Molecular Characterization of NMDA-Like Receptors in Aplysia and Lymnaea: Relevance to Memory Mechanisms
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1 The Whitney Laboratory for Marine Bioscience
2 Department of Neuroscience, 9505 Ocean Shore Blvd., St. Augustine, Florida 32080
3 Evelyn F. & William McKnight Brain Institute of the University of Florida, 9505 Ocean Shore Blvd., St. Augustine, Florida 32080
Abstract
The N-methyl-D-aspartate (NMDA) receptor belongs to the group of ionotropic glutamate receptors and has been implicated in synaptic plasticity, memory acquisition, and learning in both vertebrates and invertebrates, including molluscs. However, the molecular identity of NMDA-type receptors in molluscs remains unknown. Here, we cloned two NMDA-type receptors from the sea slug Aplysia californica, AcNR1-1 and AcNR1-2, as well as their homologs from the freshwater pulmonate snail Lymnaea stagnalis, LsNR1-1 and LsNR1-2. The cloned receptors contain a signal peptide, two extracellular segments with predicted binding sites for glycine and glutamate, three recognized transmembrane regions, and a fourth hydrophobic domain that makes a hairpin turn to form a pore-like structure. Phylogenetic analysis suggests that both the AcNR1s and LsNR1s belong to the NR1 subgroup of ionotrophic glutamate receptors. Our in situ hybridization data indicate highly abundant, but predominantly neuron-specific expression of molluscan NR1-type receptors in all central ganglia, including identified motor neurons in the buccal and abdominal ganglia as well as groups of mechanosensory cells. AcNR1 transcripts were detected extrasynaptically in the neurites of metacerebral cells of Aplysia. The widespread distribution of AcNR1 and LsNR1 transcripts also implies diverse functions, including their involvement in the organization of feeding, locomotory, and defensive behaviors.
Abbreviations: APV, 2-amino-5-phosphonovalerate ? CGC, cerebral giant cells in Lymnaea (MCC homolog) ? ER, endoplasmic reticulum ? LTP, long-term potentiation ? LYC, light yellow cells ? MCC, metacerebral cells in Aplysia; NMDA, N-methyl-D-aspartate ? NMDAR, ionotropic NMDA-type glutamate receptor ? PDZ, acronym from the postsynaptic density 95 (PSD-95), the Drosophila septate junction protein Discs-large and the epithelial tight junction protein ZO-1
Introduction
The N-methyl-D-aspartate (NMDA) type receptors are nonselective cation channels that form a class of ionotropic glutamate receptors (Gasic and Hollmann, 1992; Dingledine et al., 1999; Mayer and Armstrong, 2004). Since their discovery in the 1970s and the eventual cloning of the first NMDA receptor in 1991 (Moriyoshi et al., 1991), NMDA receptors have been a major target for learning and memory research owing to four distinct characteristics: voltage-dependent magnesium block, calcium permeability, relatively slow kinetics, and requirements for cofactors (glycine/ D-serine) (Bliss and Collingridge, 1993; Squire et al., 2002). These properties make the NMDA receptor a prominent candidate to assess mechanisms of associative learning at the cellular level. First, the voltage-dependent magnesium block implies that NMDA receptors can function as coincidence detectors, since the channel will open only when binding of glutamate coincides with cell depolarization, resulting in removal of the Mg2+ block at the resting potential (Mayer et al., 1984; Nowak et al., 1984), therefore detecting concurrence of spike activity in pre- and post-synaptic neurons. Second, calcium permeability of NMDA receptors implies that the entry of Ca2+, acting as a second messenger, can trigger a signal transduction cascade that results in post-translational modification of multiple proteins and changes in gene expression (MacDermott et al., 1986). Third, the slower kinetics (compared to other ionotropic glutamate receptors) of ionic currents mediated by NMDA receptors also make temporal summation of multiple synaptic inputs possible (Collingridge et al., 1988). As a result, the state of an NMDA receptor at any given time can be viewed as a record of the immediate past, and it enables the cell to respond differentially even to the same input again, according to the past history of cell activity. Lastly, NMDA receptors also require glycine, D-serine, or both as cofactors (Collingridge et al., 1988; Fadda et al., 1988; Kleckner and Dingledine, 1988; Wolosker et al., 1999; Mothet et al., 2000), providing an additional regulatory component for precise tuning of NMDA-mediated signaling and coupling of neuronal and glial activity (Danysz and Parsons, 1998; Schell, 2004). Finally, NMDA receptors also have additional recognition sites for polyamines (e.g., spermine that promotes receptor activation), for Zn2+ (Peters et al., 1987; Westbrook and Mayer, 1987; Hollmann et al., 1993; Choi and Lipton, 1999; Fayyazuddin et al., 2000; Erreger and Traynelis, 2005; Hatton and Paoletti, 2005; Rachline et al., 2005), and for H+ (that acts to inhibit ion flux [Traynelis et al., 1995; Low et al., 2000]), as well as cysteine sites that can act as redox sensors (Sullivan et al., 1994; Choi et al., 2001). These characteristics of NMDA receptors, along with their widespread distribution in the mammalian nervous system, make them one of the major research targets of modern neuroscience (Cotman et al., 1988; Constantine-Paton et al., 1990; Daw et al., 1993; Sugiura et al., 2001; Wenthold et al., 2003).
Recent identification of NMDA-type receptors in Drosophila melanogaster (Ultsch et al., 1993; Pellicena-Palle and Salz, 1995; Xia et al., 2005) and Caenorhabditis elegans (Brockie et al., 2001a,b; Mellem et al., 2002) stresses the universality of their contribution to behavioral plasticity, development, and sensory functions among insects, nematodes, and mammals. Furthermore, in Drosophila, it has recently been shown that knock-out of dNR1 genes specifically disrupted olfactory learning and that subsequent induction of transgene dNR1+ rescued the phenotype, thereby providing direct evidence that NMDA receptors play a critical role in learning and memory in insects (Xia et al., 2005). However, real-time physiological measurements following long-term plasticity at the level of characterized neurons are difficult in these model organisms.
In contrast, the marine mollusc Aplysia californica is one of the best-known experimental paradigms for study of the cellular basis of behavior owing to the presence of large and functionally identified neurons in many networks. For example, a simple memory-forming network associated with defensive behavior has been identified by Kandel and his colleagues; this model contributed substantially to our understanding of learning and memory mechanisms (for a review, see Kandel, 2001). Many aspects of long-term plasticity have been reproduced and analyzed using a conceptually simplified version of this network reduced to only two components—a pair of monosynaptically connected sensory-motor neurons (Rayport and Schacher, 1986). It was suggested that glutamate is one of the primary transmitters in mechanosensory neurons of this network (Dale and Kandel, 1993; Levenson et al., 2000; Drake et al., 2005). The presynaptic facilitation of this transmission by 5-HT released from modulatory interneurons is a key step in a molecular cascade of events leading to long-term plasticity of the sensory-motor synapse. The system represents a well-characterized cellular analog of conditioning known to be similar to the observed behavioral changes in the siphon- and gill-withdrawal reflex (reviewed in Kandel, 2001). In recent years there has been increasing evidence that postsynaptic components are also involved (Lin and Glanzman, 1994; Bao et al., 1997, 1998; Murphy and Glanzman, 1997; Antonov et al., 2003); i.e., the long-term facilitation of the sensory-motor synapse depends upon the rise in intracellular calcium concentrations in motor neurons when depolarization of the postsynaptic neuron is paired with presynaptic spike activity. This led to a hypothesis that classical conditioning in Aplysia is mediated, in part, by Hebbian-type long-term potentiation (LTP) due to the hypothetical activation of NMDA-related receptors located at the postsynaptic neuron (Lin and Glanzman, 1994; Murphy and Glanzman, 1997; Antonov et al., 2003); a situation that is conceptually similar to LTP in mammals. This hypothesis was further explored in recent reviews and textbooks (Bailey et al., 2000; Squire et al., 2002; Roberts and Glanzman, 2003; Byrne and Roberts, 2004), with the proposed key role of NMDA receptors as universal components of signal transduction paralleled both in Aplysia and mammals.
In spite of persuasive pharmacological data (e.g., with mammalian inhibitors of NMDA receptors) suggesting a role for NMDA receptors in memory-forming networks (Lin and Glanzman, 1994; Murphy and Glanzman, 1997, 1999), there has been no direct molecular evidence for their existence in Aplysia or any other mollusc. Yet, evidence for the presence of NMDA-like receptors that share some biophysical characteristics with mammalian receptors has been provided for a related freshwater gastropod species, Lymnaea stagnalis (Moroz et al., 1993). Nevertheless, Moroz et al. (1993) also demonstrated that both the electrophysiological and pharmacological characterizations of Lymnaea NMDA-like receptors reveal substantial differences compared to their vertebrate counterparts. For example, 2-amino-5-phosphonovalerate (APV) was not a blocker of Lymnaea’s NMDA receptors, and NMDA-induced currents were not affected by Mg2+. Finally, although other types of ionotropic glutamate receptors were cloned from selected gastropods and cephalopods (Hutton et al., 1991; Yung et al., 2002; Battaglia et al., 2003), genes for NMDA receptors have not been identified in any molluscan species.
Here we report the cloning of an NMDA-like receptor (AcNR1-1) from Aplysia, along with a splice variant AcNR1-2, and also its homolog from Lymnaea stagnalis, LsNR1-1, and a splice variant LsNR1-2. Using PCR, we detected that the AcNR1-1 transcript can be translocated to neurites of Aplysia neurons. With an in situ hybridization technique, we also found that molluscan NR1 transcripts are abundant and expressed in central neurons, including motor neurons and mechanosensory neurons, in both species. Furthermore, we constructed a phylogram of all invertebrate and several vertebrate NR1, NR2, and NR3 subunits and conclude that the Aplysia and Lymnaea NR subunits structurally belong to the NR1 gene family because they cluster with all other NR1 subunits with high bootstrap support. These data appeared elsewhere (Ha et al., 2003) in abstract form.
