The noncompetitive antagonists of the vertebrate NMDA receptor dizocilpine (MK 801) and phencyclidine (PCP), delivered in food, were found to induce a marked and reversible inhibition of locomotor activity in Drosophila larvae. To determine the site of action of these antagonists, an in vitro preparation of the Drosophila third-instar larva was used, preserving the central nervous system and segmental nerves with their connections to muscle fibers of the body wall. Intracellular recordings were made from ventral muscle fibers 6 and 7 in the abdominal segments. In most larvae, long-lasting (>1 h) spontaneous rhythmic motor activities were recorded in the absence of pharmacological activation. After sectioning of the connections between the brain and abdominal ganglia, the rhythm disappeared, but it could be partially restored by perfusing the muscarinic agonist oxotremorine, indicating that the activity was generated in the ventral nerve cord. MK 801 and PCP rapidly and efficiently inhibited the locomotor rhythm in a dose-dependent manner, the rhythm being totally blocked in 2 min with doses over 0.1 mg/mL. In contrast, more hydrophilic competitive NMDA antagonists had no effect on the motor rhythm in this preparation. MK 801 did not affect neuromuscular glutamatergic transmission at similar doses, as demonstrated by monitoring the responses elicited by electrical stimulation of the motor nerve or pressure applied glutamate. The presence of oxotremorine did not prevent the blocking effect of MK 801. These results show that MK 801 and PCP specifically inhibit centrally generated rhythmic activity in Drosophila, and suggest a possible role for NMDA-like receptors in locomotor rhythm control in the insect CNS (Cattaert, 2001)
To determine whether the cloned dNR1 and dNR2 subunits associate to form functional ionotropic receptor channels, they were coexpressed in Xenopus oocytes and the resulting electrophysiological properties were examined. Coexpression of dNR1 and dNR2-2 induced robust NMDA-selective responses, whereas dNR2-1 in combination with dNR1 induced no NMDA-dependent responses in oocytes, suggestive of some functional difference between the two dNR2 isoforms. Coexpression of dNR1 and dNR2-3 has not been tested yet. The oocytes, expressing both dNR1 and dNR2-2, exhibited significant inward currents upon application of NMDA but not AMPA, and the NMDA-activated responses were concentration dependent. This suggests that dNR1 and dNR2 can form a functional ion channel in oocytes, which selectively responds to NMDA. Mammalian NMDA receptors are modulated by glycine (Kleckner, 1998). This also is the case for fly NMDA receptors, although application of glutamate in the presence of glycine appears much less effective than NMDA alone, which may reflect the facts that the relevant structural domains for glycine and glutamate binding are not completely conserved in dNR1 and dNR2 or that residual glycine may alter the response in this heterologous system. Mammalian NMDA receptors are activated by L-aspartate as well as glutamate (Patneau, 1990). Consistent with this observation, fly NMDA receptors are activated by various concentrations of aspartate. When expressed in oocytes, however, conductance through fly NMDA receptors is not voltage dependent. Consequently, dNR1 and dNR2 was also coexpressed in Drosophila S2 cells, thereby revealing a voltage-dependent conductance that is blocked by external Mg2+. Thus, this eletrophysiological profile of coexpressed dNR1 and dNR2 reveals most of the distinguishing characteristics of vertebrate NMDARs (Xia, 2005).
To examine expression of the dNR1 protein, a rabbit anti-dNR1 polyclonal antibody was generated. The antibody recognized a single protein of the appropriate size on Western blot. dNR1 seems to be weakly expressed throughout the entire brain (see dNR1 and dNR2 protein expression in the adult brain). Higher expression levels were observed in some scattered cell bodies and part of their fibers, including those from several pairs of DPM (dorsal-posterior-medial) neurons surrounding the calyx, DAL (dorsal-anterior-lateral) and DPL (dorsal-posterior-lateral) neurons in the lateral protocerebrum (LP), VAL (ventral-anterior-lateral) neurons in the anterior protocerebrum, and two pairs of VP (ventral-posterior) neurons in the posterior protocerebrum. Many cell bodies in the optic lobes also were labeled preferentially. Notably, punctuate staining was detected in many brain regions including the superior medial protocerebrum, suggesting synaptic localization of dNR1 (Xia, 2005).
The anti-dNR1 antibody does not preferentially label MB neurons. This is notable because MBs are critically required for olfactory learning. Instead, preferential dNR1 expression was detected in 12 pairs of cell bodies surrounding the MB calyx. Interestingly, a pair of DPM2 (dorsal-paired-medial 2) neurons are located just next to the previously identified DPM neurons in which no dNR1 expression is detectable. The DPM neurons innervate all the MB lobes and appear involved in early memory. Three additional pairs of DPM3 neurons with cell bodies smaller than DPM2 also showed strong immunolabeling. The spatial distributions of these neurons are highly symmetrical. Four other DPM4 neurons are located medially to the MB calyx and send descending fibers along a common tract. DPM4 neurons are clustered together in some flies but scattered in others. Another two pairs of neurons, DPM5 and DPL (dorsal-posterior-lateral), are located above the MB calyx. They appear to project descending fibers together with DPM4 neurons . The cell bodies of the VP (ventral-posterior) neurons are located beneath the MB calyx. DAL (dorsal-anterior-lateral) neurons are located in the LP region. LP receives extensive olfactory projections through the antennalglomerular tract of the antennal lobe, which itself receives olfactory input from antennae. The function of LP in olfaction and olfactory learning is largely unknown. dNR1 appears only weakly expressed in antennal lobes and central complex (Xia, 2005).
One of the mouse monoclonal anti-dNR2 antibodies allowed evaluation of the distribution of dNR2 proteins in adult brain. This antibody labels two bands with molecular weights close to the deduced sizes of dNR2 proteins. Similarly to dNR1, weak expression of dNR2 was detected in most, if not all, brain neurons. Again, preferential expression was found in several pairs of large neurons. Notably, dNR1 and dNR2 colocalized in four cell bodies of DPM4 neurons. Both proteins also colocalized in many synapse-like punctuate structures including those along the fibers of DPM4 neurons. Nevertheless, not all dNR1-positive neurons appear to express dNR2 at equivalent levels or verse visa. dNR2 is strongly expressed in a pair of DAL2 neurons and two pairs of VAL2 neurons, for instance, whereas dNR1 is strongly expressed in DAL and VAL neurons. These observations suggest that NR1 and NR2 may be regulated differentially during development or by experience or that these subunits may partner in vivo with other unknown subunits to form functional NMDARs (Xia, 2005).
The 3D staining patterns of dNR1 and dNR2 were superimposed into a volume model of adult fly brain to analyze NR-positive fibers in more detail. VAL appears to be the only neurons sending dNR1-positive projections to the front of contralateral MB calyx. Remarkably, all other NR-positive neurons do not appear to send projections to MBs. DPL and DPM5 are descending neurons and project in parallel with DPM4 neurons to the ventral-posterior ipsilateral protecerebrum and then extend anteriorly. The NR-positive fibers from other neurons surrounding the MB calyx do not enter the calyx or lobes of MBs. This, however, does not exclude the possibility that they may contact MBs through presynaptic fibers where no dNR proteins are expressed. DAL projects ascending fibers toward the superior medial protocerebrum with dNR1 protein distributed at the cell bodies and synapse-like puncta along its fibers. Thus, at least in DAL neurons, dNR1 appears to localize both pre- and postsynaptically (Xia, 2005).
