10.1. INTRODUCTION

NMDA receptors (NMDARs), a subtype of ionotropic glutamate receptors, mediate the vast majority of excitatory neurotransmission in the central brains of vertebrates. NMDARs form heteromeric complexes usually comprised of a principal NR1 subunit and various NR2 subunits [1,2]. The NMDAR channel is highly permeable to Ca²⁺ and Na⁺, and its opening requires simultaneous binding of glutamate and postsynaptic membrane depolarization [1,3,4]. Once activated, the NMDAR channel allows calcium influx into postsynaptic cells, where calcium triggers a cascade of biochemical events resulting in synaptic changes. NMDARs play diverse roles in normal central nervous system activity and development including regulation of synaptic development and function, and refinement of synaptic connections with experience and synaptic plasticity. NMDARs have also been widely investigated as targets for pharmacological management of seizures, pain, and a variety of neurological disorders including Schizophrenia, Parkinson’s, Alzheimer’s, and Huntington’s diseases [2,5–14].

Various studies in invertebrates suggest the existence of functional NMDA-like receptors and their requirement for synaptic and behavioral plasticity [15–37]. This chapter highlights the recent characterization of Drosophila NMDARs [25,29–33], with emphasis on their physiological role during memory processing after Pavlovian olfactory conditioning—a well-defined and widely used elemental learning paradigm [38].

10.2. NMDA RECEPTOR HOMOLOGUES IN DROSOPHILA

Three mammalian families of NMDAR subtypes have been identified: NR1, NR2, and NR3 subunits. Eight functional isoforms of the NR1 subunit are generated by alternative splicing of a single NR1 gene, while four distinct NR2 (A through D) subunits and two NR3 (A and B) subunits are encoded by six different genes [2,6,39,40]. The consensus is that most native NMDARs function as heteromeric tetramers composed of two NR1 subunits and two NR2 subunits [2]. The NR1 subunit is the essential constituent of NMDARs, expressed ubiquitously in the central nervous system [41]. The NR2 subunits regulate the biophysical and pharmacological properties of the NMDAR channel, including its high affinity for glutamate, modulation by glycine, Mg²⁺ block, and channel kinetics [2,6,39,40]. The NR3 subunits that appear not to be essential components of most native NMDARs may coassemble with the NR1 and NR2 complexes and thus regulate channel function [6,39].

The situation is much simpler in Drosophila where homologues of the NR1 and NR2 subunits have been characterized. dNR1, composed of 15 exons, appears to be the only gene encoding the fly homologue of the NR1 subunit [30,33]. Although two different transcripts are generated by alternative splicing of the noncoding exon 1, they differ only in the 5′ untranslated region and contain the same coding sequence [33]. Therefore, dNR1 encodes a single NR1 subunit, different from the rodent NR1 gene that encodes multiple NR1 isoforms [2,6,39]. dNR2 may also be the only gene encoding the Drosophila homologue of the NR2 subunit [33]. Consistent with the fact that most NR2 genes are subject to alternative splicing in vertebrates [2,6,39,40], dNR2 also undergoes alternative splicing, generating eight different transcripts that may encode three different protein isoforms [33].

The major structural features of NMDARs are well conserved in both dNR1 and dNR2 [30,33], including three hydrophobic transmembrane regions (TM1, TM3, and TM4), one hydrophobic pore-forming segment (TM2) in the carboxyl terminal half [2], and two ligand binding domains (S1 and S2) with high homology to bacterial amino acid–binding proteins [42,43]. Also conserved are the major determinants for ligand binding including amino acid residues in dNR1 (F430, Y432, D491, F494, V699, S702, D747, and F769) for coagonist glycine binding [42,44–46] and those in dNR2 (E511, K591, S618, R625, T792, T798, and V841) for glutamate binding [47,48]. Finally, many of the binding determinants for the noncompetitive or competitive antagonists are conserved, including the critical amino acids in dNR1 (W626, N631, and A660) for binding of dizocilpine (MK-801) and phencyclidine [49] and those in dNR2 (K591, S618, T798, and V841) for binding of D-2-amino-5-phosphonopentanoate (AP5) and 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP) [47,48].