Materials and Methods
Animals and tissue collection
Specimens of Aplysia californica weighing 50–120 g were obtained from the National Resource for Aplysia at the University of Miami; individuals weighing 200–500 g were collected in the wild by Marinus (Long Beach, CA). Animals were anesthetized by injection of 60% (volume/body weight) isotonic MgCl2 (337 mM) prior to removal of the central nervous system (CNS). Specimens of Lymnaea stagnalis were obtained from Prof. Ken Lukowiak, University of Calgary (Canada) and maintained in aerated fresh water for up to 1 week before experiments.
Cloning of full-length cDNA encoding NMDA-like receptors
We identified an NMDA-like cDNA fragment among BLAST results from a collection of random sequenced clones (ESTs) derived from a metacerebral (MCC) neuron-specific cDNA library following the targeted search for ionotropic receptors (Moroz et al., 2004). A 424-bp-long EST that had high similarity to an NMDA-like receptor was obtained from the MCC neuron cDNA library. The amplified cDNA library was constructed from MCC neurons of Aplysia as described elsewhere (Matz, 2002; Moroz et al., 2004). The MCC library was constructed from a pool of MCCs isolated from four 60-g animals. The CNS library was constructed from a single 100-g animal. Both 5' and 3' RACE were performed to obtain the full-length copy of the coding sequence (Matz et al., 2003). A full-length cDNA sequence called AcNR1-1 (Genbank accession number AY163562) was obtained using terminal primers: 5'-TCTTCGGGCGGACAGGATGCAT-3' and 5'-CTTACTGTCACAGTGTTGCTTAA-3' from an amplified cDNA library. Three clones were isolated and sequenced from the MCC cDNA library and, independently, four from a whole CNS library. Sequencing was done at the Whitney Laboratory or by SeqWright (Houston, TX). The full-length copy of the coding sequence of AcNR1-1 was amplified from the MCC cDNA library of Aplysia and cloned into pCR 4-TOPO (Invitrogen). An additional consensus sequence was also obtained that corresponded to a splice variant, AcNR1-2 (Genbank accession number AY234809), and the full-length cDNA for this form was also cloned and sequenced.
Primers from a highly conserved area of NR1 sequences were generated from the Aplysia sequences 5'-CCGCTTCTCTCCCTTTGGGCGC-3' and 5'-GCATTGTTGCTATTAAATTT-3' and used in PCR to amplify a 1011-bp fragment from Lymnaea cerebral giant cell (CGC) and CNS cDNA libraries that showed the highest identity to the putative AcNR1-1. A full-length cDNA sequence called LsNR1-1 (Genbank accession number AY571900) was obtained using terminal primers—5'-ACCGAGGCAGTGCATTAGCG-3' and 5'-GCTCTAACAGTAGCATTTAATC-3'—with amplified cDNA libraries. Three clones were isolated and sequenced from a CGC cDNA library, and another three were independently sequenced from the whole CNS cDNA library. The CGC library was constructed from a pool of CGCs isolated from four animals. The CNS library was constructed from a single animal. The full-length copy of the coding sequence of LsNR1-1 was cloned into pCR 4-TOPO (Invitrogen). An additional consensus sequence was also obtained that corresponded to a splice variant, LsNR1-2 (Genbank accession number DQ295538), and the full-length cDNA for this form was also cloned and sequenced.
In-situhybridization of AcNR1 and LsNR1 in A. californica and L. stagnalis
Cloned full-length cDNA from AcNR1-1 and LsNR1-1 was used for the preparation of in situ probes. The two isoforms of AcNR1s and LsNR1s vary only by a 71-base insert and will not be distinguished by in situ technique; therefore, both AcNR1-1 and AcNR1-2 probes will be referred to as AcNR1 and LsNR1. The antisense probe was generated by digestion of cDNA from AcNR1-1 with Not I (New England Biolabs), then transcription with T3 polymerase from the DIG (digoxigen) RNA labeling kit (Roche Diagnostics). The control sense probe was produced by the same protocol but used Pme1 (New England Biolabs) to digest the cDNA and T7 polymerase for transcription. The DIG-labeled antisense probes were hybridized to NR1 mRNA in whole-mount CNS preparations, and the neurons containing the probe-target duplex were localized and visualized with alkaline phosphatase-conjugated anti-DIG antibody fragments (Boehriger Mannheim). The detailed in situ hybridization protocol has been described (Jezzini and Moroz, 2004; Walters et al., 2004; Jezzini et al., 2005).
Expression of AcNR1 was investigated in central ganglia of 16 experimental and 5 control CNS preparations. Expression of LsNR1 was investigated in 7 whole-mount CNS preparations and 3 control preparations. Control in situ hybridization experiments with full-length "sense" probes revealed no specific and selective staining in the CNS under identical conditions and labeling protocols in both species.
Imaging
Images were acquired with a Nikon Digital Sight DS-5M digital camera mounted on an upright Olympus SZX12 microscope. Figures were prepared using Corel Draw 11 and Adobe Photoshop.
Tree construction and sequence analysis
The phylogenetic tree was generated using default parameters and 10,000 iterations of the maximum likelihood algorithm implemented in the program TREE-PUZZLE ver. 5.0 (Schmidt et al., 2002). The initial multiple alignment was done using ClustalX ver. 1.83 (Thompson et al., 1997; Jeanmougin et al., 1998) with default parameters; all gaps were removed manually in GeneDoc (Nicholas et al., 1997) prior to tree construction. The graphical output was generated using Treeview (Page, 1996). All protein predictions were determined with Prosite (Gattiker et al., 2002) and SMART (Letunic et al., 2006).
Results
Molecular analysis of putative Aplysia and Lymnaea NR-like subunits
We report here the cloning of two putative NR1 subunits from Aplysia and Lymnaea. Two splice forms of AcNR1 were identified in an amplified cDNA library from MCC neurons of Aplysia; the first, named AcNR1-1 (AY163562), contains a 2895-bp open reading frame that encodes a putative 964 amino acid protein; the second, AcNR1-2 is 2646-bp long, encoding an 881 amino acid protein (Fig. 1). AcNR1-2 (AY234809) differs from AcNR1-1 only at the C-terminal domain because of a 71-bp insertion, which produces a frame-shift in the open reading frame that results in an early termination. There are also two amino acid substitutions between AcNR1-1 and AcNR1-2. The AcNR1-2 predicted protein has isoleucine substituted for valine at residue 31 and proline substituted for serine at residue 541. The two splice forms show 90% identity. Simultaneously, J. Boulter, K. Martin, and D.L. Glanzman reported to NCBI the cloning of a NMDA-type glutamate receptor (Accession number Y315153) having 99.5% identity to our reported AcNR1-1 with 4 amino acid substitutions.
In Lymnaea, two splice forms of NR-like subunits were initially identified in an amplified cDNA library from the CGC neurons that are homologous to MCCs in Aplysia. The first, named LsNR1-1 (AY571900), contains a 2892-bp open reading frame that encodes a putative 963 amino acid protein; the second, LsNR1-2, is 2640-bp long and encodes an 879 amino acid protein (Fig. 1). LsNR1-2 (DQ295538), like the AcNR1-2, contains a 71-bp insertion with a similar position for termination. The putative AcNR1-1 and LsNR1-1 show 82% identity, and AcNR1-2 and LsNR1-2 share 83% identity. In mammals, splice variants of NMDAR differ considerably in their electrophysiological and pharmacological properties as well as in their localization (Dingledine et al., 1999).
The deduced amino acid sequences for both the Aplysia and Lymnaea NR1s contain a signal peptide; two extracellular segments (S1 and S2) that are predicted to form binding sites for glutamate and glycine; three transmembrane regions (TM1, TM3, and TM4); and a fourth hydrophobic domain (TM2) that forms a hairpin turn (much like a pore structure) in the membrane, which is indicative of all other NR1 receptors (Fig. 1; Dingledine et al., 1999). In the pore, the putative amino acid residues involved in Ca+2 permeability and voltage-dependent Mg+2 block—tryptophan 595 and asparagine 603 (indicated by closed arrowheads)—are present in the NR1s for both Aplysia and Lymnaea (Fig. 1; Williams et al., 1998; Dingledine et al., 1999). Both species contain a substitution at amino acid residue leucine 469 (indicated by an open circle) that has been shown in other NMDA receptors to decrease glycine binding (Fig. 1; Ballard et al., 2002). Furthermore, both AcNR1 and the LsNR1 have evolutionarily conserved cysteine residues at positions 81, 305, 732, and 789 (indicated by a closed square) that have been shown to be involved in the redox modulation of the receptor (Fig. 1; Choi et al., 2001).
Both AcNR1s and the LsNR1s contain multiple predicted post-translational modification sites for N-glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation, and cAMP/cGMP-dependent protein kinase A phosphorylation (Fig. 2). Also, the AcNR1s and the LsNR1s share high identity in their post-translational modification except at the C-terminal. The C-terminals of AcNR1-1 and LsNR1-1 contain two putative cAMP/cGMP-dependent protein kinase A phosphorylation sites not present in the shorter isoforms AcNR1-2 and LsNR1-2.
