The NMDA subtype of glutamate receptor is important for synaptic plasticity and nervous system development and function. Genetic and electrophysiological methods were used to demonstrate that NMR-1, a Caenorhabditis elegans NMDA-type ionotropic glutamate receptor subunit, plays a role in the control of movement and foraging behavior. nmr-1 mutants show a lower probability of switching from forward to backward movement and a reduced ability to navigate a complex environment. Electrical recordings from the interneuron AVA show that NMDA-dependent currents are selectively disrupted in nmr-1 mutants. A slowly desensitizing variant of a non-NMDA receptor can rescue the nmr-1 mutant phenotype. It is proposed that NMDA receptors in C. elegans provide long-lived currents that modulate the frequency of movement reversals during foraging behavior (Brockie, 2001).
The C. elegans polymodal ASH sensory neurons detect mechanical, osmotic, and chemical stimuli and release glutamate to signal avoidance responses. To investigate the mechanisms of this polymodal signaling, the role of postsynaptic glutamate receptors in mediating the response to these distinct stimuli was characterized. By studying the behavioral and electrophysiological properties of worms defective for non-NMDA (GLR-1 and GLR-2) and NMDA (NMR-1) receptor subunits, it has been shown that while the osmotic avoidance response requires both NMDA and non-NMDA receptors, the response to mechanical stimuli only requires non-NMDA receptors. Furthermore, analysis of the EGL-3 proprotein convertase provides additional evidence that polymodal signaling in C. elegans occurs via the differential activation of postsynaptic glutamate receptor subtypes (Mellem, 2002; full text of article).
Nitrous oxide (N2O, also known as laughing gas) and volatile anesthetics (VAs), the original and still most widely used general anesthetics, produce anesthesia by ill-defined mechanisms. Electrophysiological experiments in vertebrate neurons have suggested that N2O and VAs may act by distinct mechanisms; N2O antagonizes the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors, whereas VAs alter the function of a variety of other synaptic proteins. However, no genetic or pharmacological experiments have demonstrated that any of these in vitro actions are responsible for the behavioral effects of either class of anesthetics. By using genetic tools in C. elegans, whether the action of N2O requires the NMDA receptor in vivo and whether its mechanism is shared by VAs was tested. Distinct from the action of VAs, N2O produced behavioral defects highly specific and characteristic of that produced by loss-of-function mutations in both NMDA and non-NMDA glutamate receptors. A null mutant of nmr-1, which encodes a C. elegans NMDA receptor, was completely resistant to the behavioral effects of N2O, whereas a non-NMDA receptor-null mutant is normally sensitive. The N2O-resistant nmr-1(null) mutant is not resistant to VAs. Likewise, VA-resistant mutants have wild-type sensitivity to N2O. Thus, the behavioral effects of N2O require the NMDA receptor NMR-1, consistent with the hypothesis formed from vertebrate electrophysiological data that a major target of N2O is the NMDA receptor (Nagele, 2004; full text of article).
Fertilization in the female reproductive tract depends on intercellular signaling mechanisms that coordinate sperm presence with oocyte meiotic progression. To achieve this coordination in C. elegans, sperm release an extracellular signal, the major sperm protein (MSP), to induce oocyte meiotic maturation and ovulation. MSP binds to multiple receptors, including the VAB-1 Eph receptor protein-tyrosine kinase on oocyte and ovarian sheath cell surfaces. Canonical VAB-1 ligands called ephrins negatively regulate oocyte maturation and MPK-1 mitogen-activated protein kinase (MAPK) activation. MSP and VAB-1 regulate the signaling properties of two Ca2+ channels that are encoded by the NMR-1 N-methyl D-aspartate type glutamate receptor subunit and ITR-1 inositol 1,4,5-triphosphate receptor. Ephrin/VAB-1 signaling acts upstream of ITR-1 to inhibit meiotic resumption, while NMR-1 prevents signaling by the UNC-43 Ca2+/calmodulin-dependent protein kinase II (CaMKII). MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation, and UNC-43 acts redundantly in oocytes to promote oocyte maturation and MAPK activation. These results support a model in which VAB-1 switches from a negative regulator into a redundant positive regulator of oocyte maturation upon binding to MSP. NMR-1 mediates this switch by controlling UNC-43 CaMKII activation at the oocyte cortex (Corrigan, 2005).
Learning and memory are essential processes of both vertebrate and invertebrate nervous systems that allow animals to survive and reproduce. The neurotransmitter glutamate signals via ionotropic glutamate receptors (iGluRs) that have been linked to learning and memory formation; however, the signaling pathways that contribute to these behaviors are still not well understood. A genetic and electrophysiological analysis of learning and memory was undertaken in the nematode Caenorhabditis elegans. This study shows that two genes, nmr-1 and nmr-2, are predicted to encode the subunits of an NMDA-type (NMDAR) iGluR that is necessary for memory retention in C. elegans. nmr-2 was cloned, a deletion mutation was generated in the gene, and it was shown that like nmr-1, nmr-2 is required for in vivo NMDA-gated currents. Using an associative-learning paradigm that pairs starvation with the attractant NaCl, it was also shown that the memory of a learned avoidance response is dependent on NMR-1 and NMR-2 and that expression of NMDARs in a single pair of interneurons is sufficient for normal memory. These results provide new insights into the molecular and cellular mechanisms underlying the memory of a learned event (Kano, 2008).
In vertebrates, the NMDA receptors (NMDAR) appears to play a role in neuronal development, synaptic plasticity, memory formation, and pituitary activity. However, functional NMDAR have not yet been characterized in insects. Immunohistochemically glutamatergic nerve terminals have been demonstrated in the corpora allata of an adult female cockroach, Diploptera punctata. Cockroach corpus allatum (CA) cells, exposed to NMDA in vitro, exhibit elevated cytosolic (Ca2+), but not in culture medium nominally free of calcium or containing NMDAR-specific channel blockers: MK-801 and Mg2+. Sensitivity of cockroach corpora allata to NMDA changed cyclically during the ovarian cycle. Highly active glands of 4-day-old mated females, exposed to 3 microM NMDA, produced 70% more juvenile hormone (JH) in vitro, but the relatively inactive glands of 8-day-old mated females showed little response to the agonist. The stimulatory effect of NMDA was eliminated by augmenting the culture medium with MK-801, conantokin, or high Mg2+. Having obtained substantive evidence of functioning NMDAR in insect corpora allata, RTPCR was used to demonstrate two mRNA transcripts, DNMDAR1 and DNMDAR2, in the ring gland and brain of last-instar Drosophila melanogaster. Immunohistochemical labeling, using mouse monoclonal antibody against rat NMDAR1, showed that only one of the three types of endocrine cells in the ring gland, CA cells, expressed rat NMDAR1-like immunoreactive protein. This antibody also labeled two brain neurons in the lateral protocerebrum, one neuron per brain hemisphere. Finally, the same primers for DNMDAR1 were used to demonstrate a fragment of putative NMDA receptor in the corpora allata of Diploptera punctata. These results suggest that the NMDAR has a role in regulating JH synthesis and that ionotropic-subtype glutamate receptors became specialized early in animal evolution (Chiang, 2002).
In contrast to vertebrates the involvement of glutamate and N-methyl-D-aspartate (NMDA) receptors in brain functions in insects is both poorly understood and somewhat controversial. This study examined the behavioural effects of two noncompetitive NMDA receptor antagonists, memantine (low affinity) and MK-801 (high affinity), on learning and memory in honeybees (Apis mellifera) using the olfactory conditioning of the proboscis extension reflex (PER). Memory deficit was induced by injecting harnessed individuals with a glutamate transporter inhibitor, L-trans-2,4-pyrrolidine dicarboxylate, that impairs long-term (24 h), but not short-term (1 h), memory in honeybees. L-trans-2,4-PDC-induced amnesia is 'rescued' by memantine injected either before training, or before testing, suggesting that memantine restores memory recall rather than memory formation or storage. When injected alone memantine has a mild facilitating effect on memory. The effects of MK-801 are similar to those of L-trans-2,4-PDC. Both pretraining and pretesting injections lead to an impairment of long-term (24 h) memory, but have no effect on short-term (1 h) memory of an olfactory task (Si, 2004).
Ionotropic glutamate receptor (iGluR) subunits contain a large N-terminal domain (NTD) that precedes the agonist-binding domain (ABD) and participates in subunit oligomerization. In NMDA receptors (NMDARs), the NTDs of NR2A and NR2B subunits also form binding sites for the endogenous inhibitor Zn2+ ion. Although these allosteric sites have been characterized in detail, the molecular mechanisms by which the NTDs communicate with the rest of the receptor to promote its inhibition remain unknown. This study identified the ABD dimer interface as a major structural determinant that permits coupling between the NTDs and the channel gate. The strength of this interface also controls proton inhibition, another form of allosteric modulation of NMDARs. Conformational rearrangements at the ABD dimer interface thus appear to be a key mechanism conserved in all iGluR subfamilies, but have evolved to fulfill different functions: fast desensitization at AMPA and kainate receptors, allosteric inhibition at NMDARs (Gielen, 2008)
Chapsyn-110, a member of the membrane-associated putative guanylate kinase (MAGUK) family and related to Drosophila DLG, binds directly to the N-methyl-D-aspartate (NMDA) receptor and Shaker K+ channel subunits. In rat brain, chapsyn-100 protein shows a somatodendritic expression pattern that overlaps partly with PSD-95 but that contrasts with the axonal distribution of SAP97, other MAGUK proteins. Chapsyn-110 associates tightly with the postsynaptic density in brain, and mediates the clustering of both NMDA receptors and K+ channels in heterologous cells. Chapsyn-110 andd PSD-95 can heteromultimerize with each other and are recruited into the same NMDA receptor and K+ channel clusters. Thus, chapsyn-110 and PSD-95 may interact at postsynaptic sites to form a multimeric scaffold for the clustering of receptors, ion channels, and associated signalling proteins (Kim, 1996)
PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 binds to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins (Irie, 1997).
Fyn, a member of the Src-family protein-tyrosine kinase (PTK), is implicated in learning and memory that involves N-methyl-D-aspartate (NMDA) receptor function. Analysis of the physical and functional interaction between Fyn and NMDA receptors was carried out to see how Fyn participates in synaptic plasticity. Tyrosine phosphorylation of NR2A, one of the NMDA receptor subunits, is reduced in fyn-mutant mice. NR2A is tyrosine-phosphorylated in 293T cells when coexpressed with Fyn. Therefore, NR2A would be a substrate for Fyn in vivo. Results also show that PSD-95, which directly binds to and coclusters with NMDA receptors, promotes Fyn-mediated tyrosine phosphorylation of NR2A. Different regions of PSD-95 associate with NR2A and Fyn, respectively; therefore, PSD-95 could mediate complex formation of Fyn with NR2A. PSD-95 also associates with other Src-family PTKs: Src, Yes, and Lyn. These results suggest that PSD-95 is critical for regulation of NMDA receptor activity by Fyn and other Src-family PTKs, serving as a molecular scaffold for anchoring these PTKs to NR2A (Tezuka, 1999).
The postsynaptic density (PSD) can be visualized as an ultrastructural thickening of the postsynaptic membrane that is characteristic of excitatory synapses. Among the glutamate receptor complexes, the NMDA receptor/PSD-95 complex is the one most tightly associated with the PSD. In biochemical preparations of the PSD, NMDA receptors and PSD-95 are highly enriched and resistant to extraction by Triton X-100 and sarkosyl detergents, while AMPA receptors/GRIP and mGluRs/Homer (see Drosophila Homer) are relatively soluble. It is possible that the components of the NMDA receptor/PSD-95 complex comprise the major constituents of the core PSD, which remains after extraction with strong detergents. Because they are likely to play critical roles in the structural organization of the synapse and in the transduction of NMDA receptor signals, these core PSD proteins are important to define and study. A family of proteins (termed GKAP, SAPAP, or DAP) has been characterized that is highly concentrated in the PSD and that binds to the guanylate kinase (GK) domain of PSD-95. GKAP appears to be tightly associated with PSD-95; it can be immunoprecipitated from the brain in a complex with PSD-95 family proteins, and it is consistently colocalized with PSD-95 in neurons, even in the absence of associated NMDA receptors. The GKAP family of proteins contains at least four members and undergoes complex alternative splicing, but the physiological roles of these variants are unknown. To gain insight into GKAP function, a screen was carried out for binding partners of GKAP, hoping to extend the network of protein interactions emanating from NMDA receptors into the PSD (Naisbitt, 1999 and references).