The mushroom bodies (MBs) are paired brain centers located in the insect protocerebrum involved in olfactory learning and memory and other associative functions. Processes from the Kenyon cells (KCs), their intrinsic neurons, form the bulk of the MB's calyx, pedunculus and lobes (see Mushroom body is a quadruple structure). In young adult Drosophila, the last-born KCs extend their processes in the alpha/beta lobes as a thin core (alpha/beta cores) that is embedded in the surrounding matrix of other mature KC processes. A high level of L-glutamate (Glu) immunoreactivity is present in the alpha/beta cores (alpha/betac) of recently eclosed adult flies. In a Drosophila model of fragile X syndrome, the main cause of inherited mental retardation, treatment with metabotropic Glu receptor (mGluR) antagonists can rescue memory deficits and MB structural defects. To address the role of Glu signaling in the development and maturation of the MB, the time course of Glu immunoreactivity was compared with the expression of various glutamatergic markers at various times, that is, 1 hour, 1 day and 10 days after adult eclosion. It was observed that last-born alpha/betac KCs in young adult as well as developing KCs in late larva and at various pupal stages transiently express high level of Glu immunoreactivity in Drosophila. One day after eclosion, the Glu level was already markedly reduced in the alpha/betac neurons. Glial cell processes expressing glutamine synthetase and the Glu transporter dEAAT1 were found to surround the Glu-expressing KCs in very young adults, subsequently enwrapping the alpha/beta lobes to become distributed equally over the entire MB neuropil. The vesicular Glu transporter DVGluT was detected by immunostaining in processes that project within the MB lobes and pedunculus, but this transporter is apparently never expressed by the KCs themselves. The NMDA receptor subunit dNR1 is widely expressed in the MB neuropil just after eclosion, but was not detected in the alpha/betac neurons. In contrast, evidence is provided that DmGluRA, the only Drosophila mGluR, is specifically expressed in Glu-accumulating cells of the MB alpha/betac immediately and for a short time after eclosion. The distribution and dynamics of glutamatergic markers indicate that newborn KCs transiently accumulate Glu at a high level in late pupal and young eclosed Drosophila, and may locally release this amino acid by a mechanism that would not involve DVGluT. At this stage, Glu can bind to intrinsic mGluRs abundant in the alpha/betac KCs, and to NMDA receptors in the rest of the MB neuropil, before being captured and metabolized in surrounding glial cells. This suggests that Glu acts as an autocrine or paracrine agent that contributes to the structural and functional maturation of the MB during the first hours of Drosophila adult life (Sinakevitch, 2010).
In Drosophila and other arthropods, Glu is well characterized as the excitatory neurotransmitter of the neuromuscular junction. However, this amino acid has important signaling functions in the Drosophila brain as well. The Drosophila genome was predicted to encode 30 iGluR subtypes, including 3 AMPA- and 15 kainate-like, 2 NMDA-like, 4 δ-like and 6 divergent receptors. For now, the best characterized of these are the postsynaptic iGluRs expressed at the neuromuscular junction. Drosophila NMDA-like receptors are expressed in the central nervous system and have been implicated in learning and memory and locomotor control. The Drosophila genome encodes a single functional mGluR, DmGluRA, an ortholog of vertebrate group II mGluRs (Parmentier, 1996). This mGluR is presynaptic and expressed at the periphery of the active zones at the glutamatergic neuromuscular junctions, where it modulates both synapse excitability and fine structure (Bogdanik, 2004). DmGluRA is also expressed in the brain, in particular in lateral clock neurons, where it regulates circadian locomotor behavior (Hamasaka, 2007; Sinakevitch, 2010 and references therein).
The mushroom bodies (MBs) are paired centers located in the protocerebrum of Drosophila and other dicondylic insects that play essential roles in olfactory learning and memory and other brain functions, such as the control of locomotor activity, courtship behavior, courtship conditioning, visual context generalization, and sleep. The intrinsic structure of the MB is provided by the Kenyon cells (KCs), which have their cell bodies in the brain cortex and their dendrites in the MB calyx, where they receive input from the antennal lobe projection neurons. Axon-like processes of KCs project anteriorly and ventrally in the peduncle to form the vertical and medial lobes, which are subdivided into discrete parallel entities, the vertical α, α' and the medial β, β' and γ lobes. In addition to the KCs, there are other MB intrinsic neurons and several classes of MB extrinsic neurons that connect the MB to other areas of the brain neuropil. Emerging evidence suggests that different subtypes of MB KCs may be involved in distinct mechanisms of memory formation due to their connections to different MB extrinsic neurons (Sinakevitch, 2010).
Developmental studies have shown that the KCs are produced in each hemisphere of the brain by the division of four neuroblasts born early during the embryonic stage. The division of these neuroblasts sequentially produces the three morphologically and spatially distinct subtypes of KCs: γ, α'/β' and α/β. The γ neurons are generated up to the mid-third instar larval stage; they form the larval dorsal and medial lobe. The next KC subtype to be generated is the α'/β' neuron, which continues to be produced until puparium formation. Lastly, the α/β neurons are generated from the time of puparium formation until adult eclosion. In the α/β lobes, the KCs are organized in concentric layers. The youngest axon-like processes situated in the inner layer of the lobes are successively displaced outwards as they differentiate and newer α/β processes are added to the structure from the most recently born KCs (Kurusu, 2002). This volume of the α/β lobes into which grow the last-born axons contains densely packed and extremely thin fibers that are rich in actin filaments. This subset of processes has been named the α/β core (α/βc) (Sinakevitch, 2010).
An increased response to mGluR activation may play a prominent role in the fragile X syndrome (FXS), the most common form of inherited mental retardation and the predominant cause of autism. Mutations in dFmr1, the Drosophila homologue of the gene implicated in FXS, lead both to learning deficits and altered development of the MB, of which the most common feature is a failure of β lobes to stop at the brain midline. These behavioral and developmental phenotypes can be successfully rescued in Drosophila by treatment with mGluR antagonists (McBride, 2005), implicating Glu in the pathology, as is the case in mammalian models. Recent studies showed that dFmr1 interacts with DmGluRA in the regulation of synaptic architecture and excitability at glutamatergic synapses (Gatto, 2008; Repicky, 2009). However, until now the precise role of Glu and mGluRs in FXS and MB development has remained obscure (Sinakevitch, 2010).
This study presents evidence that Glu and its receptor DmGluRA are directly involved in construction of the MB neural circuits. Previous studies suggested that the Drosophila last-born α/βc KCs are immunoreactive to anti-Glu antibodies. The present study shows that these neurons express a high level of Glu-like immunoreactivity in newly eclosed adult flies. Interestingly, newborn KCs in late larval and pupal stages also appear to express as a rule a high level of Glu. To understand further the role and fate of Glu during KC maturation, the dynamics of Glu, DmGluRA and other Glu signaling-associated proteins were analyzed in the MB of young adult Drosophila from the time of their eclosion until 10 days post-eclosion. The results indicate that a transient Glu release likely regulates functional maturation of newborn KCs by a paracrine action during Drosophila post-embryonic development and the first hours after adult eclosion (Sinakevitch, 2010).
One intriguing question in neuroscience is how newborn neurons establish a functional network during their period of growth and maturation. This work describes a study of the late maturation of a subset of the α/β intrinsic MB KCs, the α/βc neurons, during a short period after adult eclosion. Glu-like immunoreactivity has been observed in the ingrowth lamina of the cockroach MB, which contains axons of the youngest KCs. Similarly in Drosophila, Glu accumulates in the α/βc, which contains newly generated neurons, whereas taurine-expressing neurons were found in the outer α/βc and aspartate-expressing neurons in the rest of the α/β lobes. It has been shown in vertebrates that Glu can have a strong influence on cone motility and induce rapid filopodia protrusion from hippocampal neurites or cultured astrocytes. In the present study an extensive analysis was performed of the distribution of various glutamatergic markers in the MBs of young adult Drosophila. The results suggest that the α/βc neurons are not simply glutamatergic. Rather, the evidence provided in this study indicates that these newborn KCs may transiently use Glu as a paracrine agent to favor interactions with glial cell processes and become mature neurons forming functional circuits (Sinakevitch, 2010).