Fly and rodent NMDARs exhibit several interesting differences [33]. Three asparagine residues present in the channel-forming TM2 domains of NMDA subunits control Ca²⁺ permeability and voltage-dependent Mg²⁺ block [2,50,51]. One such asparagine residue is conserved in dNR1 (N631), but the other two are not conserved in dNR2, suggesting that Mg²⁺ block may be relatively weak for Drosophila NMDARs. Fly NMDARs may mainly interact with PDZ domain-containing proteins through dNR1 but not dNR2, which is usually the case in mammals [52,53]. PDZ domains are found on the basis of sequence repeats in PSD-95, Dlg, and ZO-1 proteins [54]. They can bind the carboxyl terminal sequences of proteins through a consensus sequence present in many glutamate receptors [55]. The type I PDZ binding motif (X–S/T–X–V) is not present in dNR2 although it is well conserved in all mammalian NR2 homologues [52,53]. Interestingly, dNR1 has a putative type II PDZ domain-binding motif (X–Ψ–X–Ψ in which Ψ is a hydrophobic amino acid) at its C terminus (and a potential type I PDZ-binding motif preceding this type II motif), suggesting that fly NMDARs may interact with PDZ domain-containing proteins via dNR1 subunits. Finally, although the entire size and domain structures of dNR2 show high homology to its vertebrate counterpart, its active pharmacological and physiological sites only moderately mimic its mammalian counterparts [33].

10.3. FUNCTIONAL EXPRESSION OF FLY NMDA RECEPTORS

An initial attempt to express cloned dNR1 cDNA in Xenopus oocytes failed to generate a reliable NMDA-selective response [30]. Xia et al. [33] thus re-cloned dNR1 and showed that it alone could produce weak but significant NMDA-dependent responses in oocytes (Figure 10.1A). This weak response appears to support the notion that dNR1 alone can form functional NMDARs in oocytes. Notably, dNR1 has a RSS (retention signal sequence) motif at its C terminus, similar to its mammalian homologues [33]. The motif regulates the insertion of NMDARs in cell membranes by retaining the NR1 subunit in the endoplasmic reticulum (ER) when not assembled in functional receptors [56,57].

FIGURE 10.1

Coexpression of dNR1 and dNR2-2 yields a functional NMDAR. A: NMDA response in Xenopus oocytes expressing both dNR1 and dNR2-2. Oocytes injected with dNR1 and dNR2-2 cRNAs exhibited inward currents upon application of NMDA (10 mM) but not upon application (more...)

dNR1 thus may be largely kept at the ER rather than inserted in membrane surfaces, making it possible to generate a weak response even if it can form a functional channel. Similarly, mammalian functional NMDARs may be formed by expression of NR1 alone in oocytes, exhibiting many of the properties of native NMDARs [41,58]. Nevertheless, one should be cautious in concluding that NR1 alone can form a homomeric functional channel. Xenopus oocytes express endogenous XenU1, a glutamate receptor subunit that can assemble with mammalian NR1 to form functional NMDARs [59]. This appears to reinforce the notion that NR1 must assemble with one or more NR2 subunits to form functional channels. However, NMDARs formed by NR1 in oocytes in fact do not contain the XenU1 subunit [60], prompting further investigation why NR1 alone can form functional channels in oocytes but not in mammalian cell lines [41,58].

Coexpression of dNR1 and dNR2 in Xenopus oocytes generates much stronger NMDA-selective responses (Figure 10.1A), consistent with the formation of highly potent NMDAR channels when the NR1 subunit is coexpressed with NR2 [2,3,61]. Combined expression of dNR1 and dNR2 also exhibits several physiological features (Figure 10.1A and B) that distinguish NMDARs from other ionotropic glutamate receptors, including selective activation by NMDA and L-asparate [3,62] and modulation by glycine as the coagonist for glutamate [63].

The NMDA-selective response, however, is not sensitive to Mg²⁺ blockade in oocytes [33]. This observation highlights certain facts. First, replacement of the asparagine residue in the channel-forming TM2 domain of the NR2 subunit disrupts Mg²⁺ block for mammalian NMDARs [51,64]. This crucial asparagine residue is replaced by glutamine in the dNR2 subunit, suggesting that Mg²⁺ block may be relatively weak for Drosophila NMDARs. TM1 and TM4 domains are also important for Mg²⁺ block [65], but both domains are poorly conserved in dNR2 [33].