A novel family of postsynaptic density (PSD) proteins, termed Shank, is described that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton (Naisbitt, 1999).
Originally identified as a substrate of Src tyrosine kinase, cortactin is an F-actin-binding protein enriched in cell-matrix contact sites, membrane ruffles and lammelipodia of cultured cells, and in growth cones of neurons. The translocation of cortactin to the cell periphery is stimulated by the small GTPase Rac1, and its F-actin cross-linking activity is inhibited by Src tyrosine phosphorylation. Thus, a large body of evidence implicates cortactin in regulation of the actin cytoskeleton in dynamic regions of the cell periphery. This study suggests that cortactin may also play a role in neuronal synapses, based on the following findings: biochemically, cortactin is loosely associated with the PSD, and immunocytochemically, it colocalizes with Shank in a subset of synapses. Most interestingly, a significant redistribution of cortactin to synaptic sites in response to glutamate stimulation has been demonstrated. The glutamate-induced synaptic localization of cortactin is reminiscent of cortactin recruitment to the cortical cytoskeleton by growth factor stimulation of nonneural cells. Their coexistence in growth cones supports the suggestion that Shank and cortactin may function at sites of active cytoskeletal remodeling in neurons. In mature synapses, it is speculated that a regulated Shank-cortactin interaction may be a mechanism for linking NMDA receptor activation to the control of the postsynaptic actin cytoskeleton. Shank is highly related to CortBP1, a protein isolated by yeast two-hybrid screening with the SH3 domain of cortactin. CortBP1 has been shown to colocalize with cortactin in membrane ruffles of cultured cells and in growth cones of cultured neurons, analogous to the colocalization of Shank and cortactin in growth cones and synapses. Based on their similarity in primary structure and cell biological properties, it seems reasonable to consider CortBP1 and Shank as members of the same family of proteins (Naisbitt, 1999 and references).
The efficiency with which N-methyl-D-aspartate receptors (NMDARs) trigger intracellular signaling pathways governs neuronal plasticity, development, senescence, and disease. Excessive Ca influx triggers excitotoxicity, damaging neurons in diverse neurological disorders. Rapid Ca2+-dependent neurotoxicity is triggered most efficiently when Ca2+ influx occurs through NMDARs, and cannot be reproduced by loading neurons with equivalent quantities of Ca2+ through non-NMDARs or voltage-sensitive Ca2+ channels (VSCCs). This suggests that Ca2+ influx through NMDAR channels is functionally coupled to neurotoxic signaling pathways. In cultured cortical neurons, suppressing the expression of the NMDAR scaffolding protein PSD-95 (postsynaptic density-95) selectively attenuates excitotoxicity triggered via NMDARs, but not by other glutamate or calcium ion (Ca2+) channels. NMDAR function is unaffected, because receptor expression, NMDA currents, and 45Ca2+ loading are unchanged. Suppressing PSD-95 blocks Ca2+-activated nitric oxide production by NMDARs selectively, without affecting neuronal nitric oxide synthase expression or function. Thus, PSD-95 is required for efficient coupling of NMDAR activity to nitric oxide toxicity, and imparts specificity to excitotoxic Ca2+ signaling. This raises the possibility that the preferential activation of neurotoxic Ca2+ signals by NMDARs is determined by the distinctiveness of NMDAR-bound MAGUKs, or of the intracellular proteins that they bind (Sattler, 1999).
Enduring forms of synaptic plasticity are thought to require ongoing regulation of adhesion molecules, such as N-cadherin, at synaptic junctions. Little is known about the activity-regulated trafficking of adhesion molecules. This study demonstrates that surface N-cadherin undergoes a surprisingly high basal rate of internalization. Upon activation of NMDA receptors (NMDAR), the rate of N-cadherin endocytosis is significantly reduced, resulting in an accumulation of N-cadherin in the plasma membrane. β-catenin, an N-cadherin binding partner, is a primary regulator of N-cadherin endocytosis. Following NMDAR stimulation, β-catenin accumulates in spines and exhibits increased binding to N-cadherin. Overexpression of a mutant form of ▀-catenin, Y654F, prevents the NMDAR-dependent regulation of N-cadherin internalization, resulting in stabilization of surface N-cadherin molecules. Furthermore, the stabilization of surface N-cadherin blocks NMDAR-dependent synaptic plasticity. These results indicate that NMDAR activity regulates N-cadherin endocytosis, providing a mechanistic link between structural plasticity and persistent changes in synaptic efficacy (Tai, 2007).
Appropriate trafficking and targeting of glutamate receptors (GluRs) to the postsynaptic density is crucial for synaptic function. mPins (mammalian homologue of Drosophila Partner of inscuteable) interacts with SAP102 and PSD-95 (two PDZ proteins present in neurons), and functions in the formation of the NMDAR - MAGUK (N-methyl-D-aspartate receptor - membrane-associated guanylate kinase) complex. mPins enhances trafficking of SAP102 and NMDARs to the plasma membrane in neurons. Expression of dominant-negative constructs and short-interfering RNA (siRNA)-mediated knockdown of mPins decreases SAP102 in dendrites and modifies surface expression of NMDARs. mPins changes the number and morphology of dendritic spines and these effects depend on its Galphai interaction domain, thus implicating G-protein signalling in the regulation of postsynaptic structure and trafficking of GluRs (Sans, 2005).
mPins is a ubiquitously expressed protein that is critical for the regulation of mitotic spindle organization in dividing cells. mPins interacts with several functionally distinct proteins, including NuMA, Ras, LKB1 and Galphai. The finding that mPins interacts with the PSD-95 family adds another group of important proteins to those whose trafficking depends on mPins. Drosophila Pins is required for asymmetric division of sensory organ precursor cells (pI) and dividing neuroblasts. Whereas the roles of Pins in cell division are relatively well-characterized, the function of mPins in the mature mammalian central nervous system remains enigmatic. The related protein, AGS3, may affect cocaine-induced plasticity by regulating G-protein signalling in the prefrontal cortex. The data show that mPins and AGS3 are both expressed in the developing hippocampus but have different subcellular localizations, perhaps because mPins, but not AGS3, interacts with SAP102. Moreover, AGS3 is down-regulated in adult hippocampus and seems to be absent from the PSD, whereas mPins is expressed throughout development and is enriched in synaptic membranes. mPins and AGS3 are found in different domains throughout the cell body and dendrites in primary cultures of hippocampal neurons. mPins, but not AGS3, redistributes into punctate structures after ionomycin or NMDA treatment, suggesting that calcium signalling functions in trafficking of mPins complexes. These findings strongly suggest that these two orthologues of Drosophila Pins have different functions in neurons (Sans, 2005).
The MAGUKs do not compete with the other known interacting proteins of mPins suggesting that the association of these other interacting proteins may indirectly influence the trafficking of the MAGUK and its associated proteins, such as NMDARs. Both Ras and Galphai are particularly interesting in this context. Ras has been implicated in the trafficking of GluRs. Characterized as molecular switches that alternate between GTP-bound ('on') and GDP-bound ('off') forms, these proteins are involved in the reorganization of synaptic structure. G-proteins, such as Galphai, influence NMDAR trafficking through metabotropic GluRs. In this study, it is shown that Galphai proteins function in NMDAR trafficking through a direct interaction with the mPins-SAP102 complex. mPins mediates G-protein signalling through binding to Galphai1-3GDP, thereby inhibiting binding of Galphai to Gßγ (and consequently enhancing Gßγ signalling in the absence of a G-protein-coupled receptor). mPins shifts between a closed state, when the N- and C-terminal halves of the protein bind to one another, and an open state when NuMA binds to mPins to switch it open, allowing the binding of Galphai. SAP102, similarly to SAP97, may exist in the cytoplasm as a folded molecule in which the GK domain is folded onto the SH3 domain. The data suggest that SAP102 binds to mPins in its closed state, as the two proteins localized in ring-like structures in COS cells. Therefore, mPins could be required upstream of, or in parallel to, the NR2B-SAP102 interaction. It is also shown that SAP102-mPins complexes have a different fate from that of NR2B-SAP102-mPins complexes, since the three proteins form clusters in COS cells and synaptic clusters in spines. These data suggest that NMDARs can open the SAP102-mPins complexes. Interestingly, cotransfection of the linker region of mPins with NR2B and SAP102 results in the formation of ternary complexes that are rapidly degraded, suggesting that interaction of Galphai with GoLoco domains (or an unidentified protein with TPR domains) is important for stabilization of the complex. mPins can bind four Galphai molecules, and it is unclear at present whether all of the sites need to be occupied for proper folding and targeting of mPins. As a modulator of G-protein signalling, the possibility cannot be excluded that Galphai binds to the NMDAR-MAGUK-mPins complex at synapses after activation of a G-protein-coupled receptor. Studies have suggested that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) can exhibit some of their effects through interactions with heterotrimeric G-proteins in addition to their ionic channel function. For instance, it has been shown that AMPA can induce dissociation of Galphai1 from the Galphai1/ß heterotrimeric complex and its association with GluR1 through an adaptor protein. In light of the current data, the possibility exists that Galphai signalling proteins may also be recruited to certain MAGUK-mPins complexes through simultaneous dissociation from AMPARs (Sans, 2005).
The results suggest that the NMDAR associates indirectly through SAP102 with two molecular complexes -- the exocyst and mPins-Galphai complexes -- and that these associations are necessary for proper trafficking of receptors in neurons. The results also suggest that this complex is formed in the ER in heterologous cells and early in the secretory pathway in neurons. Although this has not been demonstrated directly for native proteins, an association of MAGUK with AMPARs in the ER (or cis-Golgi) has been shown for native AMPARs in brain by using the endo-H sensitivity of immature AMPARs, so such an association is not unprecedented. These results suggest that NMDARs are trafficked as part of a large complex from their site of synthesis in the cell body to the postsynaptic membrane, presumably in a transport vesicle. The identification of other components of the SAP102 cargo complex (containing NMDARs, the exocyst and mPins-Galphai complexes) will undoubtedly help to clarify the steps involved in trafficking of NMDARs from assembly and ER exit to transport in dendrites and spines in normal and disease states (Sans, 2005).
Synaptic NMDA-type glutamate receptors are anchored to the second of three PDZ (PSD-95/Discs large/ZO-1) domains in the postsynaptic density (PSD) protein PSD-95. Citron, a protein target for the activated form of the small GTP-binding protein Rho, preferentially binds the third PDZ domain of PSD-95. In GABAergic neurons from the hippocampus, citron forms a complex with PSD-95 and is concentrated at the postsynaptic side of glutamatergic synapses. Citron is expressed only at low levels in glutamatergic neurons in the hippocampus and is not detectable at synapses onto these neurons. In contrast to citron, both p135 SynGAP (an abundant synaptic Ras GTPase-activating protein that can bind to all three PDZ domains of PSD-95) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) are concentrated postsynaptically at glutamatergic synapses on glutamatergic neurons. SynGAP, a Ras GTPase activating protein, is nearly as abundant in the PSD fraction as PSD-95 itself. SynGAP can be phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the PSD fraction and its GAP activity is reduced after phosphorylation. Thus, SynGAP and CaM kinase II constitute a signal transduction complex associated with the NMDA receptor. CaM kinase II is not expressed and p135 SynGAP is expressed in less than half of hippocampal GABAergic neurons. Segregation of citron into inhibitory neurons does not occur in other brain regions. For example, citron is expressed at high levels in most thalamic neurons, which are primarily glutamatergic and contain CaM kinase II. In several other brain regions, citron is present in a subset of neurons that can be either GABAergic or glutamatergic and can sometimes express CaM kinase II. Thus, in the hippocampus, signal transduction complexes associated with postsynaptic NMDA receptors are different in glutamatergic and GABAergic neurons and are specialized in a way that is specific to the hippocampus (Zhang, 1999).