Although the last-born α/βc KCs show a high level of Glu immunoreactivity a few hours prior and after adult eclosion, Glu immunostaining is dramatically reduced in these cells 24 hours after eclosion and is entirely absent a few days later. Disappearance of this signal could result from the release or intracellular metabolism of this amino acid. Similarly, it was observed in cockroach MBs that newborn KCs loose Glu immunoreactivity when they become mature and establish contacts with extrinsic neurons. This study also presents the first evidence that Glu transiently accumulates at a high level in developing newborn KCs of Drosophila in late larva and during pupal stages. Therefore, transient Glu expression could correlate with KC growth and maturation not only in the α/βc around eclosion time but also in other lobes during earlier stages of MB development (Sinakevitch, 2010).
Three subtypes of vesicular Glu transporters (VGluTs) have been identified in the mammalian nervous system with similar Glu transport functionality. Two of these (VGluT1 and VGluT2) present complementary distribution in central glutamatergic neurons. The third isoform, VGluT3, appears to be primarily expressed in neurons that release another transmitter (serotonin, dopamine, acetylcholine or GABA), where it may be required for efficient synaptic transmission. In the present study, neither the α/βc neurons nor any other intrinsic MB KCs were found to express the Drosophila vesicular transporter DVGluT. This may indicate that the Glu that is accumulated in the inner α/βc neurons is not stored in synaptic vesicles. However, the possibility cannot be excluded that these cells express another vesicular Glu transporter not yet identified in Drosophila. DVGluT immunoreactivity was observed in the MBs, particularly in the γ lobe and spur region and in the α lobes, but the punctuate labeling and localization suggest that this distribution corresponds to glutamatergic synapses belonging to extrinsic neurons (Sinakevitch, 2010).
Can the Glu transiently stored in the newborn MB neurons be released into the extracellular space? In the absence of DVGluT or another similar transporter, this could involve a non-vesicular release of Glu. Non-conventional release of Glu from immature neurons has been demonstrated in the developing rat hippocampus where Glu release exerts a paracrine action that seems to particularly affect the migration of neighboring maturing neurons. To address this question indirectly, the presence in the MB of other proteins known to be involved in the recycling and degradation of Glu at glutamergic synapses was sought (Sinakevitch, 2010).
An important role of glial cells is to capture Glu released from the synapse with specific transporters and then convert Glu to glutamine with GS. The only Drosophila high-affinity Glu transporter, dEAAT1, is expressed in subtypes of glial cells and is associated with Glu-release sites. GS2 is similarly expressed in glial cells in the Drosophila nervous system. This study shows that glial cells expressing dEAAT1 and GS surround the Drosophila MB lobe neuropiles, closely enwrapping the α/β lobes, thus isolating them from other lobes, and sending a mesh-like system of extensions inside these lobes. Enwrapping and invading of the MB β lobes by glia was also observed to occur in cockroach MBs, where glial cells are implicated in the removal of degenerating transient KC processes that occur during their establishment of mature connections with extrinsic cell dendrites. The high levels of glial dEAAT1 and GS within the Drosophila MB lobes suggest that this neuropil is tightly cordoned off from other parts of the brain and regulates the extracellular Glu level between the axons (Sinakevitch, 2010).
These data show that GS expression is highly dynamic in the MB during the first day of adult life, suggesting that glial cells play a role in establishing the MB's functional network. During the first hour after eclosion, the meshwork of glial processes expressing Glu signaling-associated molecules (GS and dEAAT1) is not present in the inner α/βc region, but within 24 hours this area becomes covered by glial extensions. These glial elements are possibly guided towards the α/βc area by the gradient of Glu released by the last born KCs. Glia could be involved in reducing Glu concentration in this area and play a role in axonal guidance and final maturation of KCs. Evidence that Glu transporters are required for coordinated brain development has been previously reported for mice: the absence of two glial Glu transporters resulted in excess of extracellular Glu and abnormal formation of the neocortex (Sinakevitch, 2010).
Assuming Glu is released by the newborn MB neurons, it has to interact with specific receptors. Therefore, the expression was sought of Glu receptors in MB neuropiles of young adult Drosophila, particularly those receptors that are likely to regulate neuronal growth and maturation through second-messenger pathways. Once activated by simultaneous Glu binding and membrane depolarization, the NMDAR channel allows calcium influx into the postsynaptic cell, where this ion triggers a cascade of biochemical events resulting in synaptic maturation and plasticity. Available antibodies against the constitutive dNR1 subunit of the Drosophila NMDAR were used. Immediately after eclosion, many processes in the MB neuropil were found to be dNR1-positive, with the exception of the α/βc neurons. The Glu released from either these α/βc neurons, or the surrounding glial cells, or extrinsic MB glutamatergic neurons may activate these NMDAR receptors. Thus, a widespread localization of NMDAR characterizes the MB immediately after eclosion, at the beginning of adult life when the MB is expected to receive the least inputs from sensory interneurons. Subsequently, with increasing sensory data being received and relayed to projection neurons, there is a dramatic and concomitant restructuring of NMDAR signaling: the majority of MB neurons no longer express these receptors. It is only those neurons that receive constant glutamatergic signaling that still address the dNR1 subunit in the vicinity of glutamatergic synapses expressing DVGluT. This occurs in particular within the spur region of the MB and the lateral horn. Such developmentally related regulation of NMDAR expression in the MBs of young adult flies may relate to adaptations of synaptic activity in response to sensory experience (Sinakevitch, 2010).
mGluRs are neuromodulatory G-protein-coupled receptors that are involved in many aspects of brain physiology, including neuronal development, synaptic plasticity, and neurological diseases. Whereas eight distinct mGluRs are present in the mammalian genome, a single functional mGluR is expressed in Drosophila, DmGluRA. The fly mGluR is structurally and pharmacologically closer to the mammalian group II mGluRs, which are mainly presynaptic receptors negatively coupled to adenylate cyclase. Attempts to locate DmGluRA with the commercially available monoclonal antibody 7G11 were not successful because the antibody produced by the hybridoma clone recently lost its binding specificity. To monitor DmGluRA distribution, a new GAL4 line was used that carries an enhancer trap insertion close to the mGluR start site of transcription, keeping in mind that expression of this GAL4 reporter may, in part, differ from the mGluR pattern. Strikingly, the DmGluRA-GAL4 line was found to express GFP selectively in the Glu-accumulating α/βc KCs of newly eclosed adult flies. This is in contrast to commonly used MB GAL4 driver lines (17d-, c739- and 201Y-GAL4) that do not express GFP in these neurons immediately after eclosion. Ten days later, the GFP staining in the DmGluRA-GAL4 line appeared strongly reduced in the α/βc; in contrast, the MB drivers now expressed GFP in these neurons (Sinakevitch, 2010).
Because the GAL4 reporter method reveals whole neurons, it could not be determined where the receptor is addressed restrictively in cell bodies, dendrites or axons. A previous study performed with an active lot of 7G11 antibody indicated that DmGluRA is present in nearly all neuropiles of the mature adult fly brain, including the MB calyces, but not in the MB lobes (Devaud, 2008). However, thas study did not report on the localization of DmGluRA in newly eclosed Drosophila. Further work is required to precisely locate the subcellular localization of DmGluRA in the newborn α/βc neurons, either with a new antibody or a DmGluRA-GFP fusion gene. The source of Glu binding to this mGluR receptor may be the neighboring glial cells or newborn KCs themselves, or both. Through activation of these receptors, Glu is likely to have a transient paracrine action on the α/βc neurons during the first day after eclosion that could be required for dendrite growth or synaptic maturation (Sinakevitch, 2010).