It has been shown that a variety of proteins, including many proteins with PDZ domains, bind to NR1 subunits (Kornau et al., 1995; Sheng and Pak, 1999; Husi and Grant, 2001). Proteins of the PSD-95 (postsynaptic density 95) family, all of which contain PDZ domains, have been shown to bind to NR1s (Kornau et al., 1995; Kennedy, 1997). While no PDZ-binding interaction domains are detected in LsNR1-1 or the AcNR1-2 and LsNR1-2 isoforms, AcNR1-1 has a putative Class I PDZ-binding interaction domain (XS/TXV) at amino acid residues 893–896, and a Class III PDZ-binding interaction domain (XDXV) at the last four terminal amino acid residues (Fig. 2B; Nourry et al., 2003). Also, AcNR1-1 and LsNR1-1 contain an additional binding domain, called a proline-rich motif, at amino acid residues 930–941 and 924–935, respectively (Zarrinpar et al., 2003). These motifs have been shown to play critical roles in assembly and regulation of many intracellular signaling complexes.
Both the Aplysia and Lymnaea NR1s contain predicted ER retention signals. These are dibasic motifs, di-arginine at residues 856-7 for AcNR1-1 and at residues 853-4 for LsNR1-1 or di-lysine at residues 857-8 for AcNR1-2 and at residues 854-5 for LsNR1-2, located at their C-terminals (Teasdale and Jackson, 1996). It has been shown that unassembled NR1 and NR2 subunits are being retained in the ER until assembly, so ER retention may serve as a quality control mechanism for ensuring the proper mixing of subunits, which can then regulate expression and targeting (Standley et al., 2000).
We also constructed a phylogram of all invertebrate and several vertebrate NR1, NR2, and NR3 subunits (Fig. 3). Our phylogenetic analysis shows that the Aplysia and Lymnaea NR subunits belong to the NR1 gene family because they cluster with all other NR1 subunits with high bootstrap support. The two human glutamate receptors were used as an outgroup. In conclusion, based on predicted amino acid sequence analysis, motif and structure analysis, and the architecture of regulatory domains and phylogenetic analysis, we conclude that both the Aplysia and Lymnaea NR1s belong to the NR1 gene family (Figs. 1, 2, and 3).
Expression and the distribution of AcNR1 and LsNR1 in the CNS of Aplysia and Lymnaea
The AcNR1 transcripts are widely distributed in the majority of central neurons and are among the most abundant transcripts in the CNS of adult Aplysia (Figs. 4A–D); their expression was also detected in peripheral tissues and earlier larval stages of the animals (pre- and post-metamorphic, not shown). Yet, using in situ hybridization labeling with specific probes, we found that the AcNR1 expression is in some degree neuron-specific, as some neurons showed very low or no detectable level of AcNR1 transcripts (e.g., some neurons from a group of neurosecretory cells known as R3–R13). We found reproducible expression of NR1 in paired serotonergic modulatory MCC neurons involved in feeding arousal (Kupfermann et al., 1979; Kupfermann and Weiss, 1982) from which the initial NR1 clone was obtained (Fig. 4C). Using reverse transcriptase–PCR with the cDNA library derived from pure MCC neuronal processes, we have confirmed the presence of NMDA receptor transcripts in neurites of MCC neurons in cell culture.
We also confirm the presence of AcNR1 in key motoneurons of a gill- and siphon-withdrawal reflex (e.g., L7 and LFs). Interestingly, we did detect the AcNR1 transcripts in pleural sensory neurons known to be presynaptic for the tail/pedal motoneurons and involved in the withdrawal reflex (Fig. 4A; Byrne and Kandel, 1996; Mauelshagen et al., 1996).
The most intense staining invariably appeared in specific neurons of the pedal and buccal ganglia, and taken as a whole, the lowest expression level was detected in the cerebral ganglia. Nevertheless, each ganglion has heterogeneous populations of neurons with different intensities of staining. For example, AcNR1 was unambiguously expressed in several visually identified neurons such as R2 and LP1, as well as in buccal and pedal motoneurons.
Predominant clustering of intensely stained populations were seen in the caudal parts of the cerebral ganglia in the area of the A/B clusters, near to the MCC neurons and E-clusters, as well as in many buccal motoneurons (Fig. 4B, C). Slightly less intense staining was seen in the abdominal ganglion, with detectable variations in expression levels between preparations. Control ganglia incubated with full-length sense probes showed no staining (Fig. 4F).
Interestingly, expression of AcNR1 was detected in all major clusters of mechanosensory neurons located in buccal (S cluster), cerebral (J and K clusters), pleural (VC), and abdominal (selected neurons in LE and RE clusters) ganglia. In addition, it appears that these expression levels can differ within the same population of sensory neurons, as shown for pleural and buccal clusters, respectively (Fig. 4A, B).
In summary, at least 6000–8000 cells out of about 10,000 neurons in the adult CNS were estimated to express AcNR1, with detectable variation in expression levels across entire neuronal populations. Similarly, LsNR1 was expressed in more than 80% of the neurons of the CNS in Lymnaea, including LYC (light yellow cells) from the right parietal ganglia where NMDA-type receptors were initially characterized electrophysiologically (Fig. 4E; (Moroz et al., 1993).
Discussion
Since the discovery of long-term potentiation (LTP) (Bliss and Lomo, 1973; Bliss and Collingridge, 1993) and distinct characteristics of NMDA receptors, NMDA-receptor-dependent LTP has been prominently regarded as a mechanism to explain cellular events underlying associative learning and memory acquisition (Bliss and Lomo, 1973; Bliss and Collingridge, 1993; Tsien et al., 1996; Kiyama et al., 1998; Martin and Morris, 2002). Even though some remain skeptical of the link between LTP and learning mechanisms in intact animals (Sanes and Lichtman, 1999), a role for NMDA receptors in long-term plasticity such as place learning and fear conditioning has been confirmed in multiple independent studies. For example, in the Morris water-maze task, blockade of NMDA receptors disrupted hippocampal LTP and spatial learning in rats (Morris et al., 1986). By utilizing region-specific transgenic technologies and transient modulation of NR2B receptors, it was possible to generate "smart mice" that showed greater learning ability (Rampon et al., 2000; Shimizu et al., 2000). However, the system functions of NMDA receptors are still under active investigation (Bannerman et al., 1995; Sanes and Lichtman, 1999; Lisman, 2003) because of the complexity of the underlying network organization and the presence of various examples of NMDA-independent long-term plasticity.
For these reasons the simpler nervous systems of various invertebrate models might be advantageous, both for exploring the role of NMDA receptors in elementary behavioral plasticity and for testing the hypothesis that the unified mechanisms underlying associative learning and memory are universally linked to the NMDA-type receptors. Although an evolutionarily conserved role for NMDA receptors in associative learning and memory has been suggested for many invertebrate groups, it was positively confirmed for flies only (Xia et al., 2005). Support for this hypothesis in other invertebrates is inadequate because of the limited knowledge about the molecular identity and biophysical characterization of NMDA receptors or because of technical difficulties accessing target neurons for detailed electrophysiological analysis.
So far, NMDA receptors have been cloned from representatives of only three invertebrate phyla (out of more than 30): arthropods (insects), nematodes, and molluscs (the present study, see Fig. 3). Thus, it is still difficult to acquire an overview of the diverse functions of NMDA receptors in different animal groups; there is, however, an obvious bias toward exploration of their role in memory.
In insects, Drosophila NMDA receptors were first cloned in 1993, and have been used to show that disrupting the expression of dNR1 disrupts olfactory learning and that expression of the dNR1 transgene rescues the deficit in learning (Ultsch et al., 1993; Lin, 2005; Xia et al., 2005). A similar role for NMDA receptors was also shown by using NMDA-receptor antagonists in honeybees (Si et al., 2004). However, the role of NMDA receptors in insects and other arthropods is not restricted to memory mechanisms. In Drosophila larvae, noncompetitive NMDA-receptor antagonists block the central locomotory pattern generator (Cattaert and Birman, 2001). It also appears that arthropod NMDA receptors are associated with functions not obviously related to associative learning, such as neuromuscular transmission and development (e.g., Pfeiffer-Linn and Glantz, 1991; Pellicena-Palle and Salz, 1995; Chiang et al., 2002; Begum et al., 2004; Liu et al., 2005).
Similarly, in C. elegans, the reported functions of NMDA receptors are also very diverse; they are required for proper wiring of sensory neurons and sensory signaling as well as for modulation of locomotion (Brockie et al., 2001a,b; Mellem et al., 2002). Interestingly, in mammals, NR1 subunits are widely distributed and expressed in many non-neuronal tissues including heart, testis, kidney, and bone marrow (Genever et al., 1999; Gill and Pulido, 2001), further suggesting the multiplicity of functions mediated by NMDA-type receptors.
NMDA receptors in molluscs and their relevance to memory mechanisms
Although NMDA receptors were shown to display a diversity of functions in C. elegans and Drosophila, the small size of the neurons in these organisms makes it more difficult to obtain real-time physiological measurements. In contrast, in gastropod molluscs we can benefit from the simpler organization of their CNS, which contains about 10,000–20,000 central neurons of relatively large size. In Aplysia, the involvement of NMDA receptors in synaptic transmission and long-term facilitation in the sensory-motor synapse of the gill- and siphon-withdrawal reflex was proposed more than 10 years ago (e.g., Dale and Kandel, 1993; Lin and Glanzman, 1994). That proposal is mainly based upon the fact that mammalian NMDA antagonists block the induction of long-term facilitation (Murphy and Glanzman, 1997). However, there has been no molecular evidence for the existence of NMDA receptors in these motoneurons until now. Here, we confirm the hypothesis that AcNR1 is expressed in motoneurons of the gill- and siphon-withdrawal reflex (L7 and LFs). Even though this finding lends credibility to the role of NMDA receptors in mediation of long-term plasticity in the sensory-motor synapse of Aplysia, we should be cautious in interpreting the pharmacological experiments without a detailed characterization of the Aplysia NMDA channel itself.
