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

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 α-type, 1 is of the β-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 α-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 ∼10% 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. 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 α-type, 1 is of the β-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 α-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 ∼10% 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) 3The abbreviations used are: nAChRnicotinic acetylcholine receptorCNScentral nervous systemLnAChRLymnaea nAChR subunitLGICligand-gated ion channelGABAγ-aminobutyric acidLBDligand-binding domain5-HT5-hydroxytryptaminentnucleotideTMtransmembraneCDCcaudodorsal cellAChacetylcholineAChBPacetylcholine-binding proteinqPCRquantitative PCRLVAChTLymnaea vesicular ACh transporterLYClight yellow cellsLGClight green cellsfwdforwardrevreverseALanterior lobeISHin situ hybridization. belong to the Cys loop family of pentameric ligand-gated ion channels (LGICs) together with the 5-HT3, GABA type A/C, and glycine receptors. nAChRs consist of an extracellular ligand-binding domain (LBD), a transmembrane ion channel, and an intracellular domain (1Corringer P.J. Le Novere N. Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar). In the mammalian central nervous system (CNS), eight α-type (α2–7 and α9–10) and three β-type (β2–4). 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. 2Alkondon M. Pereira E.F. Albuquerque E.X. Brain Res. 1998; 810: 257-263Crossref PubMed Scopus (233) Google Scholar) and are to a limited extent involved in direct, fast synaptic transmission (for example see Refs. 3Grady S.R. Meinerz N.M. Cao J. Reynolds A.M. Picciotto M.R. Changeux J.P. McIntosh J.M. Marks M.J. Collins A.C. J. Neurochem. 2001; 76: 258-268Crossref PubMed Scopus (135) Google Scholar, 4Clarke P.B. Reuben M. Br. J. Pharmacol. 1996; 117: 595-606Crossref PubMed Scopus (239) Google Scholar, 5Kaiser S. Wonnacott S. Mol. Pharmacol. 2000; 58: 312-318Crossref PubMed Scopus (203) Google Scholar, 6Reuben M. Clarke P.B. Neuropharmacology. 2000; 39: 290-299Crossref PubMed Scopus (75) Google Scholar, 7Wonnacott S. Kaiser S. Mogg A. Soliakov L. Jones I.W. Eur. J. Pharmacol. 2000; 393: 51-58Crossref PubMed Scopus (196) Google Scholar). 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 ∼20,000 large and identifiable neurons, of which many were shown to express functional nicotinic acetylcholine receptors (8Zeimal E.V. Vulfius C.A. Salanki J. Neurobiology of Invertebrates. Academiai Kiado, Budapest, Hungary1967: 255-265Google Scholar, 9Vulfius E.A. Veprintzev B.N. Zeimal E.V. Michelson M.J. Nature. 1967; 216: 400-401Crossref PubMed Scopus (11) Google Scholar). In particular, in well described neuronal networks, various nAChR subtypes were shown to mediate synaptic transmission (10Yeoman M.S. Parish D.C. Benjamin P.R. J. Neurophysiol. 1993; 70: 37-50Crossref PubMed Scopus (44) Google Scholar, 11Woodin M.A. Munno D.W. Syed N.I. J. Neurosci. 2002; 22: 505-514Crossref PubMed Google Scholar). Uniquely, molluscs possess excitatory and inhibitory nAChR subtypes conducting cations and anions, respectively (9Vulfius E.A. Veprintzev B.N. Zeimal E.V. Michelson M.J. Nature. 1967; 216: 400-401Crossref PubMed Scopus (11) Google Scholar, 12Chiarandini D.J. Gerschenfeld H.M. Science. 1967; 156: 1595-1596Crossref PubMed Scopus (22) Google Scholar, 13Chiarandini D.J. Stefani E. Gerschenfeld H.M. Science. 1967; 156: 1597-1599Crossref PubMed Scopus (29) Google Scholar, 14Chemeris N.K. Kazachenko V.N. Kislov A.N. Kurchikov A.L. J. Physiol. (Lond.). 1982; 323: 1-19Crossref Scopus (43) Google Scholar, 15Vulfius C.A. Tumina O.B. Kasheverov I.E. Utkin Y.N. Tsetlin V.I. Neurosci. Lett. 2005; 373: 232-236Crossref PubMed Scopus (25) Google Scholar). 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 (11Woodin M.A. Munno D.W. Syed N.I. J. Neurosci. 2002; 22: 505-514Crossref PubMed Google Scholar, 16Hamakawa T. Woodin M.A. Bjorgum M.C. Painter S.D. Takasaki M. Lukowiak K. Nagle G.T. Syed N.I. J. Neurosci. 