vertebrates and a7like rapidly desensitizing receptor of Aplysia [44,45]. Kehoe and McIntosh [45] described three distinct nAChRs mediating two Cl-dependent and one cation-dependent cholinergic response in Aplysia neurons. One Cl-dependent ACh-response is rapidly desensitizing and is blocked by a-CTx ImI. The other Cldependent ACh-response, which is slowly desensitizing, is unaffected by the toxin and is antagonized by Hex. With the exception of a-CTx ImI, the receptors revealed similar pharmacology because they were blocked by aBgTx, dTC, DhbE, Str, MLA and activated by cytosine, nicotine and suberyldicholine. It appears that AChRs in Lymnaea neurons have common features with cation-selective vertebrate a7-nAChRs and one type of Aplysia Clconducting receptor [46]. The FM nAChR studied here revealed also similarities to a7-nAChRs of rat intracardiac and superior cervical ganglion neurons, which also desensitize slowly and are reversibly blocked by aBgTx. The AChR of the snail tentacle muscle shares strong pharmacological properties with the aBgTxand a-CTx ImI-sensitive, a7 containing cation permeable receptor that mediates a rapidly desensitizing response in vertebrate neurons. The only exception was that the FMs of the snail tentacle did not desensitize, or did it very slowly.

Nicotinic AChRs desensitize slowly and are blocked by aBgTx
The results presented here provide the first evidence that AChevoked contractions in the FMs of the snail are attributable to nAChRs containing a a7or a7-like subunit. The a7-nAChR is the only subunit to be activated by the endogenous ligands ACh, In the cerebral ganglion (A) immunoreactivity can be seen in groups of small neurons (empty asterisks) in the mesocerebrum (mc) and procerebrum (pc). Additionally a few of immunolabeled varicose fibers (arrows) can also be observed. In the flexor muscles (B) immunoreactivity is distributed on thin muscle fiber trunks (arrows) and displayed dotted appearance (arrow heads) (B-C) which is a sign of the postsynaptic localization. In the columellar muscle (E) immunoreactivity is distributed on the surface of muscle fibers (black arrows). The immunoreactivity on the sarcolemma displays dotted appearance (insert, arrow heads). In the stem immunoreactivity (arrows) is located on the gland cells (gc) (F) and the wall of haemolymph vessels (v) (G). In both cases the immunoreactivity (arrow heads) shows dotted appearance (see inserts F, G). bar: 50 mm. doi:10.1371/journal.pone.0109538.g008 3-BrCy, PNU-282987 and ChCl. The latter was as potent as ACh, TMA and nicotine in eliciting contraction in the FMs. ChCl has received attention because it is a selective and full agonist at aBgTx-sensitive, a7 subunit containing nAChRs in vertebrates [47]. aBgTx is a potent and selective antagonist that binds with high affinity to vertebrate muscle and neuronal a7-, a8and a9containing nAChR [48]. Two groups of nAChRs exist, those that bind aBgTx with high affinity and for those that bind with low affinity. The latter group of receptors is found in the CNS of vertebrates and is referred to as neuronal nAChR. Neuronal nAChRs possess a higher permeability to Ca 2+ than muscle type nicotinic receptors and are more sensitive to ganglionic blockers [49]. Invertebrate nAChR genes isolated to date all show a greater homology to the vertebrate neuronal type nAChR than the muscle nAChRs [2]. Pharmacological studies of cholinergic neuromuscular contacts in molluscs have indicated a similarity with neuronalneuronal synapses [29].
By using degenerate PCR cloning several nAChR subunits were identified from the CNS of Lymnaea stagnalis, which formed functional receptors responding to ACh when expressed in oocytes. These subunits were divided into two groups according to their ability to conduct cations or anions: the LnAChR A and LnAChR B subunits. The cation conducting LnAChR A is a functional homopentameric receptor when expressed in oocytes, which can be activated by nicotine and choline and is sensitive to aCTx ImI and aBgTx [50]. The pharmacological properties of the FM nAChR described here display similarities to the Lymnaea LnAChR A and to vertebrate a7 subunit-containing receptors.
The presence of the a7or a7-like AChR subunit in the snail tentacle muscles is not surprising because it is thought that this subunit gene is closest to the ancestor genes that existed millions of years ago before separation of vertebrate and invertebrate lineages [40,51,52]. For example the simple nematode, Caenorhabditis elegans, possesses the most extensive known gene family of nAChR-like subunits and shows the greatest similarity with nAChR subunits of both invertebrates and vertebrates [53]. In addition the functional and pharmacological properties of the gene product are also conserved, so as to form homopentamers with high Ca-permeability and aBgTx sensitivity. 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 [35]. Unfortunately, the Western blot experiments gave dubious results revealing several peptide bands in the range of 40-140 kDa. It is described that certain commercially available a7-antibodies lack specificity because they give the same immune response in wild-type and knock-out mice although RNA analyses confirmed the disruption of the a7 allele and lack of a7 message in the knockouts [54,55].
Obscured data were compensated by our PCR experiments where the presence and expression of the Lymnaea-type (LnAChR A) nAChR subunit was assessed by PCR. PCR profiling detected expression of the LnAChR A subunit in the FM and CM and also in CNS of the snail in support of our pharmacological and immunological data. Interestingly, in the snail the a7 subunit containing nAChRs were observed mainly on muscle cells. The a4 subunit containing AChRs were detected in axons innervating the FMs, suggesting their involvement in possible feedback control of the ACh release from nerve terminals.

Conclusions
In conclusion, our work for the first time shows the existence of a7-like nAChR in FM of the snail tentacle, leading to the idea that postsynaptically a7-like nicotinic receptors may physiologically contribute to cholinergic transmission at the neuromuscular junction in molluscs. Based on pharmacological properties we conclude that the nACh receptors of the FM are cation permeable, slowly desensitizing nicotinic receptors that are sensitive to aCTx ImI and less sensitive to aBgTx and can be activated by nicotine, 3-BrCy, TMA and ChCl. Immunodetection experiments confirmed the presence of a7-like AChRs in muscle cells, and a4-AChRs in nerves innervating the muscle. Pharmacological and immune data were further supported by the presence of transcript in the FM coding for the LnAChR A subunit as assessed using PCR assays. We conclude that the nAChRs in FM of the snail tentacle more closely resemble a LnAChR A and vertebrate neuronal a7-AChRs receptor than a muscle receptor. This is the first demonstration of postsynaptic expression and an obligatory role for a functional a7-like nAChR in the molluscan periphery.