According to our previous studies on NMDA-type receptors in Lymnaea, the glutamate receptors on the light yellow cells (LYC) are activated by NMDA but not by other glutamate-receptor agonists (L. Moroz, J. Gyori, unpubl. data), are permeable to calcium ions, and are potentiated by glycine; these findings suggest that these glutamate receptors have characteristics similar to those of mammalian NMDA receptors (Moroz et al., 1993). However, the response of LYC to glutamate does not show voltage-dependent magnesium block, and other glutamate antagonists—including APV, a selective NMDA antagonist—do not block the response; these findings indicate significant discrepancies in the physiology and pharmacology between mammalian and molluscan NMDA receptors (Moroz et al., 1993)
Therefore, it is premature to draw any conclusions until we have a complete description of the biophysics, physiology, and pharmacology of NMDA receptors in Aplysia. To this end, the best option would be the expression of functional NMDA receptors in a heterologous system for physiological and pharmacological investigation. Unfortunately, our initial attempt to express an AcNR1 subunit in the Xenopus oocyte was unsuccessful. We suspect this was due to the lack of other (currently unknown) NR subunits. Indeed, functional NMDA receptors in all species studied so far are tetrameric, with two heterodimers composed of both NR1 and NR2 subunits (McIlhinney et al., 2003; Furukawa et al., 2005). In addition, cloning of NR2-type subunits in Aplysia was not a straightforward task. The difficulty can be attributed to a variety of causes, such as low abundance of the transcripts or the possible presence of pseudogenes. Although we were able to get a partial clone of the NR2-like sequence, we could not obtain a full-length cDNA sequence of predicted NR2 subunits in either Aplysia or Lymnaea. However, recent plans to sequence the Aplysia genome brighten the outlook for this endeavor to clone and characterize NR2 subunits in Aplysia (Moroz et al., 2004).
Another controversial observation regarding AcNR1 and LsNR1 is their nearly ubiquitous expression in the CNS, including in pleural sensory neurons known to be presynaptic for the tail/pedal motoneurons and involved in the withdrawal reflex (Byrne and Kandel, 1996; Mauelshagen et al., 1996). This nearly ubiquitous expression of the Aplysia NR1 subunit is also characteristic for the distribution of its ortholog in mammalian and insect brains (Xia et al., 2005). In Aplysia, we found that the AcNR1 transcripts are not only widely distributed but also highly abundant in nearly all central neurons. This is different from observations in Drosophila, where NMDA receptors are weakly expressed and show high expression levels only in some cell types (Xia et al., 2005).
This widespread expression of AcNR1 raises a possibility that glutamate may be a prevalent neurotransmitter or modulator in the CNS of Aplysia, as in mammals. Mammalian neurons are known to contain about 5 mM glutamate in the cytoplasm, and our previous studies showed that the glutamate concentration of random abdominal Aplysia neurons is close to 3 mM, which is quite comparable to mammalian systems (Nedergaard et al., 2002; Drake et al., 2005). A nearly ubiquitous expression of AcNR1 implies its possible assembly with other subunits that have more localized expression. In addition, there is still a possibility that some fraction of AcNR1 can serve functions other than those reported for "classical" NMDA glutamate receptors in mammals. For example, NR3 subunits in rats co-assemble with NR1 subunits and form excitatory glycine receptors, which are unresponsive to glutamate or NMDA application and are inhibited by D-serine (Chatterton et al., 2002). It is also important to know that some glutamate-gated ionic channels can be permeable to anions since glutamate induces hyperpolarization in a variety of neurons both in Aplysia and Lymnaea. In other words, L-glutamate is both an excitatory and an inhibitory transmitter in molluscs (e.g., Bolshakov et al., 1991), and this is quite different from the situation in the mammalian brain (where L-glutamate acts as an excitatory transmitter). Therefore, considering the ubiquitous presence of AcNR1 and LsNR1 in the CNS, it is reasonable to assume that some fraction of AcNR1 and LsNR1 may form unconventional channel proteins by associating with other, yet unidentified, subunits or proteins.
Furthermore, the abundant expression of AcNR1 in sensory neurons is quite unexpected. These pleural sensory neurons are suggested to be glutamatergic (Dale and Kandel, 1993; Levenson et al., 2000; Drake et al., 2005) but apparently are insensitive to L-glutamate. Whereas the application of glutamate with glycine (1 mM each in Mg2+ free ASW, pH 8.0) to MCC, from which we cloned AcNR1, elicits a complex biphasic response, in which typically fast depolarization is followed by slow hyperpolarization, Aplysia pleural sensory neurons did not respond to glutamate application under the same conditions (data not shown). The lack of a glutamate response by sensory neurons could be caused by factor such as inducible surface expression of functional glutamate receptors, ER retention, or lack of other NR subunits, but it still lends credibility to the idea that some AcNR1 transcripts may serve functions unrelated to memory mechanisms, such as formation of unconventional channel proteins.
On the other hand, the presence of the potential post-translational modification sites (Fig. 2) in the predicted protein suggests that the Aplysia and Lymnaea NR1s may be targets of multiple intracellular signaling pathways. The Aplysia and Lymnaea NR1-1s and NR1-2s differ not only in length at the C-terminal but also in predicted post-translational modifications. Phosphorylation of NR1s is thought to function in regulating channel activity, and may be important for synaptic plasticity as well (Carroll and Zukin, 2002). Therefore, regulation of NR1 by protein kinases can influence synaptic transmission and plasticity or lead to molecular and functional heterogeneity of the NMDA receptor family in Aplysia and Lymnaea. The putative cGMP-dependent regulatory sites in the NR1s found in MCC neurons may be involved in mediating NO signaling (e.g., from NOS containing C2 neurons [Jacklet, 1995; Koh and Jacklet, 1999; Moroz, 2006]). Nitric oxide and cGMP are also known to control NR1/NMDA channel activity (Jurado et al., 2003; Stanton et al., 2003). For example, different splice variants of NMDA receptors may be phosphorylated by cGMP-dependant protein kinase or linked to this cascade. NMDA receptors were shown to form a huge signaling complex by binding postsynaptic density 95 (PSD-95), another PDZ domain containing protein (Husi and Grant, 2001), so the difference between NR1s in regard to the presence of a PDZ interaction domain implies that they have different functions inside the cell. The conservation of these PDZ interaction domains and multiple regulatory sites between molluscan and mammalian NMDA receptors suggests an important and conserved function for this channel; however, the lack of comparative and functional data for most members of this group limits our ability to classify the functions of the channel and predict its physiological role.
The process of understanding the function of NMDA receptors in Aplysia is just beginning. Clearly, heterologous expression and functional identification of these receptors is the next step. Although the functional implications associated with NMDA receptors are purely speculative at this time, detailed studies of these receptors in identified neurons such as L7 and LFs will help us to elucidate how these receptors are involved in mediating long-term facilitation in Aplysia.
Concluding remarks
We would like to stress that the lingering controversy over the universality of the role of NMDA receptors as a key factor in learning and memory across different animal phyla, as well as over evolutionary aspects of functions for NMDA receptors (e.g., Wu, 2002; Rose et al., 2003) is primarily due to insufficient comparative data—especially data from basal metazoa (e.g., Pierobon et al., 2004; Scappaticci et al., 2004). Molecular and functional characterization of nonvertebrate homologs of these receptors would probably reveal novel regulatory mechanisms and pathways. The diversity of novel functions of NMDA receptors in molluscs is also expected (e.g., glial [Evans et al., 1991, 1992] neuromuscular [Lima et al., 2003], and epithelial [Palumbo et al., 1997]).
It is also important to note that, as a marine organism, Aplysia is exposed to extremely high concentrations of Mg2+ (about 50 mM in seawater and the hemolymph). As a result, we might also anticipate some difference in Mg2+-dependent regulation of NMDA-type channels among gastropods and many other marine groups. Differences in the pharmacology of NMDA receptors are also expected, especially in such diverse groups as molluscs. For example, NMDA can be ineffective in the mimicking of glutamate-gated currents or can even be an antagonist (as was described for the leech [Mat Jais et al., 1984]). Surprisingly, NMDA was also found as an endogenous compound in tissues of some bivalve molluscs (Sato et al., 1987; Todoroki et al., 1999; Shibata et al., 2001).
In conclusion, we have also determined the exon-intron organization of the Aplysia AcNR1-1 gene based upon the initial sequencing (2 x coverage) of the Aplysia genome (in progress). The AcNR1-1 gene consists of 18 exons and 17 introns. Exon 18 of the AcNR1-1 gene encodes the C-terminal protein cassette C2, as in the rat NR1 and chicken NR1 (Zarain-Herzberg et al., 2005). As is the case in these other species, this cassette contains an alternative splice site that includes a termination codon. The sequence and position of the exon-intron junctions for the AcNR1-1 gene are shown in Table 1, and they are compared to the NR1 gene organization of other species in Table 2. It appears that the invertebrate species examined to date have fewer exons than the vertebrate species with sequenced genomes (Table 2). However, in discussion of the evolution of NMDA receptors, it would be important to obtain genome data from cnidarians (e.g., Hydra and Nematostella), sponges (e.g., Reniera), and other basal metazoans (in progress).
Acknowledgments
This work was supported by grants to LLM from NIH, NSF, and the Evelyn F. & William McKnight Brain Research Foundations. We are grateful to Lisa Matragrano for her help in cloning the AcNR1s and to Mr. James Netherton and Mr. Sami Jezzini for careful reading of the manuscript. We also thank Prof. Ken Lukowiak for providing Lymnaea stagnalis, Dr. Peter Lovell for his help with in situ hybridization experiments on Lymnaea, and Dr. Yuri Panchin for critical comments and discussions related to the diversity of ionotropic glutamate receptors in molluscs and Clione limacina in particular.