1999; 19: 9306-9312Crossref PubMed Google Scholar). Therefore, functionally identified neurons in the Lymnaea CNS are a valuable model to explore nAChR function and diversity related to signal transmission in the nervous system. The lack of molecular information on nAChRs in molluscs, however, prevents a comprehensive analysis of cholinergic transmission. To address this issue, we identified nAChR subunits in the Lymnaea CNS, and we assessed their cellular expression by in situ hybridization and quantitative PCR. Our data underscore the postulated widespread role for fast cholinergic excitatory and inhibitory transmission in the Lymnaea CNS, involving a number of nAChR subtypes that are comparable with vertebrate species but with a functional complexity that may be much higher. nicotinic acetylcholine receptor central nervous system Lymnaea nAChR subunit ligand-gated ion channel γ-aminobutyric acid ligand-binding domain 5-hydroxytryptamine nucleotide transmembrane caudodorsal cell acetylcholine acetylcholine-binding protein quantitative PCR Lymnaea vesicular ACh transporter light yellow cells light green cells forward reverse anterior lobe in situ hybridization. Animals—Adult L. stagnalis (shell length, 28–34 mm) bred under laboratory standard conditions (17van der Steen W.J. van der Hoven N.P. Jager J.C. Neth. J. Zool. 1969; 19: 131-139Crossref Google Scholar) were used. PCR Using Degenerate Oligonucleotides—Total RNA was isolated using Trizol® reagent (Invitrogen) from freshly dissected CNS or from pooled LPeD1 (10×), RPeD1 (10×), and VD4 (6×) neurons with axons attached and isolated by mechanical suction from Lymnaea brain ganglia. For CNS preparations, mRNA was isolated using (dT)26-coated magnetic beads (Dynal, Oslo, Norway). RNA was reverse-transcribed using hexanucleotide primers and Moloney murine leukemia virus-reverse transcriptase according to the manufacturer's protocol (Promega, Madison, WI). 3′- and 5′-directed degenerate oligonucleotides (Isogen Bioscience, Amsterdam, The Netherlands) were synthesized with an EcoRI or HindIII restriction site, respectively, at the 5′-ends. The 5′-directed oligonucleotides include the following: primer 1, 5′-cgggaattcaaytayaaymmiytigarmgnccngt-3′; primer 2, 5′-cggaattccarathhtigaygtigaygaraaraayca-3′; primer 5, 5′-cggaattcaarttyggiwsitggwsitaysrngg-3′; primer 7a, 5′-aagaattccngayrtistictitayaayaaygcnga-3′; primer 7b, 5′-aagaattccngayrtnstictitayaaywsigcnga-3′. The 3′-directed oligonucleotides include the following: primer 3, 5′-cgcaagcttswrttrtaiariariayrtcnggyttcca-3′; primer 4, 5′-cgcaagcttcantkytgiwcrtcraanggraacca-3′; primer 6, 5′-cgcaagcttayrtcirbrtaiggytcikkrcarca-3′; primer 8, according to were used on a CNS or on of identified neurons cell for VD4 and cell for in an PCR for for and for using a primers were used in a PCR for for and for using of the PCR as were on and of were with EcoRI and and For were by of an of the CNS of L. stagnalis was isolated by and primer were on nAChR subunit primer were used together with primer and or and in with for for and for using was on and PCR to or were and were using J. T. Res. 2000; PubMed Scopus Google Scholar). were by three PCR on of pooled Lymnaea For of were into of were by of were by data for the are as and were with Res. PubMed Scopus Google Scholar). of protein were using J. T. Res. 2000; PubMed Scopus Google Scholar). were from the data using L. N. A. L. M. A. 2002; PubMed Scopus Google Scholar, A. E. A. 2002; Google Scholar). of was by using N. S. S. J. Mol. 1999; PubMed Scopus Google Scholar). analysis was by using the of to and of were from the were and as described M. S. A. PubMed Scopus Google Scholar). were in the by that was by and were from or were by the cell at a and the or by to in the ACh were by the of the as a function of the of the cell in of the of the CNS, and was isolated as described by a according to the manufacturer's and by and was using and of Moloney murine leukemia transcriptase (Promega, Madison, WI). RNA from pooled VD4 (6×) and LPeD1 (6×) neurons, for was and was according to van and van J. van in Google Scholar). were and in of primers were using to as and and and and and and and and and and and and and and and and of primers and were as described Munno D.W. van W.J. Syed N.I. J. PubMed Scopus Google Scholar). were used to the expression to Lymnaea number or Lymnaea number For the is as were to expression for by is the of the primer For the was on expression from were as the of CNS, and neuronal expression of all was with the at least the In situ hybridization was as described van J. Syed N.I. van J. Neurosci. 