The results presented here support the notion that differential expression of PSD-95-binding proteins in different neurons helps to determine the composition of signal transduction complexes formed by association with PSD-95 at glutamatergic PSDs. The resulting distinct compositions of these complexes will likely define the nature of local biochemical signaling associated with activation of NMDA receptors. The selective localization of citron suggests that, in hippocampus, PSDs of glutamatergic synapses made onto inhibitory interneurons contain cytoskeletal regulatory machinery that is not present at glutamatergic synapses made onto excitatory principal neurons. Furthermore, CaM kinase II is not detectable in these same PSDs but is present in the postsynaptic complex of excitatory synapses made onto glutamatergic neurons in the hippocampus. CaM kinase II can phosphorylate and regulate the GluRA/1 subunit of AMPA-type glutamate receptors and the synaptic Ras GTPase-activating protein SynGAP and can phosphorylate the NR2A and NR2B subunits of the NMDA receptor. This regulation by CaM kinase II is absent from the postsynaptic side of glutamatergic synapses on hippocampal inhibitory neurons. Thus, the modes of regulation of synaptic structure (by citron) and of synaptic strength (by CaM kinase II or citron) at glutamatergic synapses will differ dramatically between excitatory and inhibitory neurons. High citron expression found only in GABAergic neurons appears to be a unique feature of the hippocampus. In other brain regions, such as the thalamus and cerebral cortex, citron and CaM kinase II are often found together in excitatory neurons. Thus, the composition of signal transduction machinery at the postsynaptic membrane of glutamatergic synapses varies among neurons throughout the brain in ways that cannot be classified simply. Furthermore, findings regarding the mechanisms of signal transduction and plasticity at hippocampal synapses may not always generalize to synapses in other areas of the brain (Zhang, 1999).
NMDA-type glutamate receptors play a critical role in the activity-dependent development and structural remodeling of dendritic arbors and spines. However, the molecular mechanisms that link NMDA receptor activation to changes in dendritic morphology remain unclear. The Rac1-GEF Tiam1 is present in dendrites and spines and is required for their development. Tiam1 interacts with the NMDA receptor and is phosphorylated in a calcium-dependent manner in response to NMDA receptor stimulation. Blockade of Tiam1 function with either RNAi or dominant interfering mutants of Tiam1 suggests that Tiam1 mediates effects of the NMDA receptor on dendritic development by inducing Rac1-dependent actin remodeling and protein synthesis. Taken together, these findings define a molecular mechanism by which NMDA receptor signaling controls the growth and morphology of dendritic arbors and spines (Tolias, 2005).
Several second-messenger-regulated protein kinases have been implicated in the regulation of N-methyl-D-aspartate (NMDA) channel function. Yet the role of calcium and cyclic-nucleotide-independent kinases, such as casein kinase II (CKII), has remained unexplored. CKII is identified as an endogenous Ser/Thr protein kinase that potently regulates NMDA channel function and mediates intracellular actions of spermine on the channel. The activity of NMDA channels in cell-attached and inside-out recordings is enhanced by CKII or spermine and is decreased by selective inhibition of CKII. In hippocampal slices, inhibitors of CKII reduce synaptic transmission mediated by NMDA but not AMPA receptors. The dependence of NMDA receptor channel activity on tonically active CKII thus permits changes in intracellular spermine levels or phosphatase activities to effectively control channel function (Lieberman, 1999).
Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depends on the NMDA receptor. Complexes of NMDA receptors with the multivalent adaptor protein PSD-95 are found in the dorsal horn of spinal cord; PSD-95 plays a key role in neuropathic reflex sensitization. Mutant mice expressing a truncated form of the PSD-95 molecule fail to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain, but develop normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes; elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr286 CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice (Garry, 2003).
A novel mechanism has been identified for modulation of the phosphorylation state and function of the N-methyl-d-aspartate (NMDA) receptor via the scaffolding protein RACK1. RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein-tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices. This study identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. Activation of the cAMP/protein kinase A pathway in hippocampal slices induces the release of RACK1 from NR2B and Fyn. This results in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. The neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)) was identified as a ligand that induces phosphorylation of NR2B and enhances NMDA receptor potentials. Finally, it was found that activation of the cAMP/protein kinase A pathway induces the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn regulates the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together these results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1 (Yaka, 2003).
At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gαs-coupled receptors and protein kinase A. However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Gαq subunits and protein kinase C (PKC). In CA1 neurons, PACAP38 enhances synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Gαq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gαq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons (Macdonald, 2005).
Glycogen synthase kinase-3 (GSK3) has been implicated in major neurological disorders, but its role in normal neuronal function is largely unknown. GSK3β mediates an interaction between two major forms of synaptic plasticity in the brain, NMDA receptor-dependent long-term potentiation (LTP) and NMDA receptor-dependent long-term depression (LTD). In rat hippocampal slices, GSK3β inhibitors block the induction of LTD. Furthermore, the activity of GSK3β is enhanced during LTD via activation of PP1. Conversely, following the induction of LTP, there is inhibition of GSK3β activity. This regulation of GSK3β during LTP involves activation of NMDA receptors and the PI3K-Akt pathway and disrupts the ability of synapses to undergo LTD for up to 1 hr. It is concluded that the regulation of GSK3β activity provides a powerful mechanism to preserve information encoded during LTP from erasure by subsequent LTD, perhaps thereby permitting the initial consolidation of learnt information (Peineau, 2007).
NMDA receptor-dependent LTD is due to the internalization of AMPA receptors and involves protein interactions directly associated with the AMPA receptor subunits, particularly GluR2. It was reasoned that GSK3β might form a complex with AMPA receptors, and thus attempts were made to investigate this by probing for an association of native GSK3β with AMPA receptors in the CA1 area of hippocampal slices. A specific antibody against GSK3β was able to coimmunoprecipitate the GluR1 and GluR2 AMPA receptor subunits, and conversely, immunoprecipitation of AMPA receptors produced coimmunoprecipitation of GSK3β. To determine the functional status of AMPA receptor-associated GSK3β, AMPA receptors were immunoprecipitated, using antibodies against either GluR1 or GluR2, and then assayed for kinase activity. GSK3β activity was readily detected in both GluR1 and GluR2 immunoprecipitates relative to the background IgG control, demonstrating that endogenous GSK3β associates with native AMPA receptors in the brain, and that the bound GSK3β is functionally active. This association of GSK3β with AMPA receptors suggests a compartmentalization of this enzyme for the efficient regulation of AMPA receptors during LTD (Peineau, 2007).
It was asked whether the GSK3β activity that is associated with AMPA receptors could be regulated. Previous work has shown that transient exposure of cultured neurons to a solution containing sucrose plus glycine leads to an NMDA receptor-dependent insertion of AMPA receptors into the plasma membrane. Interestingly, this effect is associated with an increase in AMPA receptor-associated PI3K activity. Since PI3K is an upstream regulator of GSK3β, whether this treatment also affected the AMPA receptor-associated GSK3β enzyme activity was investigated. Neurons were treated with sucrose (200 mM) plus glycine (100 ÁM) for 2 min, and this led to the insertion of AMPA receptors into the plasma membrane as determined approximately 15 min later using surface biotinylation assays. This chemically induced AMPA receptor insertion was associated with a decrease in AMPA receptor-associated GSK3β activity (Peineau, 2007).
This study identified a form of regulation of synaptic plasticity in which the transient synaptic activation of NMDA receptors, as occurs during LTP, leads to inhibition of LTD. This regulation is very powerful since LTD is fully inhibited immediately following the conditioning stimulus and the effect lasts for approximately 1 hr. Also some of the signaling pathways responsible for this potent regulation of synaptic plasticity have been identified. GSK3β activity is an absolute requirement for the induction of LTD and the conditioning stimulus inhibits its activity via activation of the PI3K-Akt pathway. Finally, there is a correlation between the phosphorylation state of GSK3β ser9 and whether NMDA receptor activation leads to the induction or inhibition of LTD (Peineau, 2007).
GSK3β is an unusual kinase that has been implicated in many diseases. However, very little is known about its normal function in the nervous system. It is important during early development and it has been shown to play a key role in cell polarity and in the growth of neuromuscular junctions. Recently, it has been shown that GSK3β is important for determining neuronal polarity during the development of hippocampal neurons. However, though GSK3β is also highly expressed in the mature brain, its function in the nervous system has, hitherto, been largely unexplored. In the nucleus of hippocampal neurons, GSK3β is involved in the regulation of gene transcription by promoting the nuclear export of the transcription factor NF-ATc4. In addition, it has been shown that overexpression of GSK3β impairs spatial learning, though the mechanism underlying this effect is unknown. This study shows that in 2-week-old rats, an age at which the expression of GSK3β is near its peak, GSK3β activity is essential for NMDA receptor-dependent LTD in the hippocampus. This form of LTD is widespread throughout the brain and has been strongly implicated in development and learning and memory. Therefore, this novel GSK3β-dependent mechanism may be of general significance in regulating the interaction between LTP and LTD throughout the brain (Peineau, 2007).
GSK3β, unlike most enzymes, possesses high basal level constitutive activity and can be bidirectionally regulated to either further increase or decrease its activity. During LTD there is additional activation of GSK3β, probably via dephosphorylation of ser9. This effect is prevented by an inhibitor of PP1/PP2A. This suggests that the activation of PP1, which is known to occur during LTD, is responsible for the activation of GSK3β, via its dephosphorylation of ser9. LTD is associated with inhibition of Akt, probably also via the activation of PP1. These data suggest that GSK3β activity is increased during LTD because the phosphatase concomitantly inhibits Akt and directly dephosphorylates ser9 of GSK3β (Peineau, 2007).
Interestingly, the alteration in the phosphorylation status of GSK3β persists beyond the delivery of low-frequency stimulation (LFS), and lithium completely blocks LTD when applied after the delivery of LFS. These data suggest that GSK3β is required for the LTD process beyond the initial induction phase. Further studies are required to determine the full time course of the involvement of GSK3β in LTD (Peineau, 2007).
GSK3β has several upstream regulators and numerous downstream targets. In the present study, two of its upstream regulators have been identified. During LTD, GSK3β is activated via an okadaic acid-sensitive protein phosphatase, which is probably PP1. During LTP, GSK3β is inhibited via the PI3K-Akt pathway. Since GSK3β is such a ubiquitous kinase, it needs mechanisms to localize its access to its substrates. This is achieved in part via direct interactions with other proteins to form complexes. For example, in the canonical Wnt pathway, GSK3β binding proteins control access of β-catenin. It seems likely that the association between GSK3β and AMPA receptors serves to localize the kinase close to substrates that are involved in the trafficking of these receptors during synaptic plasticity. Further studies are required to establish the mechanism of this interaction as well as the downstream pathways mediated by GSK3β in the regulation of LTD (Peineau, 2007).
The finding that the synaptic activation of NMDA receptors during LTP inhibits NMDA receptor-dependent LTD raises an intriguing issue: what determines whether the synaptic activation of NMDA receptors leads to the induction or inhibition of LTD? Evidence is presented that the phosphorylation state of ser9 of GSK3β is a critical determinant. Thus, during LTP, activation of the PI3K-Akt pathway results in phosphorylation of GSK3β, and hence inhibition of its activity. In contrast, during LTD, activation of PP1 results in inhibition of Akt and the dephosphorylation of GSK3β at ser9, and this leads to an increase in the enzyme's activity. The activation of PI3K-Akt and inhibition of PP1 during LTP, but inhibition of Akt during LTD as well as the selective activation of PP1 during LTD, can be explained by the differences in the magnitude and spatiotemporal properties of the Ca2+ rise associated with the synaptic activation of NMDA receptors during these two forms of synaptic plasticity (Peineau, 2007).
Previous work has described other ways in which synaptic plasticity can be powerfully influenced by the prior history of synaptic activity. However, the mechanisms involved in these forms of metaplasticity are not known. Why synapses need such regulatory mechanisms is a matter of conjecture. One intriguing possible role for the regulation described in this study is to stabilize a synaptic modification over the short term by protecting synapses from the effects of additional NMDA receptor-dependent plasticity until the information can be either consolidated or erased by NMDA receptor-independent mechanisms (Peineau, 2007).
The regulation of synaptic plasticity is further complicated by the involvement of mGluRs, which are involved in depotentiation, LTD of baseline transmission, heterosynaptic LTD, and metaplasticity. So that focus could be placed on interactions between the NMDA receptor-dependent forms of synaptic plasticity, the additional complication of mGluR-dependent synaptic plasticity were eliminated by using the broad spectrum mGluR antagonist LY341495 and by employing stimulus protocols optimized for NMDA receptor-dependent synaptic plasticity. However, given that PI3K has been implicated in a chemically induced form of mGluR-dependent LTD and heterosynaptic LTD, it will be interesting to determine whether GSK3β is also involved in these forms of synaptic plasticity. One possibility is that the PI3K-Akt-GSK3β pathway serves to inhibit NMDA receptor-dependent LTD both homosynaptically following the induction of LTP and heterosynaptically following the induction of LTD (Peineau, 2007).