Footnotes
Received 15 December 2005; accepted 21 February 2006.
* These authors have equally contributed to this paper.
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2 Department of Neuroscience, 9505 Ocean Shore Blvd., St. Augustine, Florida 32080
3 Evelyn F. & William McKnight Brain Institute of the University of Florida, 9505 Ocean Shore Blvd., St. Augustine, Florida 32080
Abstract
The N-methyl-D-aspartate (NMDA) receptor belongs to the group of ionotropic glutamate receptors and has been implicated in synaptic plasticity, memory acquisition, and learning in both vertebrates and invertebrates, including molluscs. However, the molecular identity of NMDA-type receptors in molluscs remains unknown. Here, we cloned two NMDA-type receptors from the sea slug Aplysia californica, AcNR1-1 and AcNR1-2, as well as their homologs from the freshwater pulmonate snail Lymnaea stagnalis, LsNR1-1 and LsNR1-2. The cloned receptors contain a signal peptide, two extracellular segments with predicted binding sites for glycine and glutamate, three recognized transmembrane regions, and a fourth hydrophobic domain that makes a hairpin turn to form a pore-like structure. Phylogenetic analysis suggests that both the AcNR1s and LsNR1s belong to the NR1 subgroup of ionotrophic glutamate receptors. Our in situ hybridization data indicate highly abundant, but predominantly neuron-specific expression of molluscan NR1-type receptors in all central ganglia, including identified motor neurons in the buccal and abdominal ganglia as well as groups of mechanosensory cells. AcNR1 transcripts were detected extrasynaptically in the neurites of metacerebral cells of Aplysia. The widespread distribution of AcNR1 and LsNR1 transcripts also implies diverse functions, including their involvement in the organization of feeding, locomotory, and defensive behaviors.
Abbreviations: APV, 2-amino-5-phosphonovalerate ? CGC, cerebral giant cells in Lymnaea (MCC homolog) ? ER, endoplasmic reticulum ? LTP, long-term potentiation ? LYC, light yellow cells ? MCC, metacerebral cells in Aplysia; NMDA, N-methyl-D-aspartate ? NMDAR, ionotropic NMDA-type glutamate receptor ? PDZ, acronym from the postsynaptic density 95 (PSD-95), the Drosophila septate junction protein Discs-large and the epithelial tight junction protein ZO-1
Introduction
The N-methyl-D-aspartate (NMDA) type receptors are nonselective cation channels that form a class of ionotropic glutamate receptors (Gasic and Hollmann, 1992; Dingledine et al., 1999; Mayer and Armstrong, 2004). Since their discovery in the 1970s and the eventual cloning of the first NMDA receptor in 1991 (Moriyoshi et al., 1991), NMDA receptors have been a major target for learning and memory research owing to four distinct characteristics: voltage-dependent magnesium block, calcium permeability, relatively slow kinetics, and requirements for cofactors (glycine/ D-serine) (Bliss and Collingridge, 1993; Squire et al., 2002). These properties make the NMDA receptor a prominent candidate to assess mechanisms of associative learning at the cellular level. First, the voltage-dependent magnesium block implies that NMDA receptors can function as coincidence detectors, since the channel will open only when binding of glutamate coincides with cell depolarization, resulting in removal of the Mg2+ block at the resting potential (Mayer et al., 1984; Nowak et al., 1984), therefore detecting concurrence of spike activity in pre- and post-synaptic neurons. Second, calcium permeability of NMDA receptors implies that the entry of Ca2+, acting as a second messenger, can trigger a signal transduction cascade that results in post-translational modification of multiple proteins and changes in gene expression (MacDermott et al., 1986). Third, the slower kinetics (compared to other ionotropic glutamate receptors) of ionic currents mediated by NMDA receptors also make temporal summation of multiple synaptic inputs possible (Collingridge et al., 1988). As a result, the state of an NMDA receptor at any given time can be viewed as a record of the immediate past, and it enables the cell to respond differentially even to the same input again, according to the past history of cell activity. Lastly, NMDA receptors also require glycine, D-serine, or both as cofactors (Collingridge et al., 1988; Fadda et al., 1988; Kleckner and Dingledine, 1988; Wolosker et al., 1999; Mothet et al., 2000), providing an additional regulatory component for precise tuning of NMDA-mediated signaling and coupling of neuronal and glial activity (Danysz and Parsons, 1998; Schell, 2004). Finally, NMDA receptors also have additional recognition sites for polyamines (e.g., spermine that promotes receptor activation), for Zn2+ (Peters et al., 1987; Westbrook and Mayer, 1987; Hollmann et al., 1993; Choi and Lipton, 1999; Fayyazuddin et al., 2000; Erreger and Traynelis, 2005; Hatton and Paoletti, 2005; Rachline et al., 2005), and for H+ (that acts to inhibit ion flux [Traynelis et al., 1995; Low et al., 2000]), as well as cysteine sites that can act as redox sensors (Sullivan et al., 1994; Choi et al., 2001). These characteristics of NMDA receptors, along with their widespread distribution in the mammalian nervous system, make them one of the major research targets of modern neuroscience (Cotman et al., 1988; Constantine-Paton et al., 1990; Daw et al., 1993; Sugiura et al., 2001; Wenthold et al., 2003).
Recent identification of NMDA-type receptors in Drosophila melanogaster (Ultsch et al., 1993; Pellicena-Palle and Salz, 1995; Xia et al., 2005) and Caenorhabditis elegans (Brockie et al., 2001a,b; Mellem et al., 2002) stresses the universality of their contribution to behavioral plasticity, development, and sensory functions among insects, nematodes, and mammals. Furthermore, in Drosophila, it has recently been shown that knock-out of dNR1 genes specifically disrupted olfactory learning and that subsequent induction of transgene dNR1+ rescued the phenotype, thereby providing direct evidence that NMDA receptors play a critical role in learning and memory in insects (Xia et al., 2005). However, real-time physiological measurements following long-term plasticity at the level of characterized neurons are difficult in these model organisms.
In contrast, the marine mollusc Aplysia californica is one of the best-known experimental paradigms for study of the cellular basis of behavior owing to the presence of large and functionally identified neurons in many networks. For example, a simple memory-forming network associated with defensive behavior has been identified by Kandel and his colleagues; this model contributed substantially to our understanding of learning and memory mechanisms (for a review, see Kandel, 2001). Many aspects of long-term plasticity have been reproduced and analyzed using a conceptually simplified version of this network reduced to only two components—a pair of monosynaptically connected sensory-motor neurons (Rayport and Schacher, 1986). It was suggested that glutamate is one of the primary transmitters in mechanosensory neurons of this network (Dale and Kandel, 1993; Levenson et al., 2000; Drake et al., 2005). The presynaptic facilitation of this transmission by 5-HT released from modulatory interneurons is a key step in a molecular cascade of events leading to long-term plasticity of the sensory-motor synapse. The system represents a well-characterized cellular analog of conditioning known to be similar to the observed behavioral changes in the siphon- and gill-withdrawal reflex (reviewed in Kandel, 2001). In recent years there has been increasing evidence that postsynaptic components are also involved (Lin and Glanzman, 1994; Bao et al., 1997, 1998; Murphy and Glanzman, 1997; Antonov et al., 2003); i.e., the long-term facilitation of the sensory-motor synapse depends upon the rise in intracellular calcium concentrations in motor neurons when depolarization of the postsynaptic neuron is paired with presynaptic spike activity. This led to a hypothesis that classical conditioning in Aplysia is mediated, in part, by Hebbian-type long-term potentiation (LTP) due to the hypothetical activation of NMDA-related receptors located at the postsynaptic neuron (Lin and Glanzman, 1994; Murphy and Glanzman, 1997; Antonov et al., 2003); a situation that is conceptually similar to LTP in mammals. This hypothesis was further explored in recent reviews and textbooks (Bailey et al., 2000; Squire et al., 2002; Roberts and Glanzman, 2003; Byrne and Roberts, 2004), with the proposed key role of NMDA receptors as universal components of signal transduction paralleled both in Aplysia and mammals.
In spite of persuasive pharmacological data (e.g., with mammalian inhibitors of NMDA receptors) suggesting a role for NMDA receptors in memory-forming networks (Lin and Glanzman, 1994; Murphy and Glanzman, 1997, 1999), there has been no direct molecular evidence for their existence in Aplysia or any other mollusc. Yet, evidence for the presence of NMDA-like receptors that share some biophysical characteristics with mammalian receptors has been provided for a related freshwater gastropod species, Lymnaea stagnalis (Moroz et al., 1993). Nevertheless, Moroz et al. (1993) also demonstrated that both the electrophysiological and pharmacological characterizations of Lymnaea NMDA-like receptors reveal substantial differences compared to their vertebrate counterparts. For example, 2-amino-5-phosphonovalerate (APV) was not a blocker of Lymnaea’s NMDA receptors, and NMDA-induced currents were not affected by Mg2+. Finally, although other types of ionotropic glutamate receptors were cloned from selected gastropods and cephalopods (Hutton et al., 1991; Yung et al., 2002; Battaglia et al., 2003), genes for NMDA receptors have not been identified in any molluscan species.
Here we report the cloning of an NMDA-like receptor (AcNR1-1) from Aplysia, along with a splice variant AcNR1-2, and also its homolog from Lymnaea stagnalis, LsNR1-1, and a splice variant LsNR1-2. Using PCR, we detected that the AcNR1-1 transcript can be translocated to neurites of Aplysia neurons. With an in situ hybridization technique, we also found that molluscan NR1 transcripts are abundant and expressed in central neurons, including motor neurons and mechanosensory neurons, in both species. Furthermore, we constructed a phylogram of all invertebrate and several vertebrate NR1, NR2, and NR3 subunits and conclude that the Aplysia and Lymnaea NR subunits structurally belong to the NR1 gene family because they cluster with all other NR1 subunits with high bootstrap support. These data appeared elsewhere (Ha et al., 2003) in abstract form.