2001; PubMed Google Scholar). RNA were from to of sequence of in and number In situ hybridization for nAChR subunits were to that the intracellular loop that a of sequence of nAChR from the CNS of L. the of nAChR subunits, we a PCR using degenerate primers on various of identified different that showed similarity to a of the of nAChR subunits In addition, Lymnaea nAChR subunits were from hybridization of a CNS for nAChR and A. and from an Lymnaea CNS L. of sequence information of the was by PCR using a Lymnaea CNS In 12 were identified that were Lymnaea nAChR subunits sequence for not be For a was identified sequence diversity in the intracellular loop between and The in a protein of Lymnaea nAChR subunits Lymnaea nAChR subunits were identified by PCR using degenerate oligonucleotides in of on various by to three of the to as and and were and and and and of single RNA and were from a and an L. of in a analysis of the 12 protein similarity with as well as with functionally nAChR subunits not with subunits indicates the with nAChR subunits described Lymnaea GABA receptor subunits E. E.A. J. PubMed Scopus Google Scholar, E.A. Lett. 1993; PubMed Scopus Google Scholar) not sequence with the identified protein identified possess transmembrane with a at the and at the that are to the in nAChR the subunits by which are for to the Cys loop family of Together, the of the identified as Lymnaea nAChR of the of the Cys in the loop of the of and are classified as α-type nAChR subunits, and because of the of Cys is classified as β-type in loop the subunit an the of Lymnaea nAChR all identified subunits, and the of sequence and the For nAChR subunits sequence of not the sequence with the and nAChR subunits the is for and the subunit with sequence of Lymnaea and nAChR subunits of sequence of subunits and Lymnaea number of of and nAChR The intracellular loop was from and light the and for on sequence in a on sequence on sequence we three of subunits with and and and and of the subunits a to a molluscan protein by cells to the nAChR Syed N.I. van J. J. van der van K. Nature. 2001; PubMed Scopus Google Scholar, K. van W.J. M. van der J. Nature. 2001; PubMed Scopus Google Scholar) the sequence with the subunit The groups of subunits that include the and and the and subunits between receptors and nAChR subunits on seems as from subunits as it is from nAChR a to the vertebrate subunit that and are related to subunits to assemble into of in the contribution of subunits to functional we subunits in a subunits were for nAChR subtypes to be of functional receptors was with or subunits and with subunits that express the or subunits to of ACh but not to of 3, and or glycine not of or subunits are to nicotinic and and and A. S. van J. J. Neurosci. 2005; PubMed Scopus Google Scholar). are expressing the subunit to ACh or receptors are to and glycine not The of many subunits to express as functional receptors as well as the of the by receptors of the subunit that subunits are for functional expression. a subunit were on the subunit and similarity of expression in the CNS of the β-type subunit with subunits, the in mammalian neuronal nAChRs or not in functional be with subunits and that the subunits in the CNS of that with or not be from of expressing not into with subunits not To the of the were and in or shown in of the of the in to In a large of was the was with which that the receptors is by of display comparable of channels not We that to and to receptors. of in the Lymnaea and of expression of subunits was using and real time analysis on from various and showed that subunits are in the CNS In the CNS the for the identified subunits are in neurons as shown by was in cells in the CNS and and in of in the CNS We that all identified Lymnaea nAChR subunits are neuronal nAChR of subunits and in the Lymnaea The expression of subunits and of the in the central of the Lymnaea CNS was using and indicates the expression in the different as by under The an of the and the of neurons with the number of not to the number of The of neurons with on of the Lymnaea the the identified RPeD1 was The abbreviations used are as and and of subunits and in the Lymnaea The expression of subunits and of the in the central of the Lymnaea CNS was using and indicates the expression in the different as by under The an of the and the of neurons with the number of not to the number of The of neurons with on of the Lymnaea the the identified RPeD1 was The abbreviations used are as and and In the CNS, subunits are at different expression and to the subunit expression in the CNS to a are and subunits that together of the subunit expression. subunits, and together with the and subunits, for the of nAChR subunit expression in the are in the contribution of subunits to expression at the of ganglia. In or the subunits, in in the subunits display a high contribution in a as in the in the and and in the and subunits contribution to expression in of the ganglia. The by are in with using of subunit can be in all of the Lymnaea The and subunits are the CNS as indicated by neurons that ∼10% of the neurons in all ganglia. of the ACh in the Lymnaea the extent of transmission