Although the α/βc KCs represent a minor part of the α/β lobe neurons, the maturation of these cells appears to be essential for proper MB functioning. Selective expression of the rutabaga (rut)-encoded adenylate cyclase in the α/βc neurons with 17d-GAL4 was shown to partially restore olfactory learning and memory in 2- to 5-day-old rut mutant flies. In contrast, no rescue of the rut defect was observed with c739-GAL4, which expresses in more peripheral α/β neurons at this stage. Therefore, the network involved in olfactory learning and memory apparently requires the α/βc neurons and is already functional in 2- to 5-day-old flies. Furthermore, treatment with mGluR antagonists restored courtship behavior, memory deficits and MB structural defects in DFmr1 mutants, a Drosophila model of FXS. These positive effects are even stronger when the pharmacological treatment is applied both during larval development and after eclosion. This suggests that these behavioral defects relate to an abnormally high level or prolonged duration of DmGluRA expression in the α/βc neurons of DFmr1 mutants. Further study should determine the distribution of Glu and DmGluRA during MB development in Drosophila FXS models (Sinakevitch, 2010).
The ubiquitin-proteasome system is one of the major conserved cellular pathways controlling protein turnover in eukaryotic cells. Substrate protein ubiquitination plays important roles in neuronal differentiation, axonal targeting, synapse formation and plasticity. In addition to strong Glu immunolabeling in the inner α/βc KCs, a high level of anti-ubiquitin immunoreactivity was also observed in these neurons immediately after eclosion. Such a high staining level was no longer detected in 10-day-old flies. In contrast, the spur region of the MB showed a constant high ubiquitin immunoreactivity that did not change with the age of the animal. This could suggest that synaptic plasticity is particularly active in this MB area (Sinakevitch, 2010).
Similarly, labeling of the cockroach MB β lobe with anti-ubiquitin showed, at specific stages in each developmental instar, as well as at an early adult stage, consistent staining of newly generated KC axons. Anti-ubiquitin also labeled the extending transiently Glu-immunoreactive collateral processes from developing KCs in the ingrowth zone, the hemimetabolous homologue of Drosophila's core KCs. This study showed that ubiquitin expression precedes degeneration of these collaterals and their subsequent removal by scavenging glial cells. Glu receptors can be endocytosed by an ubiquitin-dependent mechanism. The down-regulation of Glu and its receptor protein, possibly mediated by ubiquitin, thus appear to be important steps in the maturation and differentiation of the α/βc KCs (Sinakevitch, 2010).
In conclusion the present study suggests that the Glu accumulated in the α/βc KCs of young adult Drosophila is used for cell growth and maturation rather than for neurotransmission. The distribution and dynamics of glutamatergic markers indicates that Glu released from newborn KCs can bind to intrinsic mGluRs in the α/β cores and to NMDARs in the rest of the MB neuropil before being captured and metabolized by surrounding glial cells. As an autocrine or paracrine agent, Glu is likely to play a role in pathway finding within the lobe, namely, interactions between maturing KCs and extrinsic neuron dendrites, guidance of glial cell outgrowth and glial process targets into and around the relevant lobes, and maturation of synaptic networks required for a functional MB. Further study of the paracrine function of Glu in wild-type flies and in the Drosophila FXS model may shed light on similar actions of this neurotransmitter in the developing human brain in normal and pathological conditions (Sinakevitch, 2010).
The dNR1 gene consists of 15 exons scanning more than 24 kb of genomic DNA. The 5′ end overlaps with Itp-r83A, the fly homolog of an inositol 1,4,5-tris-phosphate receptor. Flies homozygous for an F-element insertion in the third intron of dNR1 are subviable and female-sterile. Two independent EP element insertions also lie in dNR1 or nearby. EP3511 inserts in the first intron of the dNR1 gene, 718 bp upstream of the start codon in exon 2. EP331 is inserted 425 bp downstream of the 3′ end of the dNR1 transcription unit. Expression levels of dNR1 protein are reduced but not eliminated in homozygous EP3511/EP3511 or EP331/EP331 flies, indicating that both EP insertions represent hypomorphic mutations of dNR1. EP3511/EP3511 or EP331/EP331 homozygotes are viable, which allowed evaluation olfactory learning. Compared to wild-type flies, learning was reduced in both homozygotes (Xia, 2005).
The learning defects of EP3511 or EP331 mutants were rescued by cosmids containing genomic DNA from the dNR1 region. Cosmid-A contains the full-length Itp-r83A coding sequence and upstream elements that include only partial coding sequence of dNR1. Conversely, Cosmid-B and Cosmid-C contain all of the dNR1 transcription unit and only part of Itp-r83A. Cosmid-A, but not Cosmid-B or Cosmid-C, rescues the lethality associated with two different mutations of Itp-r83A, whereas Cosmid-B and Cosmid-C, but not Cosmid-A, rescued the learning defect of the EP3511 and EP331 mutants. These results establish that the learning defects of the EP mutants are due to disruption of the dNR1 gene not the Itp-r83A gene (Xia, 2005).
EP331 also allowed the use the EP-element to control the expression of dNR1 conditionally. The EP element in EP331 flies is inserted downstream of, and in an opposite orientation to, the transcription start site of dNR1. When combined with a GAL4 driver, this EP element yields an antisense transcript of dNR1. In transheterozygous EP331/+, hs-GAL4/+ flies, an anti-dNR1 message was induced by heat shock and was still detected 15 hr later, leading to a significant reduction in dNR1 protein. This antisense message was also detected before heat shock in EP331/+, hs-GAL4/+ flies but absent in heterozygous EP331/+ flies, suggesting some leaky expression of hs-GAL4 was driving low-level expression of anti-dNR1. This leaky expression did not produce any measurable effect on NR1 protein levels from Western blot analysis (Xia, 2005).
The disruption of dNR1 in EP331/+, hs-GAL4/+ flies was further confirmed with immunohistochemistry. Anti-dNR1 immunostaining was diminished throughout the entire brain after heat shock as compared with no heat shock. This reduction in dNR1 was quantified in a pair of dorsal-anterior-lateral (DAL) and a pair of ventral-anterior-lateral (VAL) neurons, where the protein is expressed at high levels. In both DAL and VAL neurons, the immunofluorescence intensity was reduced significantly 15 hr after heat shock (Xia, 2005).
Accordingly, learning was severely disrupted 15 hr after heat shock. In contrast, learning was disrupted only mildly in EP331/+, hs-GAL4/+ flies in the absence of heat shock. This mild disruptive effect is consistent with the observation that hs-GAL4 yields some leaky expression of anti-dNR1 message through development, though a concommitant reduction in NR1 protein was not detected. Alternatively, this transgenic line might harbor slight, nonspecific differences in genetic background (Xia, 2005).
Whether dNR1 was required for long-lasting memory produced by extended training was evaluated. EP331/+, hs-GAL4/+ flies were subjected to spaced or massed training 15 hr after heat shock and then tested for 1-day memory. In the absence of heat shock, 1-day memory after both spaced and massed training was normal. When trained 15 hr after heat shock, 1-day memory after massed training was normal, whereas that after spaced training was significantly reduced. Typically, 1-day memory after spaced training is composed of 50% LTM and 50% ARM (Anesthesia-Resistant Memory), and LTM specifically is disrupted in transgenic flies inducibly overexpressing CREB repressor. 1-day memory after massed training, in contrast, is composed only of ARM. Accordingly, these results suggest that ARM is normal and LTM is completely abolished in EP331/+, hs-GAL4/+ flies after acute disruption of dNR1. The observation that 1-day memory after massed training was normal also suggested that extended training might overcome the learning defect (after one training session) observed for EP331/+, hs-GAL4/+ flies subjected to heat shock. Indeed, this was the case for both spaced and massed training (Xia, 2005).
A modified massed training protocol was used, in which flies sat in the training chamber for 150 min before training began. With this protocol, massed training ends at the same time as spaced training, but 1-day memory after massed training is slightly higher than that after the standard protocol, which does not include pretraining exposure to the training chamber. Hence, the above experiments were repeated with the original massed training protocol with only heat-shocked wild-type and EP331/+, hs-GAL4/+ flies. Here again, 1-day memory after massed training was normal, whereas that after spaced training was disrupted (Xia, 2005).