The finding that in the normal brain activation of GSK3β is essential for NMDA receptor-dependent LTD, and that its activity can be regulated by LTP, may offer clues to the pathological role of this enzyme in neurological disorders. For example, the primary therapeutic action of lithium in bipolar disorders may be via inhibition of GSK3β. Indeed, specific inhibition of GSK3β has recently been shown to produce antidepressive-like activity in vivo. Overactivity of GSK3β may, therefore, lead to this mood disorder by affecting the balance and interplay between NMDA receptor-dependent LTP and LTD (Peineau, 2007).
The mossy fiber to CA3 pyramidal cell synapse (mf-CA3) provides a major source of excitation to the hippocampus. Thus far, these glutamatergic synapses are well recognized for showing a presynaptic, NMDA receptor-independent form of LTP that is expressed as a long-lasting increase of transmitter release. This study shows that in addition to this 'classical' LTP, mf-CA3 synapses can undergo a form of LTP characterized by a selective enhancement of NMDA receptor-mediated transmission. This potentiation requires coactivation of NMDA and mGlu5 receptors and a postsynaptic calcium rise. Unlike classical LTP, expression of this mossy fiber LTP is due to a PKC-dependent recruitment of NMDA receptors specifically to the mf-CA3 synapse via a SNARE-dependent process. Having two mechanistically different forms of LTP may allow mf-CA3 synapses to respond with more flexibility to the changing demands of the hippocampal network (Kwon, 2008).
The noradrenergic system in the prefrontal cortex (PFC) is involved in many physiological and psychological processes, including working memory and mood control. To understand the functions of the noradrenergic system, regulation of NMDA receptors , key players in cognition and emotion, by alpha1- and alpha2-adrenergic receptors (alpha1-ARs, alpha2-ARs) was studied in PFC pyramidal neurons. Applying norepinephrine or a norepinephrine transporter inhibitor reduced the amplitude but not paired-pulse ratio of NMDAR-mediated excitatory postsynaptic currents (EPSC) in PFC slices. Specific alpha1-AR or alpha2-AR agonists also decreased NMDAR-EPSC amplitude and whole-cell NMDAR current amplitude in dissociated PFC neurons. The alpha1-AR effect depended on the phospholipase C-inositol 1,4,5-trisphosphate-Ca(2+) pathway, whereas the alpha2-AR effect depended on protein kinase A and the microtubule-based transport of NMDARs that is regulated by ERK signaling. Furthermore, two members of the RGS family, RGS2 and RGS4, were found to down-regulate the effect of alpha1-AR on NMDAR currents, whereas only RGS4 was involved in inhibiting alpha2-AR regulation of NMDAR currents. The regulating effects of RGS2/4 on alpha1-AR signaling were lost in mutant mice lacking spinophilin, which binds several RGS members and G protein-coupled receptors, whereas the effect of RGS4 on alpha2-AR signaling was not altered in spinophilin-knockout mice. This work suggests that activation of alpha1-ARs or alpha2-ARs suppresses NMDAR currents in PFC neurons by distinct mechanisms. The effect of alpha1-ARs is modified by RGS2/4 that are recruited to the receptor complex by spinophilin, whereas the effect of alpha2-ARs is modified by RGS4 independent of spinophilin (Liu, 2006).
Neuregulin-1 (NRG1) signaling participates in numerous neurodevelopmental processes. Through linkage analysis, nrg1 has been associated with schizophrenia, although its pathophysiological role is not understood. The prevailing models of schizophrenia invoke hypofunction of the glutamatergic synapse and defects in early development of hippocampal-cortical circuitry. This study shows that the erbB4 receptor, as a postsynaptic target of NRG1, plays a key role in activity-dependent maturation and plasticity of excitatory synaptic structure and function. Synaptic activity leads to the activation and recruitment of erbB4 into the synapse. Overexpressed erbB4 selectively enhances AMPA synaptic currents and increases dendritic spine size. Preventing NRG1/erbB4 signaling destabilizes synaptic AMPA receptors and leads to loss of synaptic NMDA currents and spines. These results indicate that normal activity-driven glutamatergic synapse development is impaired by genetic deficits in NRG1/erbB4 signaling leading to glutamatergic hypofunction. These findings link proposed effectors in schizophrenia: NRG1/erbB4 signaling perturbation, neurodevelopmental deficit, and glutamatergic hypofunction (Li, 2007).
The NMDA receptor NR1 subunit has four splice variants that differ in their C-terminal, cytoplasmic domain. The contribution of the C-terminal cassettes, C0, C1, C2, and C2', to trafficking of NR1 in heterologous cells and neurons was investigated. An ER retention signal (RRR) was identified in the C1 cassette of NR1, which is similar to the RXR motif in ATP-sensitive K(+) channels. Surface expression of NR1-3, which contains C1, is due to a site on the C2' cassette, which includes the terminal 4 amino acid PDZ-interacting domain. This site suppresses ER retention of the C1 cassette and leads to surface expression. These findings suggest a role for PDZ proteins in facilitating the transition of receptors from an intracellular pool to the surface of the neuron (Standley, 2000).
NMDA receptors play major roles in synaptic transmission and plasticity, as well as excitotoxicity. NMDA receptors are thought to be tetrameric complexes mainly composed of NMDA receptor (NR)1 and NR2 subunits. The NR1 subunits are required for the formation of functional NMDA receptor channels, whereas the NR2 subunits modify channel properties. Biochemical and functional studies indicate that subunits making up NMDA receptors are organized into a dimer of dimers, and the N termini of the subunits are major determinants for receptor assembling. This study used a biophysical approach, fluorescence resonance energy transfer, to analyze the assembly of intact, functional NMDA receptors in living cells. The results showed that NR1, NR2A, and NR2B subunits could form homodimers when they were expressed alone in HEK293 cells. Subunit homodimers were also found existing in heteromeric NMDA receptors formed between NR1 and NR2 subunits. These findings are consistent with functional NMDA receptors being arranged as a dimer of dimers. In addition, the data indicated that the conformation of NR1 subunit homodimers is affected by the partner NR2 subunits during the formation of heteromeric receptor complexes, which might underlie the mechanism by which NR2 subunits modify NMDA receptor function (Qiu, 2005).
Excitatory neurotransmission mediated by NMDA receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. This study reports crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation (Furukawa, 2005).
Subunits of the NMDA receptor (NMDAR) associate with many postsynaptic proteins that substantially broaden its signaling capacity. Although much work has been focused on the signaling of NR2 subunits, little is known about the role of the NR1 subunit. This study set out to elucidate the role of the C terminus of the NR1 subunit in NMDAR signaling. By introducing a C-terminal deletion mutant of the NR1 subunit into cultured neurons from NR1(-/-) mice, it was found that the C terminus was essential for NMDAR inactivation, downstream signaling, and gene expression, but not for global increases in intracellular Ca2+. Therefore, whereas NMDARs can increase Ca2+ throughout the neuron, NMDAR-dependent signaling, both local and long range, requires coupling through the NR1 C terminus. Two major NR1 splice variants differ by the presence or absence of a C-terminal domain, C1, which is determined by alternative splicing of exon 21. Analysis of these two variants showed that removal of this domain significantly reduced the efficacy of NMDAR-induced gene expression without affecting receptor inactivation. Thus, the NR1 C terminus couples to multiple downstream signaling pathways that can be modulated selectively by RNA splicing (Bradley, 2006).
NMDA receptor activity is important for many physiological functions, including synapse formation and alterations in synaptic strength. NMDA receptors are composed most commonly of NR1 and NR2 subunits. There are four NR2 subunits (NR2A-NR2D). NR2 subunit expression varies across both brain regions and developmental stages. The identity of the NR2 subunit within a functional NMDA receptor helps to determine many pharmacological and biophysical receptor properties, including strength of block by external Mg2+ (Mg(o)2+). Mg(o)2+ block confers strong voltage dependence to NMDA receptor-mediated responses and is critically important for many of the functions that the NMDA receptor plays within the CNS. This study describes the NR2 subunit dependence of the kinetics of Mg(o)2+ unblock after rapid depolarizations. Mg(o)2+ unblocks from NR1/2A and NR1/2B receptors with a prominent slow component similar to that previously described in native hippocampal and cortical NMDA receptors. Strikingly, this slow component of Mg(o)2+ unblock is completely absent from NR1/2C and NR1/2D receptors. Thus currents from NR1/2C and NR1/2D receptors respond more rapidly to fast depolarizations than currents from NR1/2A and NR1/2B receptors. In addition, the slow component of Mg(o)2+ unblock from NR1/2B receptors is consistently slower than from NR1/2A receptors. This makes rapid depolarizations, such as action potential waveforms, more efficacious at stimulating Mg(o)2+ unblock from NR1/2A than from NR1/2B receptors. These NR2 subunit differences in the kinetics of Mg(o)2+ unblock are likely to help determine the contribution of each NMDA receptor subtype to current flow during synaptic activity (Clarke, 2006).
The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C-terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, it was found that the three amino-acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino-acids following the TM4 region (NR22) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR22 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR22 subunits were cotransfected with either the NR1-4a or NR1-1aAAA mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, it was found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2B5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence specificity-dependent. Taken together, these results show that the C-terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C-terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface (Yang, 2007).
Learning is accompanied by modulation of postsynaptic signal transduction pathways in neurons. Although the neuronal protein kinase cyclin-dependent kinase 5 (Cdk5) has been implicated in cognitive disorders, its role in learning has been obscured by the perinatal lethality of constitutive knockout mice. Conditional knockout of Cdk5 in the adult mouse brain improved performance in spatial learning tasks and enhanced hippocampal long-term potentiation and NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents. Enhanced synaptic plasticity in Cdk5 knockout mice is attributed to reduced NR2B degradation, which causes elevations in total, surface and synaptic NR2B subunit levels and current through NR2B-containing NMDARs. Cdk5 facilitates the degradation of NR2B by directly interacting with both it and its protease, calpain. These findings reveal a previously unknown mechanism by which Cdk5 facilitates calpain-mediated proteolysis of NR2B and may control synaptic plasticity and learning (Hawasli, 2007).
To monitor changes in AMPA receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) is functional and is transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP is intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induces a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events require synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediated transmission observed during long-term potentiation and activity-dependent synaptic maturation (Shi, 1999).
These results build on a number of studies suggesting that the delivery of AMPA receptors to synapses contributes to activity-dependent plasticity. Inhibition of membrane fusion processes in the postsynaptic cell blocks the action of LTP. Furthermore, the COOH-termini of AMPA receptor subunits GluR2 and GluR4c bind N-ethylmaleimide-sensitive fusion protein, a protein involved in membrane fusion processes. Vesicular organelles, possibly undergoing exocytosis and endocytosis, have been detected with electron microcopy in spines. And last, dendrites can display a calcium-evoked exocytosis of trans-Golgi-derived organelles that is mediated by the calcium/calmodulin-dependent protein kinase II, an enzyme thought to mediate LTP. Other postsynaptic mechanisms, such as an increase in conductance of AMPA receptors, may also occur in parallel. These results also do not rule out a contribution by presynaptic modifications. In addition to the spine delivery of GluR1-GFP, tetanic stimulation induces the formation of clusters of the tagged receptor within dendrites. These structures may be related to the spine apparatus, membranous structures at the base of spines that appear to contain AMPA receptors. The entry of calcium through synaptic NMDA receptors may cause nucleation of AMPA receptor-containing membranes close to active synapses. Once formed, such sites may serve several functions. These sites may replenish those receptors delivered to spines during plasticity. Additionally, they may serve as a 'synaptic tag', providing a docking site for AMPA receptors synthesized at distant sites. Last, they could provide a site for local AMPA receptor synthesis. In these capacities, such clusters could represent a structural modification serving as a long-lasting memory mechanism (Shi, 1999 and references).
The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, several GRIP-associated proteins (GRASPs) have been identified that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduces the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 is rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity (Ye, 2000).
The synapse contains densely localized and interacting proteins that enable it to adapt to changing inputs. A Ca2+-sensitive protein complex is described involved in the regulation of AMPA receptor synaptic plasticity. The complex is comprised of (1) MUPPI, a multi-PDZ domain-containing protein, (2) SynGAP, a synaptic GTPase-activating protein, and (3) the Ca2+/calmodulin-dependent kinase CaMKII. In synapses of hippocampal neurons, SynGAP and CaMKII are brought together by direct physical interaction with the PDZ domains of MUPP1, and in this complex, SynGAP is phosphorylated. Ca2+CaM binding to CaMKII dissociates it from the MUPP1 complex, and Ca2+, entering the cell via the NMDAR, drives the dephosphorylation of SynGAP. Specific peptide-induced SynGAP dissociation from the MUPP1-CaMKII complex results in SynGAP dephosphorylation accompanied by P38 MAPK inactivation, potentiation of synaptic AMPA responses, and an increase in the number of AMPAR-containing clusters in hippocampal neuron synapses. siRNA-mediated SynGAP knockdown confirms these results. These data implicate SynGAP in NMDAR- and CaMKII-dependent regulation of AMPAR trafficking (Krapivinsky, 2004).
NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission (Kim, 2005).
The ERK1/2 signaling pathway is activated by calcium influx through NMDARs and plays an important role in synaptic plasticity and cell survival. NMDAR-dependent ERK activation involves the small GTPase Ras, which is stimulated by specific guanine nucleotide exchange factors (GEFs) and inhibited by GTPase activating proteins (GAPs). The RasGEF RasGRF1 is reported to bind directly to the NR2B subunit of NMDARs. SynGAP, a RasGAP highly enriched in the postsynaptic density (PSD), can associate with NMDARs through binding to PSD-95 family proteins. The exact function of these Ras regulatory proteins in synaptic plasticity has not been established, and how they are functionally coupled to NMDARs remains unclear (Kim, 2005).
Altered AMPAR trafficking has emerged as a major postsynaptic mechanism for the expression of synaptic plasticity. A prevailing model is that NMDAR-dependent LTP is mediated by the surface insertion and synaptic delivery of GluR1, that is driven by CaM kinase II and the Ras-ERK pathway. In contrast, LTD is supposed to result, at least in part, from the removal of synaptic AMPARs by the increased endocytosis and/or reduced recycling of GluR2/3 subunits (Kim, 2005).
This study investigates the links between NMDAR subtypes, Ras-ERK signaling, and AMPAR trafficking. NR2A and NR2B are found to have antagonistic actions on Ras-ERK activation and AMPAR distribution in mature neurons. NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1 -- primarily by regulating GluR1 surface insertion. Potentially accounting for this difference is that NR2B is coupled to the inhibition rather than the activation of the Ras-ERK pathway. This functional coupling is correlated with the specific biochemical association of SynGAP with NR2B-NMDARs (Kim, 2005).
The scaffold protein PSD-95 promotes the maturation and strengthening of excitatory synapses, functions that require proper localization of PSD-95 in the postsynaptic density (PSD). Phosphorylation of ser-295 enhances the synaptic accumulation of PSD-95 and the ability of PSD-95 to recruit surface AMPA receptors and potentiate excitatory postsynaptic currents. Evidence is presented that a Rac1-JNK1 signaling pathway mediates ser-295 phosphorylation and regulates synaptic content of PSD-95. Ser-295 phosphorylation is suppressed by chronic elevation, and increased by chronic silencing, of synaptic activity. Rapid dephosphorylation of ser-295 occurs in response to NMDA treatment that causes chemical long-term depression (LTD). Overexpression of a phosphomimicking mutant (S295D) of PSD-95 inhibits NMDA-induced AMPA receptor internalization and blocks the induction of LTD. The data suggest that synaptic strength can be regulated by phosphorylation-dephosphorylation of ser-295 of PSD-95 and that synaptic depression requires the dephosphorylation of ser-295 (Kim, 2007).
Under standard conditions, cultured ventral spinal neurons cluster AMPA-type (but not NMDA-type) glutamate receptors at excitatory synapses on their dendritic shafts, in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. The NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further shows that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization is preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons (Mi, 2004).
p140 Ras-GRF1 and p130 Ras-GRF2 constitute a family of calcium/calmodulin-regulated guanine-nucleotide exchange factors that activate the Ras GTPases. Studies on mice lacking these exchange factors revealed that both p140 Ras-GRF1 and p130 Ras-GRF2 couple NMDA glutamate receptors (NMDARs) to the activation of the Ras/Erk signaling cascade and to the maintenance of CREB transcription factor activity in cortical neurons of adult mice. Consistent with this function for Ras-GRFs and the known neuroprotective effect of CREB activity, ischemia-induced CREB activation is reduced in the brains of adult Ras-GRF knockout mice and neuronal damage is enhanced. Interestingly, in cortical neurons of neonatal animals NMDARs signal through Sos rather than Ras-GRF exchange factors, implying that Ras-GRFs endow NMDARs with functions unique to mature neurons (Tian, 2004).
NMDA receptor-dependent long-term potentiation and long-term depression (LTD) involve changes in AMPA receptor activity and postsynaptic localization that are in part controlled by glutamate receptor 1 (GluR1) subunit phosphorylation. The scaffolding molecule A-kinase anchoring protein (AKAP)79/150 targets both the cAMP-dependent protein kinase (PKA) and protein phosphatase 2B/calcineurin (PP2B/CaN) to AMPA receptors to regulate GluR1 phosphorylation. Brief NMDA receptor activation leads to persistent redistribution of AKAP79/150 and PKA-RII, but not PP2B/CaN, from postsynaptic membranes to the cytoplasm in hippocampal slices. Similar to LTD, AKAP79/150 redistribution requires PP2B/CaN activation and is accompanied by GluR1 dephosphorylation and internalization. Using fluorescence resonance energy transfer microscopy in hippocampal neurons, it has been demonstrated that PKA anchoring to AKAP79/150 is required for NMDA receptor regulation of PKA-RII localization and that movement of AKAP-PKA complexes underlies PKA redistribution. These findings suggest that LTD involves removal of AKAP79/150 and PKA from synapses in addition to activation of PP2B/CaN. Movement of AKAP79/150-PKA complexes from the synapse could further favor the actions of phosphatases in maintaining dephosphorylation of postsynaptic substrates, such as GluR1, that are important for LTD induction and expression. In addition, these observations demonstrate that AKAPs serve not solely as stationary anchors in cells but also as dynamic signaling components (Smith, 2006).
The neural cell adhesion molecule (NCAM) regulates synapse formation and synaptic strength via mechanisms that have remained unknown. This study shows that NCAM associates with the postsynaptic spectrin-based scaffold, cross-linking NCAM with the NMDA receptor and Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) in a manner not firmly or directly linked to PSD95 and α-actinin. Clustering of NCAM promotes formation of detergent-insoluble complexes enriched in postsynaptic proteins and resembling postsynaptic densities. Disruption of the NCAM-spectrin complex decreases the size of postsynaptic densities and reduces synaptic targeting of NCAM-spectrin-associated postsynaptic proteins, including spectrin, NMDA receptors, and CaMKIIα. Degeneration of the spectrin scaffold in NCAM-deficient neurons results in an inability to recruit CaMKIIα to synapses after NMDA receptor activation, which is a critical process in NMDA receptor-dependent long-term potentiation. The combined observations indicate that NCAM promotes assembly of the spectrin-based postsynaptic signaling complex, which is required for activity-associated, long-lasting changes in synaptic strength. Its abnormal function may contribute to the etiology of neuropsychiatric disorders associated with mutations in or abnormal expression of NCAM (Sytayk, 2007).
Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. In cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits (Chubykin, 2007).
Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca2+ flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca2+ effector protein that binds up to four Ca2+ ions. CaM with bound Ca2+ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca2+, a large fraction of free CaM is bound to fewer than four Ca2+ ions. Binding to targets increases the affinity of CaM's remaining Ca2+-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca2+ ions. To study CaM-dependent signaling in the spine, mutant CaMs were designed that bind Ca2+ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca2+-binding domains in the 'closed' Ca2+-free conformation. Their interactions with CaMKII, a major Ca2+/CaM target that mediates initiation of long-term potentiation, were measured. CaM with two Ca2+ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. These results support the idea that competition for binding of CaM with two bound Ca2+ ions may influence significantly the outcome of local Ca2+ signaling in spines and, perhaps, in other signaling pathways (Shifman, 2006).
The NMDA receptor, brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95) and phosphatidylinositol 3-kinase (PI3K) have all been implicated in long-term potentiation. This study shows that these molecules are involved in a single pathway for synaptic potentiation. In visual cortical neurons in young rodents, the neurotrophin receptor TrkB is associated with PSD-95. When BDNF is applied to cultured visual cortical neurons, PSD-95-labeled synaptic puncta enlarge, and fluorescent recovery after photobleaching (FRAP) reveals increased delivery of green fluorescent protein-tagged PSD-95 to the dendrites. The recovery of fluorescence requires TrkB, signaling through PI3K and the serine-threonine kinase Akt, and an intact Golgi apparatus. Stimulation of NMDARs mimics the PSD-95 trafficking that is induced by BDNF but requires active BDNF and PI3K. Furthermore, local dendritic contact with a BDNF-coated microsphere induces PSD-95 FRAP throughout the dendrites of the stimulated neuron, suggesting that this mechanism induces rapid neuron-wide synaptic increases in PSD-95 and refinement whenever a few robust inputs activate the NMDAR-BDNF-PI3K pathway (Yoshii, 2007).
The canonical Wnt-β-catenin signaling pathway is important for a variety of developmental phenomena as well as for carcinogenesis. In hippocampal neurons, NMDA-receptor-dependent activation of calpain induces the cleavage of β-catenin at the N terminus, generating stable, truncated forms. These β-catenin fragments accumulate in the nucleus and induced Tcf/Lef-dependent gene transcription. Fosl1, one of the immediate-early genes, was identified as a target of this signaling pathway. In addition, exploratory behavior by mice resulted in a similar cleavage of β-catenin, as well as activation of the Tcf signaling pathway, in hippocampal neurons. Both β-catenin cleavage and Tcf-dependent gene transcription are suppressed by calpain inhibitors. These findings reveal another pathway for β-catenin-dependent signaling, in addition to the canonical Wnt-β-catenin pathway, and suggest that this other pathway could play an important role in activity-dependent gene expression (Abe, 2007).
JIP scaffold proteins are implicated in the regulation of protein kinase signal transduction pathways. To test the physiological role of these scaffold proteins, the phenotype was examined of compound mutant mice that lack expression of JIP proteins. These mice were found to exhibit severe defects in NMDA receptor function, including decreased NMDA-evoked current amplitude, cytoplasmic Ca++, and gene expression. The decreased NMDA receptor activity in JIP-deficient neurons is associated with reduced tyrosine phosphorylation of NR2 subunits of the NMDA receptor. JIP complexes interact with the SH2 domain of cFyn and may therefore promote tyrosine phosphorylation and activity of the NMDA receptor. It is concluded that JIP scaffold proteins are critically required for normal NMDA receptor function (Kennedy, 2007).
JIP scaffold proteins are important for the normal activity of NMDA receptors. It is established that JIP proteins are localized at post-synaptic densities in neurons, but the mechanism that accounts for the functional interaction of JIP proteins with NMDA receptors is unclear. One possibility is that the PTB domain of JIP1 and JIP2 may contribute to this regulatory process. Indeed, several ligands for this PTB domain have been described, including the low-density lipoprotein receptor-related protein LRP8, the Rac exchange factor Tiam1, and the Ras exchange factor Ras-Grf. Interestingly, all three of these proteins (LRP8, Tiam1, and Ras-Grf) are known to bind NMDA receptors. These proteins may therefore recruit JIP/cFyn complexes to the NMDA receptor to regulate NR2 subunit tyrosine phosphorylation (Kennedy, 2007).
The interaction of LRP8 with JIP1/2 is particularly intriguing because it is established that the site of interaction of the JIP1/2 PTB domain with the cytoplasmic domain of LRP8 is encoded by exon 19 (Stockinger, 2000). This is an alternative spliced exon that is selectively included in Lrp8 mRNA in response to neuronal activity. Thus, only exon 19-positive LRP8 can bind to the JIP1/2 PTB domain. Gene targeting studies have demonstrated that ablation of Lrp8 exon 19 results in mice that exhibit defects in NMDA receptor signaling associated with markedly decreased NR2 subunit tyrosine phosphorylation (Beffert, 2005). Exon 19-positive LRP8 may therefore regulate NMDA receptor activity. Interestingly, the defect in NMDA receptor signaling and NR2 subunit phosphorylation caused by loss of Lrp8 exon 19 (Beffert, 2005) is similar to that caused by compound mutation of JIP1/2. Together, these data suggest that the physical interaction between the JIP1/2 PTB domain and the segment of the LRP8 cytoplasmic tail encoded by Lrp8 exon 19 is functionally significant. Indeed, it is possible that JIP/cFyn complexes may mediate the effects of LRP8 on NMDA receptors. Engagement of cell surface LRP8 by its ligand Reelin may trigger this regulatory pathway to control NMDA receptor function (Kennedy, 2007).