Finally, proper external ionic conditions for oocytes and insect cells are dramatically different. The appropriate Mg²⁺ concentration for fly muscle cells, for instance, is about 10 times higher than that for oocytes [66], suggesting that fly NMDARs may have evolved to be less sensitive to Mg²⁺. Nevertheless, Drosophila NMDARs may still be regulated by Mg²⁺ block in vivo. In support, MK-801, a compound requiring binding to the asparagine residue in the NR1 subunit to execute its antagonist effect [49], has been shown to abolish NMDAR-dependent locomotor rhythm in fly larvae [29]. It also suppresses NMDA-mediated juvenile hormone biosynthesis in cockroaches [25].

Consistently, coexpression of dNR1 and dNR2 in Drosophila S2 cells reveals voltage-dependent conductance blocked by external Mg²⁺ (Figure 10.1C). Therefore, the electrophysiological profile of coexpressing dNR1 and dNR2 in oocytes or S2 cells reveals most of the distinguishing characteristics of mammalian NMDARs, including the unique requirement for coagonist and voltage-dependent Mg²⁺ block [33]. This suggests that Drosophila likely has functional NMDARs consisting of two subunits, dNR1 and dNR2.

10.4. EXPRESSION OF FLY NMDA RECEPTORS IN ADULT BRAIN

The expression of dNR1 and dNR2 in adult brains has been extensively studied with multiple antibodies [32,33]. Both proteins are widely expressed throughout the entire brain, including all neuropils that consist of neural processes and dendritic regions (Figure 10.2). In the central brain, all neurons show weak expression of dNR1 and dNR2 (Figure 10.2A and B). In both cases, immunopositive signals are detected in the calyx of the mushroom body (MB, lower insets) and in the ellipsoid body (EB, upper insets), a substructure of the central complex. Both proteins are also detected throughout the optical lobes (not shown). Interestingly, many immunopositive signals are clustered as synapse-like puncta (Figure 10.2A and B, insets) and distribute along neural fibers [33]. This observation indicates that dNR1 and dNR2 may be localized to synapses, consistent with their contribution to associative learning and memory formation (see below).

FIGURE 10.2

(See color insert following page 212.) Expression of dNR1 and dNR2 proteins in adult brain. (A) Confocal imaging of dNR1 immunostaining in whole-mount adult brain with α-85S, a specific polyclonal anti-dNR1 antibody. All neurons appear to show (more...)

The dNR2 protein may be preferentially expressed in the ellipsoid body, as indicated by strong immunopositive signals in its R4m large-field neurons from two of the anti-dNR2 antibodies (Figure 10.2C and D). This is particularly interesting because the ellipsoid body is a substructure of the central complex, one prominent neuropil located in the insect central brain that forms intricate connections to a variety of brain centers and proposed to mediate communication between the two hemispheres and many behavioral outputs [67,68]. The fan-shaped body, a closely related substructure of the central complex, may house a short-term memory trace for visual learning [69] and regulate long-term memory (LTM) formation after courtship conditioning [70]. Wu et al. [32] observed that fly NMDARs function in the ellipsoid body to regulate LTM consolidation after olfactory conditioning. However, it is unclear whether dNR1 is also preferentially expressed in the ellipsoid body.

Fly NMDARs appear to be only weakly expressed in the mushroom body, as all six (two polyclonal anti-dNR1, one monoclonal, and three polyclonal anti-dNR2) antibodies do not strongly label the structure [32]. The calyx, dendritic arborization of intrinsic neurons of the mushroom body, receives efferent inputs from several regions, including projection neurons from antennal lobes [71]. The axons of these neurons project rostrally as densely packed and stalk-like structures called pedunculi to the anterior face of the brain where they split and give rise to the dorsally projecting α and α′ lobes and the medially projecting β, β′, and γ lobes [72]. Output neurons from the MB project to many parts of the central brain [73].