Although dNR1 was expressed throughout the adult brain and especially also at the lateral protocerebrum (LP), sensorimotor responses to the odors and footshock stimuli were not affected in transheterozygous EP331/+, hs-GAL4/+ flies before or after heat shock. Homozygous EP3511/EP3511 and EP331/EP331 mutants also performed normally to these sensory stimuli (Xia, 2005).
The dendrites of neurons undergo dramatic reorganization in response to developmental and other cues, such as stress and hormones. Although their morphogenesis is an active area of research, there are few neuron preparations that allow the mechanistic study of how dendritic fields are established in central neurons. Dendritic refinement is a key final step of neuronal circuit formation and is closely linked to emergence of function. This is a study of a central serotonergic neuron in the Drosophila brain, the dendrites of which undergo a dramatic morphological change during metamorphosis. Using tools to manipulate gene expression in this neuron, the refinement of dendrites during pupal life was examined. This study shows that the final pattern emerges after an initial growth phase, in which the dendrites function as 'detectors', sensing inputs received by the cell. Consistent with this, reducing excitability of the cell through hyperpolarization by expression of K(ir)2.1 results in increased dendritic length. Sensory input, possibly acting through NMDA receptors, is necessary for dendritic refinement. These results indicate that activity triggers Wnt signaling, which plays a 'pro-retraction' role in sculpting the dendritic field: in the absence of sensory input, dendritic arbors do not retract, a phenotype that can be rescued by activating Wnt signaling. These findings integrate sensory activity, NMDA receptors and Wingless/Wnt5 signaling pathways to advance understanding of how dendritic refinement is established. The maturation of sensory function is shown to interact with broadly distributed signaling molecules, resulting in their localized action in the refinement of dendritic arbors (Singh, 2010).
This study focuses on a specific phase during the metamorphosis of the dendrites of a central serotonergic neuron, in which excess growth is removed by a process that has been termed refinement. Genetic analyses using loss-of-function mutants and RNAi-mediated knockdown of specific genes has led to a postulated a link between neuronal activity, synaptic input and Wnt signaling in this process. The sparse dendrites innervating the adult antennal lobe, present on the wide-field serotonergic neurons (CSDn) during the larval stage, are removed early in pupation by pruning, followed by a period of exuberant growth. The arrival of sensory neurons at the antennal lobe correlates well with when growth of the CSDn dendrites ceases and removal of the excess branches occurs. The CSDn must be active for the refinement process to occur, as refinement fails when neuronal activity is inhibited or when the sensory neurons are absent. Phenotypes observed in the latter case can be rescued by ectopic activation of the neuron using the temperature-sensitive dTrp-A1 channel. It is suggested that activity within the CSDn, possibly together with activity in presynaptic neurons, acts to provide the correlated activity required to trigger activation of NMDARs. Knockdown of NMDARs affects the refinement process, although identifying its specific action requires further study. A possible consequence of the activity-dependent process is activation of the Wg pathway, as the phenotype observed in aristalless mutants can also be rescued by ectopic expression of Dishevelled (Dsh) in the CSDn. It seems unlikely that activity within the CSDn leads to the release of Wnt ligands, but rather that dendrites respond locally to Wnt ligands in the region of a dendrite that is receiving input. Although other interpretations of the data are possible, a hypothesis is favored whereby specific synapses are stabilized as a result of correlated neuronal activity, and that excess dendritic branches are removed by Wnt signaling (Singh, 2010).
Perturbations in neuronal activity can be compensated by changes at multiple levels, including alterations in the expression of ion channels and in synaptic strength. Tripodi (2008) provides evidence for structural homeostasis whereby alterations in afferent input during development can be compensated by changes in dendritic geometry. This suggests that dendritic arbors serve as sensors for input levels, thus allowing the self-organization of circuits that is necessary for robust behavioral outputs (Tripodi, 2008). The current studies in the CSDn support these observations: reduced activation of the cell by targeted expression of Kir2.1 results in a greatly enlarged dendritic field in the adult. This phenotype can be explained by a mechanism in which the absence of electrical activity results in a failure of the signaling mechanisms that stop growth of the arbors and that remove additional branches. Reduced excitability could also drive the homeostatic mechanisms towards making more arbors and to suppress the refinement program (Singh, 2010).
Dendritic growth and refinement are closely associated with input activity and synapse formation during development. Activity-dependent development of circuits is thought to utilize mechanisms similar to those involved in Hebbian learning and plasticity. NMDARs are ideal candidates for detecting correlated pre- and postsynaptic activity, which is crucial in the Hebbian model of learning and plasticity. Strengthening of synapses, as in this study, leads to the stabilization and extension of dendrites, whereas weakening of synapses leads to the destabilization and elimination of dendritic branches (Espinosa, 2009; Cline, 2008; Constantine-Paton, 1998). During vertebrate hippocampal development, NMDAR activation has been shown to limit synapse number and reduce dendritic complexity. The stabilization of a particular synapse or arbor possibly attenuates the formation of new branches or synapses, thus limiting further dendritic growth. In such a scenario, knocking down NMDAR levels would be expected to result in increased dendritic complexity, as indeed has been observed in this study. The mechanism by which 'appropriately connected' synapses are strengthened, whereas suboptimal contacts are eliminated, needs to be studied in thus system. In other systems, Ca2+, which is released upon NMDAR activation, impinges on various intracellular effectors that regulate dendritic morphogenesis. In addition, selective stabilization/destabilization of dendritic arbors could be affected by the local release of growth factors in response to activity (Singh, 2010).
This study shows that activity-dependent activation of the Wnt pathway facilitates retraction of dendritic arbors. Arbors that receive appropriate input are somehow protected and stabilized. These experiments suggest that Wnt-dependent refinement functions through a non-nuclear pathway and could act by impinging directly on cytoskeletal dynamics (Schlessinger, 2009; Salinas, 2008). Disruption of the microtubule cytoskeleton is a key feature of dendritic pruning in Drosophila during metamorphosis. GSK3β (Shaggy in Drosophila) an intracellular inhibitor of the Wnt pathway, has been shown to act as a sensor of inputs for neuronal activity (Chiang, 2009) and a potent regulator of microtubule dynamics in axons. In the Drosophila embryonic CNS, the Src family of tyrosine kinases (SFKs) is required for Wnt5/Drl-mediated signaling. Interestingly, SFKs seem to act as a crucial point of convergence for multiple signaling pathways that enhance NMDAR activity and hence are thought to act as molecular hubs for the control of NMDARs. It is tempting to envisage a scenario in which there is cross-talk between Wnt5/Drl signaling-mediated activation of SFKs and NMDAR signaling during refinement (Singh, 2010).