The postnatal role of LRP8 to regulate NMDA receptor activity requires Lrp8 exon 19. However, LRP8 also plays an important developmental role in determining the positioning of neurons in the brain. Mice lacking the ligand Reelin (reeler mice) or mice with targeted ablation of the Lrp8 gene exhibit marked defects in neuronal positioning. However, neither targeted ablation of the alternatively spliced Lrp8 exon 19 nor compound deficiency of JIP1/2 causes a reeler-like neuronal positioning defect during development. Thus, the JIP1/2 PTB domain interaction with the cytoplasmic tail of LRP8 does not play a role in the early developmental function of LRP8 to regulate neuronal positioning (Kennedy, 2007).
A critical role for cFyn in NMDA receptor regulation is established by the finding that cFyn-/- mice exhibit severely reduced tyrosine phosphorylation of NR2A and NR2B and display major defects in long-term potentiation and spatial learning. Nevertheless, other members of the SRC family of tyrosine kinases (including Lck, Lyn, Src, and Yes) have also been implicated in the regulation of NMDA receptor function. cFyn may therefore represent only one member of a larger group of SRC family kinases that are recruited by JIP scaffold proteins. This function of JIP proteins may be coordinated with the actions of other scaffold proteins that can bind SRC family kinases and are present within post-synaptic densities (e.g., PSD-95 or RACK1). Since Jip1-/- and Jip2-/- neurons exhibit different defects in NMDA receptor function, it is possible that these JIP scaffold proteins may cooperate by influencing different steps in the regulatory process. It is also possible that the structurally unrelated scaffold proteins JIP3 and JIP4 may also contribute to NMDA receptor regulation (Kennedy, 2007).
In conclusion, this study has demonstrated that JIP1/2 scaffold proteins represent novel components of the NMDA receptor regulatory network required for normal NMDA-mediated signal transduction (Kennedy, 2007).
Synaptic NMDA-type glutamate receptors (NMDARs) play important roles in synaptic plasticity, brain development, and pathology. In the last few years, the view of NMDARs as relatively fixed components of the postsynaptic density has changed. A number of studies have now shown that both the number of receptors and their subunit compositions can be altered. During development, the synaptic NMDARs subunit composition changes, switching from predominance of NR2B-containing to NR2A-containing receptors, but little is known about the mechanisms involved in this developmental process. This study reports that, depending on the pattern of NMDAR activation, the subunit composition of synaptic NMDARs is under extremely rapid, bidirectional control at neonatal synapses. This switching, which is at least as rapid as that seen with AMPARs, will have immediate and dramatic consequences on the integrative capacity of the synapse (Bellone, 2007).
At early ages synaptic NMDARs are at least as dynamic as AMPARs. LTP-inducing stimuli causes a switch in subunit composition within seconds and the change lasts for at least an hour. However, depotentiating stimuli rapidly reverses the change. These data indicate that the activity-dependent loss of NR2B receptors is coincident with the simultaneous recruitment of NR2A receptors. How this swapping of receptors is coordinated is unclear, but is likely to be dependent on the differences in the C terminus of the two receptor subunits and the partners with which they interact as well as to differences in phosphorylation (Bellone, 2007).
Cells in the superficial layers of primary visual cortex (area 17) are distinguished by feedforward input from thalamic-recipient layers and by massive recurrent excitatory connections between neighboring cells. The connections use glutamate as transmitter, and the postsynaptic cells contain both NMDA and AMPA receptors. The possible role of these receptor types in generating emergent responses of neurons in the superficial cortical layers is unknown. NMDA and AMPA receptors are both involved in the generation of direction-selective responses in layer 2/3 cells of area 17 in cats. NMDA receptors contribute prominently to responses in the preferred direction, and their contribution to responses in the nonpreferred direction is reduced significantly by GABAergic inhibition. AMPA receptors decrease spatial phase-selective simple cell responses and generate phase-invariant complex cell responses (Rivadulla, 2001).
By combining extracellular recording and iontophoresis of receptor blockers, the following results have been demonstrated: (1) Blocking AMPA receptors removes a proportionately larger component from nonpreferred compared with preferred responses and increases direction selectivity. The remaining responses are mediated by NMDA receptors and are overwhelmingly in the preferred direction. (2) Blocking NMDA receptors removes proportional components from preferred and nonpreferred responses and preserves directional selectivity. Because the remaining responses are mediated by AMPA receptors, these receptors are sufficient for direction selectivity. (3) Blocking inhibition preferentially enhances the contribution of NMDA receptors to nonpreferred responses and reduces direction selectivity. Thus, inhibition contributes to direction selectivity by reducing NMDA responses in the nonpreferred direction. (4) Blocking AMPA receptors increases the modulation of complex cell responses by a drifting grating stimulus. Thus, AMPA receptors decrease the selectivity of complex cells for spatial phase or the spatial location of visual stimuli. (5) Blocking NMDA receptors or inhibition has little effect on the temporal modulation of simple or complex cell responses. Together, these results allow for the proposal specific roles for NMDA and AMPA receptors in direction selectivity in the superficial layers of area 17 and in the generation of phase selectivity by simple and complex cells in these layers (Rivadulla, 2001).
Direction selectivity first appears in simple cells of layer 4 in area 17, where NMDA receptors are not present in significant proportions and contribute little to visual responses. The mechanism(s) by which direction selectivity is generated and whether the mechanism is similar in various cortical layers remain unresolved. One hypothesis is that inhibition reduces the response in the nonpreferred direction. The hypothesis is supported by pharmacological studies in cat and monkey, demonstrating that blockade of inhibition in cortical cells induces a loss of selectivity to the direction of stimulus motion. An alternative hypothesis is that there is enhancement of excitation in the preferred direction. It has been shown that simple cells in area 17 have asymmetries in the time course of the response evoked from different positions of the receptive field. Linear summation of these asymmetries allows one to predict the direction preference of the cell but also leads to an overestimation of the response in the nonpreferred direction. Recurrent excitation has been proposed as a nonlinear mechanism by which responses can be increased in the preferred direction. Recently, it has been postulated that inhibition can sculpt the spatiotemporal profile of the receptive field, accentuating spatiotemporal asymmetry and increasing direction selectivity, particularly in layer 4 (Rivadulla, 2001 and references therein).
A fundamental difference between layers 2/3 and 4 is the presence in supragranular layers of NMDA receptors, where they have been shown to participate in transmission in vivo and in vitro. AMPA and NMDA receptors both contribute to direction selectivity in supragranular layers. AMPA receptors are sufficient for generating direction selectivity, either because inputs to the superficial layers conveyed by AMPA receptors are already biased for direction or because feedforward and recurrent connections mediated by AMPA receptors generate direction selectivity within these layers. NMDA receptors by themselves can generate highly direction-selective responses, by summing and/or amplifying responses to the preferred stimulus while contributing less to nonpreferred responses because of close GABAergic control. One possibility is that NMDA receptor activation is possible only with enough excitation in the preferred direction. However, a comparison of the nonpreferred and spontaneous responses that remain after application of AMPA receptor inhibitor CNQX (spontaneous activity in this population of cells is reduced on average by only 28% under CNQX, whereas nonpreferred responses are reduced by 88%) suggests that the reduced contribution of NMDA receptors to nonpreferred responses is likely mediated by active inhibition rather than simply being a function of overall response magnitude: spontaneous activity occurs under less inhibition than nonpreferred responses and remains significantly greater after AMPA receptor blockade (Rivadulla, 2001).
Two lines of evidence indicate that GABAergic inhibition regulates the reduced contribution of NMDA receptors to nonpreferred responses. (1) Blocking inhibition by application of bicuculline decreases the direction selectivity of cells, but this effect is reversed by simultaneous application of APV, indicating that release of inhibition facilitates NMDA responses in the nonpreferred direction. (2) Blockade of inhibition concurrent with AMPA receptor blockade by CNQX reduces direction selectivity. Bicuculline preferentially increases nonpreferred responses, leaving a higher contribution of NMDA responses in the nonpreferred direction (Rivadulla, 2001).
These data confirm and extend the findings that NMDA inhibitor APV causes a proportional reduction in responses of area 17 cells to optimally oriented moving bars as stimulus contrast is increased, whereas application of NMDA increases responses by a proportional amount and quisqualate increases responses by an absolute amount at all contrasts. Importantly, responses in different directions (as also simple and complex cell responses) were studied with APV and CNQX and the modulation of NMDA and AMPA responses by inhibition. NMDA-mediated responses in the nonpreferred direction are reduced nonlinearly by inhibition. Furthermore, the contribution of AMPA receptors to complex cell responses is much more than addition of a constant response component; rather, there is a nonlinear change in the temporal modulation of the response. These observations argue for specific circuits that engage inhibition for generating direction-selective responses and AMPA receptors for generating complex cell responses (Rivadulla, 2001).
The possibility that inhibition regulates NMDA-mediated activity is consistent with other lines of evidence in area 17. The relationship between GABAergic inhibition and NMDA function is probably related to the voltage dependence of NMDA receptors; the binding of extracellular Mg2+ to the channel pore is highly dependent on membrane potential, and changes in the latter could significantly modulate NMDA receptor-mediated activity. The likely source of inhibition is GABAergic interneurons located within layer 2/3 itself (Rivadulla, 2001).
One caveat is that the iontophoresis technique does not allow definitive conclusions about whether all receptors on a cell are affected or whether other cells (either excitatory or inhibitory) in the vicinity could be modifying the responses of the recorded cell. However, the temporal effects of drug application were studied in the first and the second half of the iontophoresis period in several cells and found to be similar. Furthermore, the effect of iontophoresis does not change with ejection time, indicating that most of the affected receptors are in the volume covered by the antagonist since the start of iontophoresis (Rivadulla, 2001).
In addition to examining the role of AMPA and NMDA receptors in direction selectivity, an examination was made of their role in generating simple and complex responses in the supragranular layers by analyzing the temporal pattern of response of cells when they were stimulated with drifting gratings. CNQX caused a dramatic change in complex cell responses, causing them to increase their temporal modulation and respond in a manner similar to that of simple cells. APV does not affect the response modulation of complex cells, and the modulation of simple cell responses is unaffected by CNQX or APV (Rivadulla, 2001).
Recently, it has been proposed that complex cell responses arise as a consequence of decreasing the phase selectivity of simple cell responses by recurrent intracortical connections. The model predicts that a decrease in intracortical excitation should cause complex cells to respond like simple cells. If AMPA receptors primarily mediate short-range intracortical excitation, the results presented here agree strongly with this prediction, demonstrating that during blockade of AMPA receptors complex cells behave as simple cells when stimulated with drifting gratings (Rivadulla, 2001).
It is proposed that AMPA and NMDA receptors in layer 2/3 have different spatial distributions on cells, with both present on the same cell but in different proportions at different inputs. Both receptors mediate feedforward connections, and these afferents provide the necessary input for direction selectivity in layer 2/3. The data are consistent with spatiotemporal asymmetry and enhancement of excitation in feedforward pathways as crucial for direction selectivity in layer 2/3, with a prominent role for NMDA receptors in generating the preferred response and a role for GABAergic inhibition in reducing the nonpreferred response. In contrast to feedforward connections, local recurrent connections are mainly mediated by AMPA receptors (with a possible small contribution from NMDA receptors), and they are responsible for smearing the phase selectivity of simple cells to create phase-invariant complex cell responses (Rivadulla, 2001).
The suggestion that short-range excitation between cortical cells is mediated primarily by AMPA receptors is consistent with the fact that fast EPSCs that are evoked in supragranular layer cells in area 17 by adjacent intralaminar stimulation are not APV sensitive. In somatosensory cortex, intracellular recording of unitary EPSCs in layer 4 and the supragranular cortex indicates that both thalamocortical and intracortical EPSCs are mediated by AMPA receptors and have similar characteristics. In slices of area 17, white matter stimulation evokes EPSPs in the supragranular layers that have NMDA- and AMPA-mediated components. Because of feedforward and local recurrent connections, short-latency responses are primarily AMPA mediated, whereas long-latency responses, because of horizontal connections, have significant NMDA components. Furthermore, long-range horizontal inputs to layer 2/3 cells in area 17 can sum nonlinearly with feedforward or short-range inputs, indicative of NMDA receptor involvement in the long-range connections. Thus, it is likely that there is even finer spatial segregation of glutamate receptors associated with specific inputs on layer 2/3 cells. Together with the modulation of responses (particularly those mediated by NMDA receptors) by inhibition, the specific relationship between receptor types and anatomical connections provides a rich substrate for dynamic control of emergent responses in the cortex (Rivadulla, 2001).