The mushroom body, one of the most prominent and well-characterized neuro-pillar structures in the insect central brain, has long been shown to mediate associative learning and early memory processing [74,75]. The weak expression of dNR1 and dNR2 in the mushroom body is intriguing, as targeted dsRNA-mediated knockdown of either protein disrupts the formation of middle-term memory [2], an earlier phase of memory processing that depends on the normal function of the mushroom body [76]. Also intriguing are the strong immunopositive signals in scattered cell bodies and parts of their fibers that were detected with two of these antibodies but not with the remaining four [32,33]. Finally, as noted above, the ellipsoid body was preferentially labeled with only two of the polyclonal anti-dNR2 antibodies [32,33].

10.5. NMDA RECEPTOR-DEPENDENT LEARNING AND LONG-TERM MEMORY CONSOLIDATION

Accumulating evidence over the past two decades has established that NMDARs and their downstream signaling pathways play a crucial role in the regulation of synaptic and behavioral plasticity by mediating long-lasting changes in synapse strength [long-term depression (LTD) and long-term potentiation (LTP)] in mammalian and human brains [9,14,77–85]. Opening of the NMDAR channel requires simultaneous binding of presynaptically released neurotransmitters and postsynaptic membrane depolarization as it is subjected to a unique voltage-dependent Mg²⁺ block [1,3,4]. This suggests that NMDARs may serve as “Hebbian coincidence detectors” underlying associative learning [81,86–90]. Molecular and physiological characterization of functional NMDARs in Drosophila [33] makes it possible to extend these finding to invertebrates [91–93].

Using the Pavlovian olfactory conditioning paradigm involving well-defined odors as conditioned stimuli (CSs) and footshocks as unconditioned stimuli (US) [38], Xia et al. demonstrated that NMDARs are required acutely for associative learning and subsequent LTM consolidation in Drosophila [33]. By limiting rapid inducible knockdown of dNR1 with a specific anti-dNR1 message in adults, olfactory learning is transiently disrupted (Figure 10.3A), suggesting that NMDARs play an acute, physiological role in associative learning. This observation rules out a potential developmental explanation for adult learning deficits, something that past genetic studies did not achieve [91,93].

FIGURE 10.3

Disruption of olfactory learning and LTM consolidation by acute induction of anti-dNR1 mRNA. EP331 flies contain EP elements inserted downstream of and in an opposite orientation to the transcription start site of dNR1. The EP element yields a specific (more...)

The physiological requirement for NMDARs during olfactory learning strengthens the idea that these receptors play a central role in synaptic and behavioral plasticity, potentially by acting as coincidence detectors [81,86–90]. Extended (massed or spaced) training can overcome such an acute requirement for dNR1 [33]. Nevertheless, the acute adult-specific knockdown of dNR1 appears to abolish LTM, which is specifically induced by extended spaced training (Figure 10.3B). Considering that this acute effect is specific to LTM consolidation but not retrieval (see below), this observation suggests that NMDARs are acutely required for LTM consolidation—an interesting idea supported by past genetic but not pharmacological studies [94,95].

The relevant results also support the idea that NMDARs are involved with LTM consolidation and storage but not retrieval (Figure 10.3). The acute knockdown of dNR1, induced 15 hr before training, disrupted initial learning after one-session training, suggesting that NMDARs are involved in early encoding of olfactory memory (Figure 10.3A). Such a disruptive effect on learning was reversed when knockdown of dNR1 was induced 36 hr before training, suggesting that the dNR1 protein returns to its preinduction level (Figure 10.3A, right panels).

LTM tested 24 hours after spaced training was specifically abolished by the acute knockdown of dNR1 induced 15 hr before training (Figure 10.3B). The induction was delivered 15 hr before spaced training that lasted about 3 hr [96], and then LTM was tested 24 hr after training. Therefore, LTM was tested 42 hr (15 +24 + 3) after induction of the antisense dNR1 transcript, a time when the dNR1 protein returned to preinduction level, suggesting that LTM consolidation but not retrieval was abolished. Spaced training can overcome the learning defect (present after one training session) after the acute knockdown of dNR1 induced 15 hr before training [33], again ruling out the possibility that NMDARs are involved in memory retrieval. Therefore, Drosophila NMDARs are acutely and selectively required for early encoding and consolidation of olfactory memory but not retrieval [33], consistent with studies of mammals [83,94,97–104].