In summary, this study shows that the dendritic refinement of a central modulatory serotonergic neuron is regulated by electrical activity, NMDAR and Wnt signaling. Similar mechanisms have been implicated in dendritic growth and refinement of excitatory neurons in vertebrates. This study provides a model neuron preparation in which the dendritic growth and refinement of a modulatory neuron can be analyzed genetically. It was demonstrated that the dendrites of CSDn receive input from sensory neurons from the arista, supporting previous suggestions that mechanosensory input could alter sensitivity to odorant stimuli. In both Drosophila (Dacks, 2009) and the mammalian olfactory bulb (Petzold, 2009), serotonin gates the odor-evoked sensory response. CSDn sends projections to higher brain centers and multiglomerular projections to the contralateral antennal lobe and hence it is likely to influence the overall properties of the olfactory circuit. This study suggests that the structural and resulting functional properties of this neuron emerge from an interaction between partner neurons, together with input from intrinsic and extrinsic cues (Singh, 2010).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila
Abe, P. and Takeichi, M. (2007). NMDA-receptor activation induces Calpain-mediated β-catenin cleavages for triggering gene expression. Neuron 53: 387-397. Medline abstract: 17270735
Asai, M., et al. (2001). Visualization of mPer1 transcription in vitro: NMDA induces a rapid phase shift of mPer1 gene in cultured SCN. Curr. Biol. 11: 1524-1527. Medline abstract: 11591320
Beffert, U., et al. (2005). Modulation of synaptic plasticity and memory by Reelin involves differential splicing of the lipoprotein receptor Apoer2. Neuron 47: 567-579. PubMed citation: 16102539
Bellone, C. and Nicoll, R. A. (2007). Rapid bidirectional switching of synaptic NMDA receptors. Neuron 55(5): 779-85. Medline abstract: 17785184
Ben Mamou, C., Gamache, K. and Nader, K. (2006). NMDA receptors are critical for unleashing consolidated auditory fear memories. Nat. Neurosci. 9(10): 1237-9. Medline abstract: 16998481
Bogdanik, L., et al. (2004). The Drosophila metabotropic glutamate receptor DmGluRA regulates activity-dependent synaptic facilitation and fine synaptic morphology. J. Neurosci. 24: 9105-9116. PubMed Citation: 15483129
Bradley, J., Carter, S. R., Rao, V. R., Wang, J. and Finkbeiner, S. (2006). Splice variants of the NR1 subunit differentially induce NMDA receptor-dependent gene expression. J. Neurosci. 26(4): 1065-76. Medline abstract: 16436592
Brockie, P. J., et al. (2001). The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated currents that regulate reversal frequency during locomotion. Neuron 31: 617-630. Medline abstract: 11545720
Burgos-Robles, A., et al. (2007). Consolidation of fear extinction requires NMDA receptor-dependent bursting in the ventromedial prefrontal cortex. Neuron 53: 871-880. Medline abstract: 17359921
Burnashev, N., et al. (1992). Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor. Science 257: 1415-1419. Medline abstract: 1382314
Cammarota, M., et al. (2000). Learning-associated activation of nuclear MAPK, CREB and Elk-1, along with Fos production, in the rat hippocampus after a one-trial avoidance learning: abolition by NMDA receptor blockade, Brain Res. Mol. Brain Res. 76: 36-46. Medline abstract: 10719213
Cattaert, D. and Birman, S. (2001). Blockade of the central generator of locomotor rhythm by noncompetitive NMDA receptor antagonists in Drosophila larvae. J. Neurobiol. 48: 58-73. Medline abstract: 11391649
Chen, B. S. and Roche, K. W. (2009). Growth factor-dependent trafficking of cerebellar NMDA receptors via protein kinase B/Akt phosphorylation of NR2C. Neuron 62(4): 471-8. PubMed Citation: 19477150
Chiang, A. S., et al. (2002). Insect NMDA receptors mediate juvenile hormone biosynthesis, Proc. Natl. Acad. Sci. USA 99: 37-42. Medline abstract: 11773617
Chiang, A., et al. (2009). Neuronal activity and Wnt signaling act through Gsk3-β to regulate axonal integrity in mature Drosophila olfactory sensory neurons. Development 136: 1273-1282. PubMed Citation: 19304886
Chubykin, A. A., et al. (2007). Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2. Neuron 54(6): 919-31. Medline abstract: 17582332
Clarke, R. J. and Johnson, J. W. (2006). NMDA receptor NR2 subunit dependence of the slow component of magnesium unblock. J. Neurosci. 26(21): 5825-34. Medline abstract: 16723541
Cline, H. T. and Constantine-Paton, M. (1990). NMDA receptor agonist and antagonists alter retinal ganglion cell arbor structure in the developing frog retinotectal projection. J. Neurosci. 10: 1197-1216. PubMed Citation: 2158526
Cline, H. and Haas, K. (2008). The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: a review of the synaptotrophic hypothesis. J. Physiol. 586: 1509-1517. PubMed Citation: 18202093
Constantine-Paton, M. and Cline, H. T. (1998). LTP and activity-dependent synaptogenesis: the more alike they are, the more different they become. Curr. Opin. Neurobiol. 8: 139-148. PubMed Citation: 9568401
Corrigan, C., Subramanian, R. and Miller, M. A.(2005). Eph and NMDA receptors control Ca2+/calmodulin-dependent protein kinase II activation during C. elegans oocyte meiotic maturation. Development 132(23): 5225-37. Medline abstract: 16267094
Dacks A. M., et al. (2009). Serotonin modulates olfactory processing in the antennal lobe of Drosophila. J. Neurogenet. 23. 366-377. PubMed Citation: 19863268
Datwani, A. Iwasato, T., Itohara, S. and Erzurumlu, R. S. (2002). NMDA receptor-dependent pattern transfer from afferents to postsynaptic cells and dendritic differentiation in the barrel cortex. Mol. Cell. Neurosci. 21: 477-492. PubMed Citation: 12498788
Devaud, J. M., et al. (2008). Widespread brain distribution of the Drosophila metabotropic glutamate receptor. Neuroreport 19: 367-371. PubMed Citation: 18303583
Dingledine, R., Borges, K. Bowie, D. and Traynelis, S. F. (1999). The glutamate receptor ion channels, Pharmacol. Rev. 51: 7-61. Medline abstract: 10049997
Engert, F. and Bonhoeffer, T. (1999) Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 399: 66-70. PubMed Citation: 10331391
Espinosa, J. S., Wheeler, D. G., Tsien, R. W. and Luo, L. (2009). Uncoupling dendrite growth and patterning: single-cell knockout analysis of NMDA receptor 2B. Neuron 62(2): 205-17. PubMed Citation: 19409266
Ferrer-Montiel, A. V., Sun, W. and Montal, M. (1995). Molecular design of the N-methyl-D-aspartate receptor binding site for phencyclidine and dizolcipine, Proc. Natl. Acad. Sci. 92; 8021-8025. Medline abstract: 7644531
Furukawa, H., Singh, S. K., Mancusso, R. and Gouaux, E. (2005). Subunit arrangement and function in NMDA receptors. Nature 438(7065): 185-92. Medline abstract: 16281028
Garry, E. M., et al. (2003). Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/CaM kinase II interactions are disrupted in PSD-95 mutant mice. Curr. Biol. 13: 321-328. Medline abstract: 12593798
Gatto, C. L. and Broadie, K. (2008). Temporal requirements of the fragile X mental retardation protein in the regulation of synaptic structure. Development 135: 2637-2648. PubMed Citation: 1857967
Gielen, M., et al. (2008). Structural rearrangements of NR1/NR2A NMDA receptors during allosteric inhibition. Neuron 57(1): 80-93. PubMed citation: 18184566
Hamasaka, Y., et al. (2007). Glutamate and its metabotropic receptor in Drosophila clock neuron circuits. J. Comp. Neurol. 505: 32-45. PubMed Citation: 17729267