Materials and Methods
Animals and tissue collection
Specimens of Aplysia californica weighing 50–120 g were obtained from the National Resource for Aplysia at the University of Miami; individuals weighing 200–500 g were collected in the wild by Marinus (Long Beach, CA). Animals were anesthetized by injection of 60% (volume/body weight) isotonic MgCl2 (337 mM) prior to removal of the central nervous system (CNS). Specimens of Lymnaea stagnalis were obtained from Prof. Ken Lukowiak, University of Calgary (Canada) and maintained in aerated fresh water for up to 1 week before experiments.
Cloning of full-length cDNA encoding NMDA-like receptors
We identified an NMDA-like cDNA fragment among BLAST results from a collection of random sequenced clones (ESTs) derived from a metacerebral (MCC) neuron-specific cDNA library following the targeted search for ionotropic receptors (Moroz et al., 2004). A 424-bp-long EST that had high similarity to an NMDA-like receptor was obtained from the MCC neuron cDNA library. The amplified cDNA library was constructed from MCC neurons of Aplysia as described elsewhere (Matz, 2002; Moroz et al., 2004). The MCC library was constructed from a pool of MCCs isolated from four 60-g animals. The CNS library was constructed from a single 100-g animal. Both 5' and 3' RACE were performed to obtain the full-length copy of the coding sequence (Matz et al., 2003). A full-length cDNA sequence called AcNR1-1 (Genbank accession number AY163562) was obtained using terminal primers: 5'-TCTTCGGGCGGACAGGATGCAT-3' and 5'-CTTACTGTCACAGTGTTGCTTAA-3' from an amplified cDNA library. Three clones were isolated and sequenced from the MCC cDNA library and, independently, four from a whole CNS library. Sequencing was done at the Whitney Laboratory or by SeqWright (Houston, TX). The full-length copy of the coding sequence of AcNR1-1 was amplified from the MCC cDNA library of Aplysia and cloned into pCR 4-TOPO (Invitrogen). An additional consensus sequence was also obtained that corresponded to a splice variant, AcNR1-2 (Genbank accession number AY234809), and the full-length cDNA for this form was also cloned and sequenced.
Primers from a highly conserved area of NR1 sequences were generated from the Aplysia sequences 5'-CCGCTTCTCTCCCTTTGGGCGC-3' and 5'-GCATTGTTGCTATTAAATTT-3' and used in PCR to amplify a 1011-bp fragment from Lymnaea cerebral giant cell (CGC) and CNS cDNA libraries that showed the highest identity to the putative AcNR1-1. A full-length cDNA sequence called LsNR1-1 (Genbank accession number AY571900) was obtained using terminal primers—5'-ACCGAGGCAGTGCATTAGCG-3' and 5'-GCTCTAACAGTAGCATTTAATC-3'—with amplified cDNA libraries. Three clones were isolated and sequenced from a CGC cDNA library, and another three were independently sequenced from the whole CNS cDNA library. The CGC library was constructed from a pool of CGCs isolated from four animals. The CNS library was constructed from a single animal. The full-length copy of the coding sequence of LsNR1-1 was cloned into pCR 4-TOPO (Invitrogen). An additional consensus sequence was also obtained that corresponded to a splice variant, LsNR1-2 (Genbank accession number DQ295538), and the full-length cDNA for this form was also cloned and sequenced.
In-situhybridization of AcNR1 and LsNR1 in A. californica and L. stagnalis
Cloned full-length cDNA from AcNR1-1 and LsNR1-1 was used for the preparation of in situ probes. The two isoforms of AcNR1s and LsNR1s vary only by a 71-base insert and will not be distinguished by in situ technique; therefore, both AcNR1-1 and AcNR1-2 probes will be referred to as AcNR1 and LsNR1. The antisense probe was generated by digestion of cDNA from AcNR1-1 with Not I (New England Biolabs), then transcription with T3 polymerase from the DIG (digoxigen) RNA labeling kit (Roche Diagnostics). The control sense probe was produced by the same protocol but used Pme1 (New England Biolabs) to digest the cDNA and T7 polymerase for transcription. The DIG-labeled antisense probes were hybridized to NR1 mRNA in whole-mount CNS preparations, and the neurons containing the probe-target duplex were localized and visualized with alkaline phosphatase-conjugated anti-DIG antibody fragments (Boehriger Mannheim). The detailed in situ hybridization protocol has been described (Jezzini and Moroz, 2004; Walters et al., 2004; Jezzini et al., 2005).
Expression of AcNR1 was investigated in central ganglia of 16 experimental and 5 control CNS preparations. Expression of LsNR1 was investigated in 7 whole-mount CNS preparations and 3 control preparations. Control in situ hybridization experiments with full-length "sense" probes revealed no specific and selective staining in the CNS under identical conditions and labeling protocols in both species.
Imaging
Images were acquired with a Nikon Digital Sight DS-5M digital camera mounted on an upright Olympus SZX12 microscope. Figures were prepared using Corel Draw 11 and Adobe Photoshop.
Tree construction and sequence analysis
The phylogenetic tree was generated using default parameters and 10,000 iterations of the maximum likelihood algorithm implemented in the program TREE-PUZZLE ver. 5.0 (Schmidt et al., 2002). The initial multiple alignment was done using ClustalX ver. 1.83 (Thompson et al., 1997; Jeanmougin et al., 1998) with default parameters; all gaps were removed manually in GeneDoc (Nicholas et al., 1997) prior to tree construction. The graphical output was generated using Treeview (Page, 1996). All protein predictions were determined with Prosite (Gattiker et al., 2002) and SMART (Letunic et al., 2006).
Results
Molecular analysis of putative Aplysia and Lymnaea NR-like subunits
We report here the cloning of two putative NR1 subunits from Aplysia and Lymnaea. Two splice forms of AcNR1 were identified in an amplified cDNA library from MCC neurons of Aplysia; the first, named AcNR1-1 (AY163562), contains a 2895-bp open reading frame that encodes a putative 964 amino acid protein; the second, AcNR1-2 is 2646-bp long, encoding an 881 amino acid protein (Fig. 1). AcNR1-2 (AY234809) differs from AcNR1-1 only at the C-terminal domain because of a 71-bp insertion, which produces a frame-shift in the open reading frame that results in an early termination. There are also two amino acid substitutions between AcNR1-1 and AcNR1-2. The AcNR1-2 predicted protein has isoleucine substituted for valine at residue 31 and proline substituted for serine at residue 541. The two splice forms show 90% identity. Simultaneously, J. Boulter, K. Martin, and D.L. Glanzman reported to NCBI the cloning of a NMDA-type glutamate receptor (Accession number Y315153) having 99.5% identity to our reported AcNR1-1 with 4 amino acid substitutions.
In Lymnaea, two splice forms of NR-like subunits were initially identified in an amplified cDNA library from the CGC neurons that are homologous to MCCs in Aplysia. The first, named LsNR1-1 (AY571900), contains a 2892-bp open reading frame that encodes a putative 963 amino acid protein; the second, LsNR1-2, is 2640-bp long and encodes an 879 amino acid protein (Fig. 1). LsNR1-2 (DQ295538), like the AcNR1-2, contains a 71-bp insertion with a similar position for termination. The putative AcNR1-1 and LsNR1-1 show 82% identity, and AcNR1-2 and LsNR1-2 share 83% identity. In mammals, splice variants of NMDAR differ considerably in their electrophysiological and pharmacological properties as well as in their localization (Dingledine et al., 1999).
The deduced amino acid sequences for both the Aplysia and Lymnaea NR1s contain a signal peptide; two extracellular segments (S1 and S2) that are predicted to form binding sites for glutamate and glycine; three transmembrane regions (TM1, TM3, and TM4); and a fourth hydrophobic domain (TM2) that forms a hairpin turn (much like a pore structure) in the membrane, which is indicative of all other NR1 receptors (Fig. 1; Dingledine et al., 1999). In the pore, the putative amino acid residues involved in Ca+2 permeability and voltage-dependent Mg+2 block—tryptophan 595 and asparagine 603 (indicated by closed arrowheads)—are present in the NR1s for both Aplysia and Lymnaea (Fig. 1; Williams et al., 1998; Dingledine et al., 1999). Both species contain a substitution at amino acid residue leucine 469 (indicated by an open circle) that has been shown in other NMDA receptors to decrease glycine binding (Fig. 1; Ballard et al., 2002). Furthermore, both AcNR1 and the LsNR1 have evolutionarily conserved cysteine residues at positions 81, 305, 732, and 789 (indicated by a closed square) that have been shown to be involved in the redox modulation of the receptor (Fig. 1; Choi et al., 2001).
Both AcNR1s and the LsNR1s contain multiple predicted post-translational modification sites for N-glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation, and cAMP/cGMP-dependent protein kinase A phosphorylation (Fig. 2). Also, the AcNR1s and the LsNR1s share high identity in their post-translational modification except at the C-terminal. The C-terminals of AcNR1-1 and LsNR1-1 contain two putative cAMP/cGMP-dependent protein kinase A phosphorylation sites not present in the shorter isoforms AcNR1-2 and LsNR1-2.