Specific abolition of LTM consolidation by acute knockdown of dNR1 (Figure 10.3B) is similar to that produced by induced expression of a CREB-repressor transgene and indicates a specific disruption of cycloheximide-dependent LTM [105]. Fly NMDARs are required for CREB-dependent LTM formation, consistent with mammalian experiments revealing NMDAR-dependent activation of CREB during LTP and LTM in both amygdala and hippocampus [106–109]. The cAMP/PKA/CREB signaling pathway plays an important role in diverse processes from barrel formation and hippocampal LTP to learning and memory in invertebrates and vertebrates [48, 110–119]; but see [120,121]. Interestingly, two types of functionally distinct NMDAR signaling complexes have been identified: synaptic and extra-synaptic [122]. Synaptic NMDARs can cause sustained CREB phosphorylation and CRE-mediated gene expression. Extra-synaptic NMDARs suppress CREB activity. It seems possible that synaptic NMDAR complexes regulate memory consolidation by controlling nuclear signaling to CREB.

10.6. LOCALIZATION OF NMDA RECEPTOR-DEPENDENT MEMORIES

Memory formation after olfactory conditioning proceeds through several temporal phases, all of which have been proposed to be predominantly processed in the mushroom body [74,75]. Surprisingly, dsRNA-mediated silencing of dNR1 or dNR2 in the mushroom body disrupts middle-term memory, an earlier memory phase proposed to be processed upstream of LTM [96,123,124], without affecting LTM consolidation [32]. Because the mushroom body is required for LTM retrieval [32,124], this observation suggests that it may be involved with LTM processing via an NMDAR-independent pathway, and NMDAR-dependent middle-term memory in the structure may not be necessary for LTM consolidation.

Interestingly, aging-dependent memory impairment is regulated by the amnesiac peptide [encoding the fly homologue of PACAP (pituitary adenylate cyclase-activating polypeptide) [125,126]] and DC0 (encoding a catalytic subunit of cAMP-dependent protein kinase known as PKA [127]), and is specific to middle-term memory in Drosophila [128–130]. Considering that both PACAP and PKA are known to mediate normal NMDAR function through phosphorylation [131,132], the appearance of NMDAR-dependent middle-term memory in the mushroom body raises the interesting possibility that amnesiac peptide and PKA may regulate aging-dependent memory impairment by phosphorylation of fly NMDARs in the mushroom body.

When targeted specifically to the R4m neurons of the ellipsoid body, where dNR2 (and presumably dNR1) may be preferentially expressed (Figure 10.2), dsRNA-mediated silencing of dNR2 (or dNR1 [32]) specifically abolishes protein synthesis-dependent LTM (Figure 10.4A and B), suggesting that the ellipsoid body plays a critical role during LTM processing. The involvement of NMDARs during LTM processing is physiological rather than developmental, because induction of the dsRNA transgene is limited to adults (Figure 10.4A and B).

FIGURE 10.4

Inducible knockdown of dNR2 specifically blocks consolidation and storage but not retrieval of protein synthesis-dependent LTM. UAS-dsNR2 is a dsRNA-based transgene that can silence expression of dNR2 gene in the presence of GAL4 [32]. dsRNA triggers (more...)

The abolition is specific to LTM consolidation and storage but not retrieval (Figure 10.4C through E). This requirement for NMDARs is also specific for a memory phase (LTM only) and brain region (ellipsoid body, but not mushroom body), as initial learning and early memories are normal when NMDARs are silenced in the ellipsoid body, and LTM forms normally when these receptors are silenced in the mushroom body [32]. Therefore, functional NMDARs contribute specifically to the consolidation and storage, but not to the retrieval of protein synthesis-dependent LTM in the ellipsoid body.

These data identify for the first time a brain region outside the mushroom body for LTM consolidation and storage. The specific involvement of the R4m subtype large-field neurons in the ellipsoid body during LTM consolidation and storage along with the requirement of LTM formation for neuronal activity from the mushroom body and correlation with the appearance of an asymmetrical body near the central complex [124,133,134], supports a broader neuroanatomical circuitry involving both brain structures that subserves olfactory memory consolidation in Drosophila.