Hawasli, A. H., et al. (2007). Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation. Nature Neurosci. 10: 880-886. Medline abstract: 17529984
Husi, H., et al. (2000). Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nat. Neurosci. 3: 661-669. Medline abstract: 10862698
Irie, M. L., et al. (1997). Binding of neuroligins to PSD-95. Science 277(5331): 1511-1515. Medline abstract: 9278515
Kano, T., et al. (2008). Memory in Caenorhabditis elegans is mediated by NMDA-type ionotropic glutamate receptors. Curr. Biol. 18(13): 1010-5. PubMed Citation: 18583134
Keifer, J. (2001). In vitro eye-blink classical conditioning is NMDA receptor dependent and involves redistribution of AMPA receptor subunit GluR4. J. Neurosci. 21(7): 2434-2441. Medline abstract: 11264317
Kennedy, N. J., et al. (2007). Requirement of JIP scaffold proteins for NMDA-mediated signal transduction. Genes Dev. 21(18): 2336-46. Medline abstract: 17875667
Kim, E., et al. (1996). Heteromultimerization and NMDA receptor-clustering activity of Chapsyn-110, a member of the PSD-95 family of proteins. Neuron 17: 103-113. Medline abstract: 8755482
Kim, M. J., et al. (2005). Differential roles of NR2A- and NR2B-containing NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking. Neuron 46: 745-760. Medline abstract: 15924861
Kim, M. J., et al. (2007). Synaptic accumulation of PSD-95 and synaptic function regulated by phosphorylation of serine-295 of PSD-95. Neuron 56(3): 488-502. PubMed citation: 17988632
Kleckner, N. W. and Dingledine, R. (1988). Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes. Science 241: 835-837. Medline abstract: 2841759
Kopp, C., Longordo, F., Nicholson, J. R. and Luthi, A. (2006). Insufficient sleep reversibly alters bidirectional synaptic plasticity and NMDA receptor function. J. Neurosci. 26(48): 12456-65. Medline abstract: 17135407
Krapivinsky, G., Medina, I., Krapivinsky, L., Gapon, S. and Clapham, D. E. (2004). SynGAP-MUPP1-CaMKII synaptic complexes regulate p38 MAP kinase activity and NMDA receptor-dependent synaptic AMPA receptor potentiation. Neuron 43(4): 563-74. Medline abstract: 15312654
Kuner. T. and Schoepfer, R. (1996). Multiple structural elements determine subunit specificity of Mg2+ block in NMDA receptor channels, J. Neurosci. 16: 3549-3558. Medline abstract: 8642401
Kurusu, M., et al. (2002). Embryonic and larval development of the Drosophila mushroom bodies: concentric layer subdivisions and the role of fasciclin II. Development 129: 409-419. PubMed Citation: 11807033
Kwon, H. B. and Castillo, P. E. (2008). Long-term potentiation selectively expressed by NMDA receptors at hippocampal mossy fiber synapses. Neuron 57(1): 108-20. PubMed citation: 18184568
Larkin, A., et al. (2010). Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae. Learn Mem. 17(12): 645-53. PubMed Citation: 21106688
Lee, H. K., et al. (1998). NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus. Neuron 21(5): 1151-62. Medline abstract: 99072290
Lee, L. J., Iwasato, T., Itohara, S. and Erzurumlu, R. S. (2005). Exuberant thalamocortical axon arborization in cortex-specific NMDAR1 knockout mice. J. Comp. Neurol. 485 280-292. PubMed Citation: 15803506
Lieberman, D. N. and Mody, I. (1999). Casein kinase-II regulates NMDA channel function in hippocampal neurons. Nature Neurosci. 2(2): 125-132. Medline abstract: 10195195
Li, B., et al. (2007). The Neuregulin-1 receptor ErbB4 controls glutamatergic synapse maturation and plasticity. Neuron 54: 583-597. Medline abstract: 17521571
Liu, W., et al. (2006). Adrenergic modulation of NMDA receptors in prefrontal cortex is differentially regulated by RGS proteins and spinophilin. Proc. Natl. Acad. Sci. 103(48): 18338-43. Medline abstract: 17101972
Macdonald, D. S., et al. (2005). Modulation of NMDA receptors by pituitary adenylate cyclase activating peptide in CA1 neurons requires G alpha q, protein kinase C, and activation of Src. J. Neurosci. 25(49): 11374-84. Medline abstract: 16339032
Maletic-Savatic, Malinow, R. and Svoboda, K. (1999). Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283: 1923-1927. PubMed Citation: 10082466
McBride, S. M., et al. (2005). Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome. Neuron 45: 753-764. PubMed Citation: 15748850
McHugh, T. J., et al. (2007). Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network. Science 317(5834): 94-9. PubMed citation: 17556551
Mellem, J. E., Brockie, P. J., Zheng, Y., Madsen, D. M. and Maricq, A. V. (2002). Decoding of polymodal sensory stimuli by postsynaptic glutamate receptors in C. elegans. Neuron 36(5): 933-44. Medline abstract: 12467596
Mi, R., et al. (2004). AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons. Neuron 44(2): 335-49. Medline abstract: 15473971
Mori, H. and Mishina, M. (1995). Structure and function of the NMDA receptor channel, Neuropharmacology 34: 1219-1237. Medline abstract: 8570021
Nagele, P., Metz, L. B. and Crowder, C. M. (2004). Nitrous oxide (N2O) requires the N-methyl-D-aspartate receptor for its action in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 101(23): 8791-6. Medline abstract: 15159532
Naisbitt, S., et al. (1999). Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron 23: 569-582. Medline abstract: 99360650
Oestreich, J., Dembrow, N. C., George, A. A. and Zakon, H. H. (2006). A "sample-and-hold" pulse-counting integrator as a mechanism for graded memory underlying sensorimotor adaptation. Neuron 49(4): 577-88. Medline abstract: 16476666
Panatier, A., et al. (2006). Glia-derived D-serine controls NMDA receptor activity and synaptic memory. Cell 125(4): 775-84. Medline abstract: 16713567
Parmentier, M. L., Pin, J. P., Bockaert, J. and Grau, Y. (1996). Cloning and functional expression of a Drosophila metabotropic glutamate receptor expressed in the embryonic CNS. J. Neurosci. 16: 6687-6694. PubMed Citation: 8824309
Patneau. D. K. and Mayer, M.L. (1990). Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors. J. Neurosci. 10: 2385-2399. Medline abstract: 2165523
Peineau, S., et al. (2007). LTP inhibits LTD in the hippocampus via regulation of GSK3β. Neuron 53(5): 703-17. Medline abstract: 17329210
Petzold G. C., Hagiwara A. and Murthy V. N. (2009). Serotonergic modulation of odor input to the mammalian olfactory bulb. Nat. Neurosci. 12: 784-791. PubMed Citation: 19430472
Popescu, A. T., Saghyan, A. A. and Pare, D. (2007). NMDA-dependent facilitation of corticostriatal plasticity by the amygdala. Proc. Natl. Acad. Sci. 104(1): 341-6. Medline abstract: 17182737
Qiu, S., et al. (2005). Subunit assembly of N-methyl-D-aspartate receptors analyzed by fluorescence resonance energy transfer. J. Biol. Chem. 280(26): 24923-30. Medline abstract: 15888440
Rajan, I. and Cline, H. T. (1998). Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo. J. Neurosci. 18: 7836-7846. PubMed Citation: 9742152
Ramirez-Lugo, L., Zavala-Vega, S. and Bermudez-Rattoni, F. (2007). NMDA and muscarinic receptors of the nucleus accumbens have differential effects on taste memory formation. Learn Mem. 13(1): 45-51. Medline abstract: 16452653
Repicky, S. and Broadie, K. (2009). Metabotropic glutamate receptor-mediated use-dependent down-regulation of synaptic excitability involves the fragile X mental retardation protein. J. Neurophysiol. 101: 672-687. PubMed Citation: 19036865
Riedel, G., Platt B. and Micheau, J. Glutamate receptor function in learning and memory, Behav. Brain Res. 140: 1-47. Medline abstract: 12644276