Forming distinct representations of multiple contexts, places, and episodes is a crucial function of the hippocampus. The dentate gyrus subregion has been suggested to fulfill this role. This hypothesis was tested by generating and analyzing a mouse strain that lacks the gene encoding the essential subunit of the N-methyl-D-aspartate (NMDA) receptor NR1, specifically in dentate gyrus granule cells. The mutant mice performed normally in contextual fear conditioning, but are impaired in the ability to distinguish two similar contexts. A significant reduction in the context-specific modulation of firing rate was observed in the CA3 pyramidal cells when the mutant mice were transferred from one context to another. These results provide evidence that NMDA receptors in the granule cells of the dentate gyrus play a crucial role in the process of pattern separation (McHugh, 2007).
Using conditional genetic-engineering techniques, it has been shown that NRs in the CA3 play a crucial role in rapid learning and pattern completion-mediated recall, whereas CA1 NRs are required for the formation of both spatial and nonspatial memory. The DG-NR1 KO mice described here allowed extension of the study to the roles of DG NRs and NR-dependent plasticity. The data support the notion that DG GC NRs play an important role in rapidly forming a unique memory of a context and discriminating it from similar contexts previously encountered (pattern separation), although they are dispensable for the acquisition of contextual memory per se (McHugh, 2007).
DG-NR1 KO mice exhibited impaired context discrimination early during training in the incremental fear-conditioning paradigm and impaired context-modulated place cell activity in CA3 on the initial day of recording. Both deficits were overcome with training or experience. Together, these data suggest that NR function at perforant path (PP)-GC synapses is important for the animals' ability to discriminate similar contexts rapidly with limited experience, but not for slower acquisition of this ability over more trials. It is suggested that common mechanisms underlie the DG-NR1 KO deficits observed at both the behavioral and physiological levels, despite the different timelines of recovery to control levels. These differences may reflect differences between the cues used to define the contexts, the use of conditioning footshocks in the behavioral experiment, the contribution of non-hippocampal structures in fear conditioning, or the greater sensitivity of the readout in the place cell recordings relative to the behavioral task. The eventual acquisition of the discriminating power by DG-NR1 KO mice may be due to the gradual development of synaptic plasticity at sites downstream of the PP-GC synapses. For example, the recurrent collateral-CA3 synapses may provide a complementary site at which small differences in PP input can be amplified; this is supported by a recent study reporting a contribution of CA3 NRs to pattern separation. Although the large number of cells and sparse connectivity of the DG would provide the ultimate substrate for the pattern separation, synaptic plasticity may be the tool that allows rapid and efficient separation of representations (McHugh, 2007).
CA3 receives excitatory input from two external sources, the DG and the EC. Input from the DG is most likely to contribute to the orthogonalization of CA3 representations by virtue of the high GC number and the sparse GC-CA3 connectivity. Loss of NRs in the GCs may decrease drive from the DG to CA3. This would increase the relative proportion of EC drive to CA3, thus reducing the CA3 ensemble's ability to detect, amplify, and reflect small differences in EC activity generated in similar contexts. Indeed, rate remapping in CA3 (induced by changes in recording chamber shape or color) can occur in the absence of detectable changes in medial EC firing rates or locations. However, despite unvarying input from the EC, DG GCs did respond to contextual changes robustly and rapidly under these conditions. These data suggest that NR-mediated activity or plasticity in the GCs may underlie these changes, subsequently shaping CA3 encoding (McHugh, 2007).
It is puzzling that rate remapping in CA3 did not always affect spatial or rate coding downstream in CA1. It is possible that under different conditions, such as in the behavioral discrimination task, small differences in context-specific coding parameters, including firing rates, could be amplified by contextual salience (such as footshock) and may be manifested in CA1. It remains to be seen whether the context specificity of CA3 coding will be transferred to CA1 under the conditions of behavioral discrimination (McHugh, 2007).
Many physiological and behavioral phenomena are controlled by an internal, self-sustaining oscillator with a periodicity of approximately 24 hr. In mammals, the principal oscillator resides in the suprachiasmatic nucleus (SCN). A light pulse during the subjective night causes a phase shift of the circadian rhythm via direct glutamatergic retinal afferents to the SCN. Along with the accepted theoretical models of the clock, it is suggested that behavioral resetting of mammals is completed within 2 hr; however, the molecular mechanism has not been elucidated. The real-time image of the transcription of the circadian-clock gene mPer1 is shown in the cultured SCN by using the transgenic mice that carry a luciferase reporter gene under the control of the mPer1 promoter. The real-time image demonstrates that the mPer1 promoter activity oscillates robustly in a circadian manner and that this promoter activity is reset rapidly (within 2-3 hr) when a phase shift occurs (Asai, 2001).
Insufficient sleep impairs cognitive functions in humans and animals. However, whether long-term synaptic plasticity, a cellular substrate of learning and memory, is compromised by sleep loss per se remains unclear because of confounding factors related to sleep deprivation (SD) procedures in rodents. Using an ex vivo approach in C57BL/6J mice, it has been shown that sleep loss rapidly and reversibly alters bidirectional synaptic plasticity in the CA1 area of the hippocampus. A brief (approximately 4 h) total SD, respecting the temporal parameters of sleep regulation and maintaining unaltered low corticosterone levels, shifted the modification threshold for long-term depression/long-term potentiation (LTP) along the stimulation frequency axis (1-100 Hz) toward the right. Reducing exposure to sensory stimuli by whisker trimming did not affect the SD-induced changes in synaptic plasticity. Recovery sleep reversed the effects induced by SD. When SD was combined with moderate stress, LTP induction was not only impaired but also occluded. Both electrophysiological analysis and immunoblotting of purified synaptosomes revealed that an alteration in the molecular composition of synaptically activated NMDA receptors toward a greater NR2A/NR2B ratio accompanied the effects of SD. This change was reversed after recovery sleep. By using an unparalleled, particularly mild form of SD, this study describes a novel approach toward dissociating the consequences of insufficient sleep on synaptic plasticity from nonspecific effects accompanying SD in rodents. A framework was established for cellular models of cognitive impairment related to sleep loss, a major problem in modern society (Kopp, 2006).
The NMDA receptor is a key player in excitatory transmission and synaptic plasticity in the central nervous system. Its activation requires the binding of both glutamate and a co-agonist like D-serine to its glycine site. Since D-serine is released exclusively by astrocytes, the physiological impact of the glial environment on NMDA receptor-dependent activity and plasticity was studied. To this end, advantage was taken of the changing astrocytic ensheathing of neurons occurring in the supraoptic nucleus during lactation. Direct evidence is provided that in this hypothalamic structure the endogenous co-agonist of NMDA receptors is D-serine and not glycine. Consequently, the degree of astrocytic coverage of neurons governs the level of glycine site occupancy on the NMDA receptor, thereby affecting their availability for activation and thus the activity dependence of long-term synaptic changes. Such a contribution of astrocytes to synaptic metaplasticity fuels the emerging concept that astrocytes are dynamic partners of brain signaling (Panatier, 2006).
NMDA receptor subunit composition varies throughout the brain, providing molecular diversity in NMDA receptor function. The NR2 subunits (NR2A-D) in large part dictate the distinct functional properties of NMDA receptors and differentially regulate receptor trafficking. Although the NR2C subunit is highly enriched in cerebellar granule cells and plays a unique role in cerebellar function, little is known about NR2C-specific regulation of NMDA receptors. This study demonstrates that PKB/Akt directly phosphorylates NR2C on serine 1096 (S1096). In addition, 14-3-3epsilon was identified as an NR2C interactor, whose binding is dependent on S1096 phosphorylation. Both growth factor stimulation and NMDA receptor activity lead to a robust increase in both phosphorylation of NR2C on S1096 and surface expression of cerebellar NMDA receptors. Finally, NR2C expression, unlike NR2A and NR2B, supports neuronal survival. Thus, these data provide a direct mechanistic link between growth factor stimulation and regulation of cerebellar NMDA receptors (Chen, 2009).
Growth factors play a critical role in regulating neuronal survival during development. Cerebellar granule cells, in particular, are known to undergo a period of extensive apoptosis during development, which is precisely regulated by growth factors. IGF-1 has been demonstrated to promote cerebellar granule cell survival both in vitro and in vivo by blocking apoptosis. IGF-1 increases PI3 kinase activity leading to increased PKB activity and stimulation of downstream kinase/signaling cascades. PKB phosphorylates a variety of substrates, many of which regulate mitochondrial function and apoptosis. Mice lacking various isoforms of PKB have an overall reduction in cell number in many tissues, and specifically in the brain when the PKBγ isoform is absent. The growth factor/PKB signaling pathway has been specifically implicated in regulating cerebellar granule cell survival. Furthermore, there is evidence that PKB can potentiate NMDA receptor responses in cerebellar granule cells, suggesting a link between growth factor signaling and receptor activation in these neurons. However, until now, there was no mechanism described linking growth factor signaling, PKB activity, and NMDA receptor expression (Chen, 2009).
This study found that growth factor signaling and PKB phosphorylation increase surface expression of NR2C-containing NMDA receptors. Importantly, overexpression of NR2C was shown to protect neurons from NMDA-induced toxicity. Consistent with a role for phosphorylation of S1096 in receptor trafficking, NR2C S1096A was less effective at neuronal protection than NR2C WT, supporting a role for the PI3K/PKB pathway in neuronal survival. Overexpression of NR2C S1096A also has a protective effect compared to NR2A or NR2B, which is probably due to the increased level of NR2C surface expression due to exogenous overexpression. For example, the S1096A mutation does not completely abolish the surface expression of NR2C when overexpressed in cerebellar granule cells. These results suggest that surface-expressed NR2C S1096A, even if decreased compared to that of NR2C WT, is sufficient to support neuronal survival. How does NR2C contribute to neuronal survival? The answer probably lies in the fact that NR2C-containing NMDA receptors have unique channel properties and that NR2C may mediate specific signaling pathways to activate downstream survival molecules. In conclusion, these findings that PKB directly phosphorylates cerebellar NMDA receptors and increases their surface expression reveals a novel molecular mechanism by which growth factor signaling can directly affect NMDA receptor activity (Chen, 2009).
NR3A is the only NMDA receptor (NMDAR) subunit that downregulates sharply prior to the onset of sensitive periods for plasticity, yet the functional importance of this transient expression remains unknown. To investigate whether removal/replacement of juvenile NR3A-containing NMDARs is involved in experience-driven synapse maturation, a reversible transgenic system was used that prolonged NR3A expression in the forebrain. Removal of NR3A is required to develop strong NMDAR currents, full expression of long-term synaptic plasticity, a mature synaptic organization characterized by more synapses and larger postsynaptic densities, and the ability to form long-term memories. Deficits associated with prolonged NR3A were reversible, as late-onset suppression of transgene expression rescued both synaptic and memory impairments. These results suggest that NR3A behaves as a molecular brake to prevent the premature strengthening and stabilization of excitatory synapses and that NR3A removal might thereby initiate critical stages of synapse maturation during early postnatal neural development (Roberts, 2009).
NMDARs are heteromers containing NR1 subunits and combinations of four NR2 (A-D) and two NR3 (A-B) subunits. Most research to date has focused on the role of the developmental switch between predominantly NR2B- to NR2A-containing NMDARs because it occurs in many brain regions and can be driven by neural activity and experience. In contrast, little is known about the importance of the removal/replacement of NR3A-containing NMDARs despite growing evidence that nonconventional NR3A subunits are well positioned to be potent regulators of NMDAR function. First, inclusion of NR3A results in the formation of heteromeric receptors (NR1/NR2/NR3A) with reduced Ca2+ permeability and low sensitivity to Mg2+ blockade, thus modifying two major properties of classical NMDARs (NR1/NR2 heteromers). Second, NR3A is prominently expressed during a narrow temporal window of postnatal development that correlates with periods of intense synaptogenesis and pruning and later becomes downregulated, just prior to the onset of critical period plasticity. Third, genetic deletion of NR3A increases spine density, indicating that NR3A might participate in synapse formation or elimination. Finally, removal of NR3A-containing NMDARs can be regulated by activity via selective recruitment of the endocytic adaptor PACSIN/syndapin 1, suggesting that regulated removal might occur at the level of individual synapses rather than nonselectively along entire dendritic branches or trees. Yet, if and how NR3A subunits modulate synaptic plasticity, synapse maturation, or learning is unknown, leaving an important gap in understanding of NMDAR-mediated development. Assessing the role of NR3A may also be important for understanding human disease. For example, genetic variations in NR3A alter human brain function, and abnormal NR3A expression is associated with schizophrenia and bipolar disorder (Roberts, 2009 and references therein).