It has been shown that a variety of proteins, including many proteins with PDZ domains, bind to NR1 subunits (Kornau et al., 1995; Sheng and Pak, 1999; Husi and Grant, 2001). Proteins of the PSD-95 (postsynaptic density 95) family, all of which contain PDZ domains, have been shown to bind to NR1s (Kornau et al., 1995; Kennedy, 1997). While no PDZ-binding interaction domains are detected in LsNR1-1 or the AcNR1-2 and LsNR1-2 isoforms, AcNR1-1 has a putative Class I PDZ-binding interaction domain (XS/TXV) at amino acid residues 893–896, and a Class III PDZ-binding interaction domain (XDXV) at the last four terminal amino acid residues (Fig. 2B; Nourry et al., 2003). Also, AcNR1-1 and LsNR1-1 contain an additional binding domain, called a proline-rich motif, at amino acid residues 930–941 and 924–935, respectively (Zarrinpar et al., 2003). These motifs have been shown to play critical roles in assembly and regulation of many intracellular signaling complexes.
Both the Aplysia and Lymnaea NR1s contain predicted ER retention signals. These are dibasic motifs, di-arginine at residues 856-7 for AcNR1-1 and at residues 853-4 for LsNR1-1 or di-lysine at residues 857-8 for AcNR1-2 and at residues 854-5 for LsNR1-2, located at their C-terminals (Teasdale and Jackson, 1996). It has been shown that unassembled NR1 and NR2 subunits are being retained in the ER until assembly, so ER retention may serve as a quality control mechanism for ensuring the proper mixing of subunits, which can then regulate expression and targeting (Standley et al., 2000).
We also constructed a phylogram of all invertebrate and several vertebrate NR1, NR2, and NR3 subunits (Fig. 3). Our phylogenetic analysis shows that the Aplysia and Lymnaea NR subunits belong to the NR1 gene family because they cluster with all other NR1 subunits with high bootstrap support. The two human glutamate receptors were used as an outgroup. In conclusion, based on predicted amino acid sequence analysis, motif and structure analysis, and the architecture of regulatory domains and phylogenetic analysis, we conclude that both the Aplysia and Lymnaea NR1s belong to the NR1 gene family (Figs. 1, 2, and 3).
Expression and the distribution of AcNR1 and LsNR1 in the CNS of Aplysia and Lymnaea
The AcNR1 transcripts are widely distributed in the majority of central neurons and are among the most abundant transcripts in the CNS of adult Aplysia (Figs. 4A–D); their expression was also detected in peripheral tissues and earlier larval stages of the animals (pre- and post-metamorphic, not shown). Yet, using in situ hybridization labeling with specific probes, we found that the AcNR1 expression is in some degree neuron-specific, as some neurons showed very low or no detectable level of AcNR1 transcripts (e.g., some neurons from a group of neurosecretory cells known as R3–R13). We found reproducible expression of NR1 in paired serotonergic modulatory MCC neurons involved in feeding arousal (Kupfermann et al., 1979; Kupfermann and Weiss, 1982) from which the initial NR1 clone was obtained (Fig. 4C). Using reverse transcriptase–PCR with the cDNA library derived from pure MCC neuronal processes, we have confirmed the presence of NMDA receptor transcripts in neurites of MCC neurons in cell culture.
We also confirm the presence of AcNR1 in key motoneurons of a gill- and siphon-withdrawal reflex (e.g., L7 and LFs). Interestingly, we did detect the AcNR1 transcripts in pleural sensory neurons known to be presynaptic for the tail/pedal motoneurons and involved in the withdrawal reflex (Fig. 4A; Byrne and Kandel, 1996; Mauelshagen et al., 1996).
The most intense staining invariably appeared in specific neurons of the pedal and buccal ganglia, and taken as a whole, the lowest expression level was detected in the cerebral ganglia. Nevertheless, each ganglion has heterogeneous populations of neurons with different intensities of staining. For example, AcNR1 was unambiguously expressed in several visually identified neurons such as R2 and LP1, as well as in buccal and pedal motoneurons.
Predominant clustering of intensely stained populations were seen in the caudal parts of the cerebral ganglia in the area of the A/B clusters, near to the MCC neurons and E-clusters, as well as in many buccal motoneurons (Fig. 4B, C). Slightly less intense staining was seen in the abdominal ganglion, with detectable variations in expression levels between preparations. Control ganglia incubated with full-length sense probes showed no staining (Fig. 4F).
Interestingly, expression of AcNR1 was detected in all major clusters of mechanosensory neurons located in buccal (S cluster), cerebral (J and K clusters), pleural (VC), and abdominal (selected neurons in LE and RE clusters) ganglia. In addition, it appears that these expression levels can differ within the same population of sensory neurons, as shown for pleural and buccal clusters, respectively (Fig. 4A, B).
In summary, at least 6000–8000 cells out of about 10,000 neurons in the adult CNS were estimated to express AcNR1, with detectable variation in expression levels across entire neuronal populations. Similarly, LsNR1 was expressed in more than 80% of the neurons of the CNS in Lymnaea, including LYC (light yellow cells) from the right parietal ganglia where NMDA-type receptors were initially characterized electrophysiologically (Fig. 4E; (Moroz et al., 1993).
Discussion
Since the discovery of long-term potentiation (LTP) (Bliss and Lomo, 1973; Bliss and Collingridge, 1993) and distinct characteristics of NMDA receptors, NMDA-receptor-dependent LTP has been prominently regarded as a mechanism to explain cellular events underlying associative learning and memory acquisition (Bliss and Lomo, 1973; Bliss and Collingridge, 1993; Tsien et al., 1996; Kiyama et al., 1998; Martin and Morris, 2002). Even though some remain skeptical of the link between LTP and learning mechanisms in intact animals (Sanes and Lichtman, 1999), a role for NMDA receptors in long-term plasticity such as place learning and fear conditioning has been confirmed in multiple independent studies. For example, in the Morris water-maze task, blockade of NMDA receptors disrupted hippocampal LTP and spatial learning in rats (Morris et al., 1986). By utilizing region-specific transgenic technologies and transient modulation of NR2B receptors, it was possible to generate "smart mice" that showed greater learning ability (Rampon et al., 2000; Shimizu et al., 2000). However, the system functions of NMDA receptors are still under active investigation (Bannerman et al., 1995; Sanes and Lichtman, 1999; Lisman, 2003) because of the complexity of the underlying network organization and the presence of various examples of NMDA-independent long-term plasticity.
For these reasons the simpler nervous systems of various invertebrate models might be advantageous, both for exploring the role of NMDA receptors in elementary behavioral plasticity and for testing the hypothesis that the unified mechanisms underlying associative learning and memory are universally linked to the NMDA-type receptors. Although an evolutionarily conserved role for NMDA receptors in associative learning and memory has been suggested for many invertebrate groups, it was positively confirmed for flies only (Xia et al., 2005). Support for this hypothesis in other invertebrates is inadequate because of the limited knowledge about the molecular identity and biophysical characterization of NMDA receptors or because of technical difficulties accessing target neurons for detailed electrophysiological analysis.
So far, NMDA receptors have been cloned from representatives of only three invertebrate phyla (out of more than 30): arthropods (insects), nematodes, and molluscs (the present study, see Fig. 3). Thus, it is still difficult to acquire an overview of the diverse functions of NMDA receptors in different animal groups; there is, however, an obvious bias toward exploration of their role in memory.
In insects, Drosophila NMDA receptors were first cloned in 1993, and have been used to show that disrupting the expression of dNR1 disrupts olfactory learning and that expression of the dNR1 transgene rescues the deficit in learning (Ultsch et al., 1993; Lin, 2005; Xia et al., 2005). A similar role for NMDA receptors was also shown by using NMDA-receptor antagonists in honeybees (Si et al., 2004). However, the role of NMDA receptors in insects and other arthropods is not restricted to memory mechanisms. In Drosophila larvae, noncompetitive NMDA-receptor antagonists block the central locomotory pattern generator (Cattaert and Birman, 2001). It also appears that arthropod NMDA receptors are associated with functions not obviously related to associative learning, such as neuromuscular transmission and development (e.g., Pfeiffer-Linn and Glantz, 1991; Pellicena-Palle and Salz, 1995; Chiang et al., 2002; Begum et al., 2004; Liu et al., 2005).
Similarly, in C. elegans, the reported functions of NMDA receptors are also very diverse; they are required for proper wiring of sensory neurons and sensory signaling as well as for modulation of locomotion (Brockie et al., 2001a,b; Mellem et al., 2002). Interestingly, in mammals, NR1 subunits are widely distributed and expressed in many non-neuronal tissues including heart, testis, kidney, and bone marrow (Genever et al., 1999; Gill and Pulido, 2001), further suggesting the multiplicity of functions mediated by NMDA-type receptors.
NMDA receptors in molluscs and their relevance to memory mechanisms
Although NMDA receptors were shown to display a diversity of functions in C. elegans and Drosophila, the small size of the neurons in these organisms makes it more difficult to obtain real-time physiological measurements. In contrast, in gastropod molluscs we can benefit from the simpler organization of their CNS, which contains about 10,000–20,000 central neurons of relatively large size. In Aplysia, the involvement of NMDA receptors in synaptic transmission and long-term facilitation in the sensory-motor synapse of the gill- and siphon-withdrawal reflex was proposed more than 10 years ago (e.g., Dale and Kandel, 1993; Lin and Glanzman, 1994). That proposal is mainly based upon the fact that mammalian NMDA antagonists block the induction of long-term facilitation (Murphy and Glanzman, 1997). However, there has been no molecular evidence for the existence of NMDA receptors in these motoneurons until now. Here, we confirm the hypothesis that AcNR1 is expressed in motoneurons of the gill- and siphon-withdrawal reflex (L7 and LFs). Even though this finding lends credibility to the role of NMDA receptors in mediation of long-term plasticity in the sensory-motor synapse of Aplysia, we should be cautious in interpreting the pharmacological experiments without a detailed characterization of the Aplysia NMDA channel itself.