Rivadulla, C., Sharma, J. and Sur, M. (2001). Specific roles of NMDA and AMPA receptors in direction-selective and spatial phase-selective responses in visual cortex. J. Neurosci. 21(5): 1710-1719. Medline abstract: 11222660
Roberts, A. C. and Glanzman, D. L. (2003). Learning in Aplysia: looking at synaptic plasticity from both sides. Trends Neurosci. 26: 662-670. Medline abstract: 14624850
Roberts, A. C., et al. (2009). Downregulation of NR3A-containing NMDARs is required for synapse maturation and memory consolidation. Neuron 63(3): 342-56. PubMed Citation: 19679074
Rondi-Reig, L., et al. (2006). Impaired sequential egocentric and allocentric memories in forebrain-specific-NMDA receptor knock-out mice during a new task dissociating strategies of navigation. J. Neurosci. 26(15): 4071-81. Medline abstract: 16611824
Ruthazer, E. S., Akerman, C. J. and Cline, H. T. (2003). Control of axon branch dynamics by correlated activity in vivo. Science 301: 66-70. PubMed Citation: 12843386
Sachse, S., Rueckert, E., Keller, A., Okada, R., Tanaka, N. K., Ito, K. and Vosshall, L. B. (2007). Activity-dependent plasticity in an olfactory circuit. Neuron 56: 838-850. PubMed Citation: 18054860
Salinas, P. C. and Zou Y. (2008). Wnt signaling in neural circuit assembly. Annu. Rev. Neurosci. 31: 339-358. PubMed Citation: 18558859
Sans, N., et al. (2005). mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression. Nat. Cell Biol. 7(12): 1079-90. Medline abstract: 16299499
Sattler, S., et al. (1999). Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science 284(5421): 1845-8. Medline abstract: 10364559
Schlessinger, K., Hall A. and Tolwinski, N. (2009). Wnt signaling pathways meet Rho GTPases. Genes Dev. 23: 265-277. PubMed Citation: 19204114
Schulz, S., Siemer, H., Krug, M. and Hollt, V. (1999). Direct evidence for biphasic cAMP responsive element-binding protein phosphorylation during long-term potentiation in the rat dentate gyrus in vivo, J. Neurosci. 19: 5683-5692. Medline abstract: 10377374
Sheng, M. and Sala, C. (2001). PDZ domains and the organization of supramolecular complexes. Annu. Rev. Neurosci. 24: 1-29. Medline abstract: 11283303
Shi, S.-H., et al. (1999). Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Medline abstract: 99294858
Shifman, J. M., et al. (2006). Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by calmodulin with two bound calciums. Proc. Natl. Acad. Sci. 103(38): 13968-73. Medline abstract: 16966599
Si, A., Helliwell, P. and Maleszka, R. (2004). Effects of NMDA receptor antagonists on olfactory learning and memory in the honeybee (Apis mellifera), Pharmacol. Biochem. Behav. 77: 191-197. Medline abstract: 14751445
Sin, W. C., et al. (2002). Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature 419: 475-480. PubMed Citation: 12368855
Sinakevitch, I., Grau, Y., Strausfeld, N. J. and Birman, S. (2010). Dynamics of glutamatergic signaling in the mushroom body of young adult Drosophila. Neural Dev. 5: 10. PubMed Citation: 20370889
Singh, A. P., VijayRaghavan, K. and Rodrigues, V. (2010). Dendritic refinement of an identified neuron in the Drosophila CNS is regulated by neuronal activity and Wnt signaling. Development 137(8): 1351-60. PubMed Citation: 20223760
Smith, K. E., Gibson, E. S. and Dell'Acqua, M. L. (2006). cAMP-dependent protein kinase postsynaptic localization regulated by NMDA receptor activation through translocation of an A-kinase anchoring protein scaffold protein. J. Neurosci. 26(9): 2391-402. Medline abstract: 16510716
Standley, S., et al. (2000). PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants. Neuron 28: 887-898. Medline abstract: 11163274
Stockinger, W., Brandes, C., Fasching, D., Hermann, M., Gotthardt, M., Herz, J., Schneider, W.J., and Nimpf, J. (2000). The reelin receptor ApoER2 recruits JNK-interacting proteins-1 and -2. J. Biol. Chem. 275: 25625-25632. PubMed citation: 10827199
Sytnyk, V., et al. (2007). NCAM promotes assembly and activity-dependent remodeling of the postsynaptic signaling complex. J. Cell Biol. 174: 1071-1085. Medline abstract: 17000882
Tai, C.-Y., et al. (2007). Activity-regulated N-cadherin endocytosis. Neuron 54: 771-785. Medline abstract: 17553425
Tashiro, A., Sandler, V. M., Toni, N., Zhao, C. and Gage, F. H. (2006). NMDA-receptor-mediated, cell-specific integration of new neurons in adult dentate gyrus. Nature 442(7105): 929-33. Medline abstract: 16906136
Tezuka, T., et al. (1999). PSD-95 promotes fyn-mediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A. Proc. Natl. Acad. Sci. 96(2): 435-40. Medline abstract: 99110908
Tian, X., et al. (2004). Developmentally regulated role for Ras-GRFs in coupling NMDA glutamate receptors to Ras, Erk and CREB. EMBO J. 23(7): 1567-75. Medline abstract: 15029245
Tolias, K. F., et al. (2005). The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines. Neuron 45(4): 525-38. Medline abstract: 15721239
Tripodi M., et al. (2008). Structural homeostasis: compensatory adjustments of dendritic arbor geometry in response to variations of synaptic input. PLoS Biol. 6: e260. PubMed Citation: 18959482
Ultanir, S. K., et al. (2007). Regulation of spine morphology and spine density by NMDA receptor signaling in vivo. Proc. Natl. Acad. Sci. 104: 19553-19558. PubMed Citation: 18048342
Ultsch, A., et al. (1993). Glutamate receptors of Drosophila melanogaster. Primary structure of a putative NMDA receptor protein expressed in the head of the adult fly. FEBS Lett. 324 (2): 171-177. Medline abstract: 93285330
Wang, H., et al. (2007). Genetic evidence for adenylyl cyclase 1 as a target for preventing neuronal excitotoxicity mediated by N-methyl-D-aspartate receptors. J. Biol. Chem. 282(2): 1507-17. Medline abstract: 17121841
Xia, S., Miyashita, T., Fu, T. F., Lin, W. Y., Wu, C. L., Pyzocha, L., Lin, I. R., Saitoe, M., Tully, T. and Chiang, A. S. (2005). NMDA receptors mediate olfactory learning and memory in Drosophila. Curr. Biol. 15(7): 603-15. Medline abstract: 15823532
Yaka, R., He, D. Y., Phamluong, K. and Ron, D. (2003). Pituitary adenylate cyclase-activating polypeptide (PACAP(1-38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1. J. Biol. Chem. 278(11): 9630-8. Medline abstract: 12524444
Yamakura, T. and Shimoji, K. (1999). Subunit- and site-specific pharmacology of the NMDA receptor channel. Prog. Neurobiol. 59: 279-298. Medline abstract: 10465381
Yang, W., et al. (2007). A three amino acid tail following the TM4 region of NR2 subunits is sufficient to overcome ER retention of NR1-1a subunit. J. Biol. Chem. 282(12): 9269-78. Medline abstract: 17255096
Ye, B., et al. (2000). GRASP-1: A neuronal RasGEF associated with the AMPA receptor/GRIP complex. Neuron 26: 603-617. Medline abstract: 20353053
Yoshii, A. and Constantine-Paton, M. (2007). BDNF induces transport of PSD-95 to dendrites through PI3K-AKT signaling after NMDA receptor activation. Nat. Neurosci. 10(6): 702-11. PubMed citation: 17515902
Zhang, S.-J., et al. (2007). Decoding NMDA receptor signaling: Identification of genomic programs specifying neuronal survival and death. Neuron 53: 549-562. Medline abstract: 17296556
Zhang, W., et al. (1999). Citron binds to PSD-95 at glutamatergic synapses on inhibitory neurons in the hippocampus. J. Neurosci. 19(1): 96-108. Medline abstract: 99088082
Zweifel, L. S., Argilli, E., Bonci, A. and Palmiter, R. D. (2008). Role of NMDA receptors in dopamine neurons for plasticity and addictive behaviors. Neuron 59(3): 486-96. PubMed Citation: 18701073
date revised: 1 November 2010
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.