The acute and specific requirement for NMDARs in the ellipsoid body for LTM consolidation and storage along with the occurrence of associative learning within or upstream of the mushroom body [74,75,135–138] raises the provocative hypothesis that the acquired olfactory experience may be transferred from the mushroom body to the ellipsoid body for LTM consolidation, in agreement with observations from other species [83,139,140]. Consistent with this hypothesis, blocking the synaptic output from the mushroom body but not from the ellipsoid body during and within the first 6 hr after training abolished the later consolidation of LTM (Figure 10.5A and B). A second consistent finding is that the synaptic output of the ellipsoid body is specifically required for LTM retrieval but not for acquisition and consolidation (Figure 10.5B and C), suggesting that NMDARs function in the ellipsoid body to support LTM consolidation and storage, while neuronal activity from the structure regulates NMDAR-independent LTM retrieval. These results reveal a distributed brain system subserving olfactory memory formation and the existence of a system-level memory consolidation in Drosophila that was previously only demonstrated in mammals [83,141–143].

FIGURE 10.5

Transference of memory from MB to EB during LTM consolidation. The UAS-shi^ts1 transgene was shown to block neuronal transmission in a temperature-dependent, dominant-negative fashion [156]. OK107 is a GAL4 driver that targets gene expression in most mushroom (more...)

10.7. NMDA RECEPTORS IN OTHER INVERTEBRATES

NMDARs have been shown to exist in several other invertebrate species including Caenorhabditis elegans (nematodes) [19,21], Aplysia californica, Lymnaea stagnalis, and Sepioteuthis sepioidea (mollusks) [16,22,23,28,34–37], Hirudo medicinalis (annelids) [24], Procambarus clarkia and Chasmagnathus (crustaceans) [15,18], and Apis mellifera and Diploptera punctata (insects) [25–27]. The characterization of those invertebrate NMDARs was achieved through electrophysiological and/or pharmacological analyses, mostly in the context of cellular recording [15–18,21,24,35–37], behavior [21–23,26], and recent molecular cloning of the NR1 homologues [21,27,28].

NMDARs in invertebrates seem to share major structural hallmarks and some biophysical and pharmacological characteristics with their vertebrate counterparts. Besides dNR1 and dNR2 [30,33], the NR1 homologues have been fully cloned in Caenorhabditis elegans [21], Lymnaea stagnalis [28], Aplysia californica [28], and Apis mellifera [27]. The partial sequence of ~400 amino acids (including the predicted transmembrane segments, pore-forming regions, and ligand-binding regions) was also cloned for the NR2 homologue (NMR-2) in Caenorhabditis elegans [19]. All seven fully or partially cloned receptor subunits contain all the signature features of NMDARs including three hydrophobic transmembrane segments (TM1, TM3, and TM4), one hydrophobic pore-forming region (TM2), two ligand-binding domains (S1 and S2) with high homology to bacterial amino acid–binding proteins [42,43], and the highly conserved SYTANLAAF amino acid sequence in TM3 [19,21,27,28,33].

Most of the amino acids for glycine binding in the NR1 homologues [21,27,28,33] and glutamate binding in the NR2 homologues are well conserved [19,33]. In addition all five fully cloned NR1 homologues contain one or more putative PDZ binding motifs [21,27,28,33], allowing them to interact with PDZ domain-containing proteins and thus form huge signaling complexes [52–55,144]. Consistently, the basic biophysical characteristics of NMDARs appear to be conserved in most if not all of these invertebrate species including selective activation by NMDA, modulation via glycine as the coagonist for glutamate, calcium permeability and even relatively slow kinetics, as supported by electrophysiological experiments [15–18,21,24,25,29,33].

Invertebrate and mammalian NMDARs exhibit certain pharmacological similarities. MK-801, for example, has been shown to block NMDA-selective responses or NMDA-dependent processes in all the invertebrate models tested [21–23,25,26,29]. It binds to the same asparagine residue in the channel-forming TM2 of the NR1 subunit that also controls the calcium permeability and voltage-dependent Mg²⁺ block [2,50,51]. This residue is conserved in all the invertebrate NR1 homologues cloned to date [21,27,28,33].