According to our previous studies on NMDA-type receptors in Lymnaea, the glutamate receptors on the light yellow cells (LYC) are activated by NMDA but not by other glutamate-receptor agonists (L. Moroz, J. Gyori, unpubl. data), are permeable to calcium ions, and are potentiated by glycine; these findings suggest that these glutamate receptors have characteristics similar to those of mammalian NMDA receptors (Moroz et al., 1993). However, the response of LYC to glutamate does not show voltage-dependent magnesium block, and other glutamate antagonists—including APV, a selective NMDA antagonist—do not block the response; these findings indicate significant discrepancies in the physiology and pharmacology between mammalian and molluscan NMDA receptors (Moroz et al., 1993)
Therefore, it is premature to draw any conclusions until we have a complete description of the biophysics, physiology, and pharmacology of NMDA receptors in Aplysia. To this end, the best option would be the expression of functional NMDA receptors in a heterologous system for physiological and pharmacological investigation. Unfortunately, our initial attempt to express an AcNR1 subunit in the Xenopus oocyte was unsuccessful. We suspect this was due to the lack of other (currently unknown) NR subunits. Indeed, functional NMDA receptors in all species studied so far are tetrameric, with two heterodimers composed of both NR1 and NR2 subunits (McIlhinney et al., 2003; Furukawa et al., 2005). In addition, cloning of NR2-type subunits in Aplysia was not a straightforward task. The difficulty can be attributed to a variety of causes, such as low abundance of the transcripts or the possible presence of pseudogenes. Although we were able to get a partial clone of the NR2-like sequence, we could not obtain a full-length cDNA sequence of predicted NR2 subunits in either Aplysia or Lymnaea. However, recent plans to sequence the Aplysia genome brighten the outlook for this endeavor to clone and characterize NR2 subunits in Aplysia (Moroz et al., 2004).
Another controversial observation regarding AcNR1 and LsNR1 is their nearly ubiquitous expression in the CNS, including in pleural sensory neurons known to be presynaptic for the tail/pedal motoneurons and involved in the withdrawal reflex (Byrne and Kandel, 1996; Mauelshagen et al., 1996). This nearly ubiquitous expression of the Aplysia NR1 subunit is also characteristic for the distribution of its ortholog in mammalian and insect brains (Xia et al., 2005). In Aplysia, we found that the AcNR1 transcripts are not only widely distributed but also highly abundant in nearly all central neurons. This is different from observations in Drosophila, where NMDA receptors are weakly expressed and show high expression levels only in some cell types (Xia et al., 2005).
This widespread expression of AcNR1 raises a possibility that glutamate may be a prevalent neurotransmitter or modulator in the CNS of Aplysia, as in mammals. Mammalian neurons are known to contain about 5 mM glutamate in the cytoplasm, and our previous studies showed that the glutamate concentration of random abdominal Aplysia neurons is close to 3 mM, which is quite comparable to mammalian systems (Nedergaard et al., 2002; Drake et al., 2005). A nearly ubiquitous expression of AcNR1 implies its possible assembly with other subunits that have more localized expression. In addition, there is still a possibility that some fraction of AcNR1 can serve functions other than those reported for "classical" NMDA glutamate receptors in mammals. For example, NR3 subunits in rats co-assemble with NR1 subunits and form excitatory glycine receptors, which are unresponsive to glutamate or NMDA application and are inhibited by D-serine (Chatterton et al., 2002). It is also important to know that some glutamate-gated ionic channels can be permeable to anions since glutamate induces hyperpolarization in a variety of neurons both in Aplysia and Lymnaea. In other words, L-glutamate is both an excitatory and an inhibitory transmitter in molluscs (e.g., Bolshakov et al., 1991), and this is quite different from the situation in the mammalian brain (where L-glutamate acts as an excitatory transmitter). Therefore, considering the ubiquitous presence of AcNR1 and LsNR1 in the CNS, it is reasonable to assume that some fraction of AcNR1 and LsNR1 may form unconventional channel proteins by associating with other, yet unidentified, subunits or proteins.
Furthermore, the abundant expression of AcNR1 in sensory neurons is quite unexpected. These pleural sensory neurons are suggested to be glutamatergic (Dale and Kandel, 1993; Levenson et al., 2000; Drake et al., 2005) but apparently are insensitive to L-glutamate. Whereas the application of glutamate with glycine (1 mM each in Mg2+ free ASW, pH 8.0) to MCC, from which we cloned AcNR1, elicits a complex biphasic response, in which typically fast depolarization is followed by slow hyperpolarization, Aplysia pleural sensory neurons did not respond to glutamate application under the same conditions (data not shown). The lack of a glutamate response by sensory neurons could be caused by factor such as inducible surface expression of functional glutamate receptors, ER retention, or lack of other NR subunits, but it still lends credibility to the idea that some AcNR1 transcripts may serve functions unrelated to memory mechanisms, such as formation of unconventional channel proteins.
On the other hand, the presence of the potential post-translational modification sites (Fig. 2) in the predicted protein suggests that the Aplysia and Lymnaea NR1s may be targets of multiple intracellular signaling pathways. The Aplysia and Lymnaea NR1-1s and NR1-2s differ not only in length at the C-terminal but also in predicted post-translational modifications. Phosphorylation of NR1s is thought to function in regulating channel activity, and may be important for synaptic plasticity as well (Carroll and Zukin, 2002). Therefore, regulation of NR1 by protein kinases can influence synaptic transmission and plasticity or lead to molecular and functional heterogeneity of the NMDA receptor family in Aplysia and Lymnaea. The putative cGMP-dependent regulatory sites in the NR1s found in MCC neurons may be involved in mediating NO signaling (e.g., from NOS containing C2 neurons [Jacklet, 1995; Koh and Jacklet, 1999; Moroz, 2006]). Nitric oxide and cGMP are also known to control NR1/NMDA channel activity (Jurado et al., 2003; Stanton et al., 2003). For example, different splice variants of NMDA receptors may be phosphorylated by cGMP-dependant protein kinase or linked to this cascade. NMDA receptors were shown to form a huge signaling complex by binding postsynaptic density 95 (PSD-95), another PDZ domain containing protein (Husi and Grant, 2001), so the difference between NR1s in regard to the presence of a PDZ interaction domain implies that they have different functions inside the cell. The conservation of these PDZ interaction domains and multiple regulatory sites between molluscan and mammalian NMDA receptors suggests an important and conserved function for this channel; however, the lack of comparative and functional data for most members of this group limits our ability to classify the functions of the channel and predict its physiological role.
The process of understanding the function of NMDA receptors in Aplysia is just beginning. Clearly, heterologous expression and functional identification of these receptors is the next step. Although the functional implications associated with NMDA receptors are purely speculative at this time, detailed studies of these receptors in identified neurons such as L7 and LFs will help us to elucidate how these receptors are involved in mediating long-term facilitation in Aplysia.
Concluding remarks
We would like to stress that the lingering controversy over the universality of the role of NMDA receptors as a key factor in learning and memory across different animal phyla, as well as over evolutionary aspects of functions for NMDA receptors (e.g., Wu, 2002; Rose et al., 2003) is primarily due to insufficient comparative data—especially data from basal metazoa (e.g., Pierobon et al., 2004; Scappaticci et al., 2004). Molecular and functional characterization of nonvertebrate homologs of these receptors would probably reveal novel regulatory mechanisms and pathways. The diversity of novel functions of NMDA receptors in molluscs is also expected (e.g., glial [Evans et al., 1991, 1992] neuromuscular [Lima et al., 2003], and epithelial [Palumbo et al., 1997]).
It is also important to note that, as a marine organism, Aplysia is exposed to extremely high concentrations of Mg2+ (about 50 mM in seawater and the hemolymph). As a result, we might also anticipate some difference in Mg2+-dependent regulation of NMDA-type channels among gastropods and many other marine groups. Differences in the pharmacology of NMDA receptors are also expected, especially in such diverse groups as molluscs. For example, NMDA can be ineffective in the mimicking of glutamate-gated currents or can even be an antagonist (as was described for the leech [Mat Jais et al., 1984]). Surprisingly, NMDA was also found as an endogenous compound in tissues of some bivalve molluscs (Sato et al., 1987; Todoroki et al., 1999; Shibata et al., 2001).
In conclusion, we have also determined the exon-intron organization of the Aplysia AcNR1-1 gene based upon the initial sequencing (2 x coverage) of the Aplysia genome (in progress). The AcNR1-1 gene consists of 18 exons and 17 introns. Exon 18 of the AcNR1-1 gene encodes the C-terminal protein cassette C2, as in the rat NR1 and chicken NR1 (Zarain-Herzberg et al., 2005). As is the case in these other species, this cassette contains an alternative splice site that includes a termination codon. The sequence and position of the exon-intron junctions for the AcNR1-1 gene are shown in Table 1, and they are compared to the NR1 gene organization of other species in Table 2. It appears that the invertebrate species examined to date have fewer exons than the vertebrate species with sequenced genomes (Table 2). However, in discussion of the evolution of NMDA receptors, it would be important to obtain genome data from cnidarians (e.g., Hydra and Nematostella), sponges (e.g., Reniera), and other basal metazoans (in progress).
Acknowledgments
This work was supported by grants to LLM from NIH, NSF, and the Evelyn F. & William McKnight Brain Research Foundations. We are grateful to Lisa Matragrano for her help in cloning the AcNR1s and to Mr. James Netherton and Mr. Sami Jezzini for careful reading of the manuscript. We also thank Prof. Ken Lukowiak for providing Lymnaea stagnalis, Dr. Peter Lovell for his help with in situ hybridization experiments on Lymnaea, and Dr. Yuri Panchin for critical comments and discussions related to the diversity of ionotropic glutamate receptors in molluscs and Clione limacina in particular.
Footnotes
Received 15 December 2005; accepted 21 February 2006.
* These authors have equally contributed to this paper.
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