in the Lymnaea CNS, we the expression of the cells by the cellular expression of the Lymnaea vesicular ACh transporter The vesicular ACh transporter been used as a of cholinergic neurons Munno D.W. van W.J. Syed N.I. J. PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar). of an 10% of the neurons the CNS The expression is for the in is with the the and the the expression of expression in the of in Lymnaea CNS various identified of which and have been shown to be in various of as and To neurons be by cholinergic transmission or ACh as the expression of subunits and of respectively, was of cells were caudodorsal cells light green cells light yellow cells function anterior lobe neurons, and neurons expression of all identified subunits in of neuronal cell in expression were and were with the expression in the ganglia. In of the high of the this indicates that the neuronal express a of subunits the neuronal display the expression of the with of the and in which is The expression of was by in situ hybridization that showed a of in the and a in the is for subunits, and as the in the and in the of in the and neurons as indicated by was not with in the expression of subunits the neuronal as by the but not by the of display a for the cells are all in to the of the and cell was in the VD4 and LPeD1 the sequence information of nAChRs subunits be used to nAChR expression by the identified neurons described in the Lymnaea of we subunit expression in LPeD1 and VD4 neurons that have been shown to express different of functional nAChRs by of (11Woodin M.A. Munno D.W. Syed N.I. J. Neurosci. 2002; 22: 505-514Crossref PubMed Google Scholar). Lymnaea expression is expression of subunits is to the of the is to expression of a number of subunit in in VD4 For subunit and in LPeD1 and and in expression can be but not in all expression of the subunit in LPeD1 be in with expression in VD4 are The and subunits that are in LPeD1 and respectively, display the of expression by a large the of the of ACh as a the receptors of ACh have been in the CNS of molluscs by of (8Zeimal E.V. Vulfius C.A. Salanki J. Neurobiology of Invertebrates. Academiai Kiado, Budapest, Hungary1967: 255-265Google Scholar, 9Vulfius E.A. Veprintzev B.N. Zeimal E.V. Michelson M.J. Nature. 1967; 216: 400-401Crossref PubMed Scopus (11) Google Scholar, 12Chiarandini D.J. Gerschenfeld H.M. Science. 1967; 156: 1595-1596Crossref PubMed Scopus (22) Google Scholar, 13Chiarandini D.J. Stefani E. Gerschenfeld H.M. Science. 1967; 156: 1597-1599Crossref PubMed Scopus (29) Google Scholar, L. Gerschenfeld H.M. Nature. PubMed Scopus Google Scholar, L. Gerschenfeld H.M. J. Neurophysiol. PubMed Scopus Google Scholar). CNS as to in mammalian that fast synaptic transmission is in the CNS and is by and nAChR subtypes (10Yeoman M.S. Parish D.C. Benjamin P.R. J. Neurophysiol. 1993; 70: 37-50Crossref PubMed Scopus (44) Google Scholar, 11Woodin M.A. Munno D.W. Syed N.I. J. Neurosci. 2002; 22: 505-514Crossref PubMed Google Scholar, J. Neurophysiol. PubMed Scopus Google Scholar, J. Neurophysiol. 1969; PubMed Scopus (22) Google Scholar, Science. 1967; PubMed Scopus Google Scholar). In this for the a comprehensive analysis of the molecular complexity of subunits involved in fast cholinergic transmission in a molluscan species is in the Lymnaea we identified 10 α-type and 1 β-type Lymnaea nAChR subunits, as well as nAChR this the of nAChR subunits in the Lymnaea CNS is however, the number of Lymnaea subunits that in For in the α-type and β-type neuronal nAChR subunits and possess α-type and β-type nAChR subunits, which all neuronal nAChR subunit M. 2005; PubMed Scopus Google Scholar, Jones K. 2005; PubMed Scopus Google Scholar). in and the nAChR subunit diversity in Lymnaea not the high number of least α-type and PubMed Scopus Google Scholar) or of the α-type and 12 PubMed Scopus Google Scholar). that we not the of the subunit a β-type nAChR subunit we were to degenerate primers that β-type subunits, which the β-type subunit was from β-type nAChR subunits to be In addition, we not of cells using which that the to an nAChR shown in to be on cells Syed N.I. van J. J. van der van K. Nature. 2001; PubMed Scopus Google Scholar) to be The functional expression of the subunit and their for ACh and indicates that the related subunits are involved in the of The however, of functional expression of the subunits indicates that for their functional expression subunits are is to that functional expression of receptors was by the of an subunit with a vertebrate β-type subunit M. M. S. Eur. J. Neurosci. PubMed Scopus Google Scholar, E. S. A. J. Neurochem. 