Mammalian NMDARs emerged as major targets for studying synaptic and behavioral plasticity since their discovery in the 1970s [2,7–9,14]. Many pharmacological studies of invertebrate NMDARs focused on their contributions to long-term synaptic plasticity and memory formation [22–24,26,35–37]. In particular NMDAR-dependent LTP of the Aplysia sensorimotor synapse mediates associative learning of the withdrawal reflexes, leading to a hypothesis that classical conditioning in Aplysia is partially mediated by Hebbian-type LTP due to the hypothetical activation of NMDARs located at postsynaptic neurons [35–37]. The hypothesis has been further elaborated, assuming that the critical role of NMDARs is paralleled during long-term synaptic plasticity both in Aplysia and mammals [145,146]. Since the molecular identities of the NR1 homologues (AcNR1-1 and AcNR1-2) were identified [28], this hypothesis may be further explored.

Nevertheless, invertebrate and vertebrate NMDARs reveal substantial differences. In particular, the two asparagine residues controlling calcium permeability and Mg²⁺ block [2,50,51] are not conserved in both dNR2 and NMR-2, leading to a less sensitive Mg²⁺ block in Drosophila [33] and possible absence of Mg²⁺ block in Caenorhabditis elegans [21]. Similarly, Mg²⁺ block is relatively weak [15,16,25] and even absent [17] in other invertebrate species, suggesting that NMDARs may have evolved to be increasingly sensitive to Mg²⁺ blockade. Also, the active pharmacological and physiological binding sites appear less conserved in invertebrate NR2 homologues [19,33]. Some antagonists including AP5 and R-CPP are less effective and showed no block effects in some experiments [17,21,29].

10.8. SUMMARY

Molecular and physiological characterizations of cloned dNR1 and dNR2 reveal functional NMDARs in Drosophila that consist of dNR1 and dNR2 subunits. Co-expression of dNR1 and dNR2 in Xenopus oocytes or Drosophila S2 cells produces an electrophysiological profile exhibiting most of the distinguishing properties specific to mammalian NMDARs including selective activation by NMDA and L-aspartate, modulation by glycine as a coagonist for glutamate and voltage- and Mg²⁺-dependent conductance.

Genetic analyses of the dNR1 gene reveal an acute and physiological role for NMDARs in associative learning and subsequent LTM consolidation. This extends genetic findings in vertebrates to invertebrates. Many intracellular signaling proteins are known to be physically associated with vertebrate NMDARs [144,147]. Obvious Drosophila homologues can be identified for most of these proteins and many have been shown to be important for associative learning and memory formation [112,148,149]. It will be important identify more of the biochemical signaling pathway from NMDAR to CREB during LTM formation and the functional genomics of NMDAR-dependent memory consolidation.

Subsequent identification of the ellipsoid body for LTM consolidation and storage supports a much broader and more complex neuronal circuitry subserving memory consolidation in Drosophila. Distinct components of this extensive neuronal circuitry seem to be independently involved with different temporal stages of memory consolidation, with the mushroom body responsible for acquisition and earlier memory processing, while the ellipsoid body specifically controls LTM consolidation and storage. This discovery implies a “transference” of memory from one anatomic location (mushroom body) to another (ellipsoid body) as consolidation progresses.

The conservation of functional NMDARs and their involvement during behavioral plasticity in invertebrates further demonstrate that a unified mechanism may underlie associative learning and memory across species. Because behavioral plasticity is tightly associated with synaptic plasticity, we speculate that similar cellular mechanisms of NMDAR-mediated long-term changes including LTP and LTD may also exist in the insect brain. We expect that Drosophila genetics will likely continue to discover additional genes and signaling pathways important for these forms of plasticity.

ACKNOWLEDGMENTS

We thank Dr. Tim Tully for valuable comments and discussion and Drs. Josh Dubnau and Glenn Turner for critical reading of the manuscript. S.X. is a senior research scientist in Dr. Tully’s laboratory at Cold Spring Harbor. This work was supported by grants to Dr. Tully from Dart Neurosciences, LLC, and to A.S.C. from the National Science Council and the Ministry of Education of Taiwan.

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