1998; PubMed Scopus Google Scholar). Also, our to express various α-type subunits together with the and β-type subunit not in a functional channel in the of subunits comparable with A. A. J. J. 2005; PubMed Scopus Google Scholar) be be to this of Lymnaea nAChR analysis that and subunits are to subunits from and on this we functional of that is related to the nAChR and nAChR J. Neurochem. PubMed Scopus Google Scholar, M. J. Mol. Pharmacol. Google Scholar) the to form functional receptors in and and are related to the of nAChR subunits and can be to in receptors. with the subunit be related to Lymnaea and β-type subunits with the β-type nAChR subunits from various with not to the nAChR subunits, the and and the and groups display similarity to mammalian or nAChR subunits are of molluscan In with and and and to of the nAChR subtypes to in molluscs (9Vulfius E.A. Veprintzev B.N. Zeimal E.V. Michelson M.J. Nature. 1967; 216: 400-401Crossref PubMed Scopus (11) Google Scholar, 12Chiarandini D.J. Gerschenfeld H.M. Science. 1967; 156: 1595-1596Crossref PubMed Scopus (22) Google Scholar, 13Chiarandini D.J. Stefani E. Gerschenfeld H.M. Science. 1967; 156: 1597-1599Crossref PubMed Scopus (29) Google Scholar, 14Chemeris N.K. Kazachenko V.N. Kislov A.N. Kurchikov A.L. J. Physiol. (Lond.). 1982; 323: 1-19Crossref Scopus (43) Google Scholar, 15Vulfius C.A. Tumina O.B. Kasheverov I.E. Utkin Y.N. Tsetlin V.I. Neurosci. Lett. 2005; 373: 232-236Crossref PubMed Scopus (25) Google Scholar). The of analysis and the of in loop of in α-type nAChR subunits to a large extent of as an α-type nAChR however, that considerably from nAChR subunits as from nAChR subunits as from receptor In addition, the in loop of of is to with an with the of an suggested for at in α-type nAChR subunits van van W.J. K. PubMed Scopus Google Scholar). In the contribution of to of to be and to The widespread of in the Lymnaea CNS that this subunit a which functional in the Lymnaea data in this for the time a of the expression of molecular involved in cellular and vesicular release of acetylcholine in L. stagnalis. on our nAChRs are by at least 10% of the neurons in the this number be from that that all neurons in the Lymnaea CNS to of ACh nicotinic receptors that are and excitatory in (8Zeimal E.V. Vulfius C.A. Salanki J. Neurobiology of Invertebrates. Academiai Kiado, Budapest, Hungary1967: 255-265Google Scholar, 9Vulfius E.A. Veprintzev B.N. Zeimal E.V. Michelson M.J. Nature. 1967; 216: 400-401Crossref PubMed Scopus (11) Google Scholar). Our data large in expression and of subunit and the of nAChR subtypes with or cell type and of subunits involved in and The expression of large of neurons in all of the Lymnaea cholinergic have been the molluscan CNS is to neurons for many of the cholinergic For and E. Science. PubMed Scopus Google Scholar) have functionally identified cholinergic neurons in the to the and of as neurons The expression of subunits by identified cell that the release of by cells is under cholinergic the subunit expression the van J. Res. PubMed Scopus Google Mol. 1993; PubMed Scopus Google and van van J. PubMed Scopus Google Scholar) neuronal to the functional diversity for the expression of neuronal For the different of for in the and with the differential expression of the nAChR subunits in Together, a cholinergic modulation of all identified neuronal cell groups the were described to express nAChRs involved in the of release A. J.C. Physiol. Scopus (22) Google Scholar, A. J.C. J. Neurophysiol. PubMed Scopus Google Scholar). into the which as the for the and from the the have the cholinergic of at to by of neurons A. 39: PubMed Scopus Google Scholar). The LPeD1 and VD4 neurons in have been shown to express excitatory and inhibitory nAChR respectively Munno van J. Syed N.I. J. Neurophysiol. PubMed Scopus (22) Google Scholar). to the of subunit expression in neurons, the of of of subunits in functionally In particular, in LPeD1 and in VD4 are for functional for RNA of subunit expression van J. J. van Mol. Neurosci. 2005; PubMed Scopus Google Scholar). of and in excitatory and inhibitory nAChR respectively, in LPeD1 and VD4 with a of in receptor channels The data described in this together with of cholinergic transmission in identified neuronal in molluscs, a widespread of cholinergic transmission by the molluscan CNS, which a number of nAChR subtypes that are comparable with as it the Lymnaea nAChR subunits can be divided into of which to the Lymnaea subunits to a and functional complexity of the receptors is for We L. of for the of Lymnaea nAChR subunit with

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