To address the role of NR3A subunits in synapse development and plasticity, transgenic mice were generated in which NR3A expression could be prolonged beyond its natural time window in postnatal forebrain neurons. Prolonging NR3A expression results in synaptic NMDAR hypofunction, deficits in long-term potentiation (LTP), reduced postsynaptic maturation at Schaffer collateral-CA1 synapses of the hippocampus, and impairs behavioral flexibility and memory consolidation. Conversely, genetic deletion of endogenous NR3A yielded a premature concentration of NMDARs at postsynaptic sites, enhancing synaptic NMDAR currents and promoting an earlier developmental onset of LTP. Both the synaptic and cognitive deficits resulting from persistent NR3A expression could be reversed by suppressing transgene expression at later stages. These findings indicate that the presence of NR3A maintains synapses in an anatomically and functionally juvenile state that is refractory to the induction of LTP and structural plasticity and incapable of supporting long-term memory storage (Roberts, 2009).
Brief bath application of N-methyl-D-aspartate (NMDA) to hippocampal slices produces long-term synaptic depression (LTD) in CA1 that is (1) sensitive to postnatal age, (2) saturable, (3) induced postsynaptically, (4) reversible, and (5) not associated with a change in paired pulse facilitation. Chemically induced LTD (chem-LTD) and homosynaptic LTD are mutually occluding, suggesting a common expression mechanism. Using phosphorylation site-specific antibodies, induction of chem-LTD is found to produce a persistent dephosphorylation of the GluR1 subunit of AMPA receptors at serine 845, a cAMP-dependent protein kinase (PKA) substrate, but not at serine 831, a substrate of protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII). These results suggest that dephosphorylation of AMPA receptors is an expression mechanism for LTD and indicate an unexpected role for PKA in the postsynaptic modulation of excitatory synaptic transmission (Lee, 1998).
The classically conditioned vertebrate eye-blink response is a model in which to study neuronal mechanisms of learning and memory. In this paradigm, an eye-blink reflex in response to a tone can be evoked when the tone is repeatedly paired with an air puff to the cornea that normally elicits the blink response. A neural correlate of this response recorded in the abducens nerve can be conditioned entirely in vitro using an isolated brainstem-cerebellum preparation from the turtle by pairing trigeminal and auditory nerve stimulation. Conditioning requires that the paired stimuli occur within a narrow temporal window of <100 msec. Conditioning is blocked by a NMDA receptor antagonist. Moreover, there is a significant positive correlation between the levels of conditioning and greater immunoreactivity with the glutamate receptor 4 (GluR4) AMPA receptor subunit in the abducens motor nuclei, but not with NMDAR1 or GluR1. It is concluded that in vitro classical conditioning of an abducens nerve eye-blink response is generated by NMDA receptor-mediated mechanisms that may act to modify the AMPA receptor by increasing GluR4 subunits in auditory nerve synapses (Keifer, 2001).
The mechanisms behind the induction of cellular correlates of memory by sensory input and their contribution to meaningful behavioral changes are largely unknown. A graded memory in the form of sensorimotor adaptation has been reported in the electromotor output of electric fish. This study shows that the mechanism for this adaptation is a synaptically induced long-lasting shift in intrinsic neuronal excitability. This mechanism rapidly integrates hundreds of spikes in a second, or gradually integrates the same number of spikes delivered over tens of minutes. Thus, this mechanism appears immune to frequency-dependent fluctuations in input and operates as a simple pulse counter over a wide range of time scales, enabling it to transduce graded sensory information into a graded memory and a corresponding change in the behavioral output. This adaptation is based on an NMDA receptor-mediated change in intrinsic excitability of the postsynaptic neurons involving the Ca2+-dependent activation of TRP channels (Oestreich, 2006).
Memories are dynamic and can change when recalled. The process that returns memories to a labile state during remembering is unclear. The presence of NMDA, but not AMPA, receptor antagonists in the amygdala prior to recall prevented the consolidated fear memory from returning to a labile state. These findings suggest that NMDA receptors in the amygdala are critical for transforming a memory from a fixed to a labile state (Ben Mamou, 2006).
The hippocampus is considered to play a role in allocentric but not in egocentric spatial learning. How does this view fit with the emerging evidence that the hippocampus and possibly related cortical areas are necessary for episodic-like memory, i.e., in all situations in which events need to be spatially or sequentially organized? Are NMDA receptor-dependent mechanisms crucial for the acquisition of spatiotemporal relationships? To address this issue, knock-out (KO) mice lacking hippocampal CA1 NMDA receptors and presenting a reduction of these receptors in the deep cortical layers (NR1-KO mice) were used. A new task (the starmaze) was designed, allowing the distinguishing of allocentric and sequential-egocentric memories. NR1-KO mice were impaired in acquiring both types of memory. These findings suggest that memories composed of multiple spatiotemporal events require intact NMDA receptors-dependent mechanisms in CA1 and possibly in the deep cortical layers (Rondi-Reig, 2006).
Emotions generally improve memory, and the basolateral amygdala (BLA) is believed to mediate this effect. After emotional arousal, BLA neurons increase their firing rate, facilitating memory consolidation in BLA targets. The enhancing effects of BLA activity extend to various types of memories, including motor learning, which is thought to involve activity-dependent plasticity at corticostriatal synapses. However, the underlying mechanisms are unknown. The NMDA-to-AMPA ratio is nearly twice as high at BLA as compared with cortical synapses onto principal striatal neurons; activation of BLA inputs greatly facilitates long-term potentiation induction at corticostriatal synapses. This facilitation is NMDA-dependent, but it occurs even when BLA and cortical stimuli are 0.5 s apart during long-term potentiation induction. Overall, these results suggest that BLA activity opens long time windows during which the induction of corticostriatal plasticity is facilitated (Popescu, 2007).
Animals recognize a taste cue as aversive when it has been associated with post-ingestive malaise; this associative learning is known as conditioned taste aversion (CTA). When an animal consumes a new taste and no negative consequences follow, it becomes recognized as a safe signal, leading to an increase in its consumption in subsequent presentations (attenuation of neophobia, AN). It has been shown that the nucleus accumbens (NAcc) has an important role in taste learning. To elucidate the involvement of NMDA and muscarinic receptors in the NAcc during safe and aversive taste memory formation, bilateral infusions of DL-2-amino-5-phosphonopentanoic acid (APV) or scopolamine were administrated in the NAcc shell or core respectively. The results showed that pre-training injections of APV in the NAcc core and shell disrupted aversive but not safe taste memory formation, whereas pre-training injections of scopolamine in the NAcc shell, but not core, disrupted both CTA and AN. These results suggest that muscarinic receptors seem to be necessary for processing taste stimuli for either safe or aversive taste memory, whereas NMDA receptors are only involved in the aversive taste memory trace formation (Ramirez-Lugo, 2007).
Extinction of conditioned fear is an active learning process requiring NMDA receptors, but the timing, location, and neural mechanisms of NMDAR-mediated processing in extinction are a matter of debate. This study shows that infusion of the NMDAR antagonist CPP into the ventromedial prefrontal cortex (vmPFC) prior to, or immediately after, extinction training impairs 24 hr recall of extinction. These findings indicate that consolidation of extinction requires posttraining activation of NMDARs within the vmPFC. Using multichannel unit recording, it is observed that CPP selectively reduces burst firing in vmPFC neurons, suggesting that bursting in vmPFC is necessary for consolidation of extinction. In support of this, it was found that the degree of bursting in infralimbic vmPFC neurons shortly after extinction predicts subsequent recall of extinction. It is suggested that NMDAR-dependent bursting in the infralimbic vmPFC initiates calcium-dependent molecular cascades that stabilize extinction memory, thereby allowing for successful recall of extinction (Burgos-Robles, 2007).
New neurons are continuously integrated into existing neural circuits in adult dentate gyrus of the mammalian brain. Accumulating evidence indicates that these new neurons are involved in learning and memory. A substantial fraction of newly born neurons die before they mature and the survival of new neurons is regulated in an experience-dependent manner, raising the possibility that the selective survival or death of new neurons has a direct role in a process of learning and memory--such as information storage--through the information-specific construction of new circuits. However, a critical assumption of this hypothesis is that the survival or death decision of new neurons is information-specific. Because neurons receive their information primarily through their input synaptic activity, whether the survival of new neurons is regulated by input activity in a cell-specific manner was investigated. A retrovirus-mediated, single-cell gene knockout technique was developed in mice and it was shown that the survival of new neurons is competitively regulated by their own NMDA-type glutamate receptor during a short, critical period soon after neuronal birth. This finding indicates that the survival of new neurons and the resulting formation of new circuits are regulated in an input-dependent, cell-specific manner. Therefore, the circuits formed by new neurons may represent information associated with input activity within a short time window in the critical period. This information-specific addition of new circuits through selective survival or death of new neurons may be a unique attribute of new neurons that enables them to play a critical role in learning and memory (Tashiro, 2006).
The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. This study reports that genetic deletion of AC1 significantly attenuates neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion does not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, neuronal excitotoxicity induced by an intracortical injection of NMDA was studied. Cortical lesions induced by NMDA are significantly reduced in AC1 but not in AC8 knock-out mice. These findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases (Wang, 2007).
NMDA receptors promote neuronal survival but also cause cell degeneration and neuron loss. The mechanisms underlying these opposite effects on neuronal fate are unknown. Whole-genome expression profiling revealed that NMDA receptor signaling is decoded at the genomic level through activation of two distinct, largely nonoverlapping gene-expression programs. The location of the NMDA receptor activated specifies the transcriptional response: synaptic NMDA receptors induce a coordinate upregulation of newly identified pro-survival genes and downregulation of pro-death genes. Extrasynaptic NMDA receptors fail to activate this neuroprotective program, but instead induce expression of Clca1, a putative calcium-activated chloride channel that kills neurons. These results help explain the opposing roles of synaptic and extrasynaptic NMDA receptors on neuronal fate. They also demonstrate that the survival function is implemented in neurons through a multicomponent system of functionally related genes, whose coordinate expression is controlled by specific calcium signal initiation sites (Zhang, 2007).
A single exposure to drugs of abuse produces an NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) of AMPA receptor (AMPAR) currents in DA neurons; however, the importance of LTP for various aspects of drug addiction is unclear. To test the role of NMDAR-dependent plasticity in addictive behavior, functional NMDAR signaling was genetically inactivated exclusively in DA neurons (KO mice). Inactivation of NMDARs results in increased AMPAR-mediated transmission that is indistinguishable from the increases associated with a single cocaine exposure, yet locomotor responses to multiple drugs of abuse were unaltered in the KO mice. The initial phase of locomotor sensitization to cocaine is intact; however, the delayed sensitization that occurs with prolonged cocaine withdrawal did not occur. Conditioned behavioral responses for cocaine-testing environment were also absent in the KO mice. These findings provide evidence for a role of NMDAR signaling in DA neurons for specific behavioral modifications associated with drug seeking behaviors (Zweifel, 2008).
N-methyl-D-aspartate receptors (NMDARs) play important functions in neural development. NR2B is the predominant NR2 subunit of NMDAR in the developing brain. This study used mosaic analysis with double markers (MADM) to knock out NR2B in isolated single cells and analyze its cell-autonomous function in dendrite development. NR2B mutant dentate gyrus granule cells (dGCs) and barrel cortex layer 4 spiny stellate cells (bSCs) have similar dendritic growth rates, total length, and branch number as control cells. However, mutant dGCs maintain supernumerary primary dendrites resulting from a pruning defect. Furthermore, while control bSCs restrict dendritic growth to a single barrel, mutant bSCs maintain dendritic growth in multiple barrels. Thus, NR2B functions cell autonomously to regulate dendrite patterning to ensure that sensory information is properly represented in the cortex. This study also indicates that molecular mechanisms that regulate activity-dependent dendrite patterning can be separated from those that control general dendrite growth and branching (Espinosa, 2009).
The dendrites of CNS neurons play a critical role in integrating synaptic inputs from a multitude of presynaptic partners and, subsequently, in determining the extent to which a neuron transmits this information to its postsynaptic partners. Characteristic dendritic arborization patterns allow neurons to perform signal processing and computation appropriate for their functions. For example, in the adult somatosensory cortex of rodents, most layer 4 stellate neurons orient their dendrites toward a single barrel center to maximize contacts with thalamocortical afferents representing a single whisker. The development of dendritic trees characteristic to specific neuronal types is believed to result from the interplay between intrinsic genetic programs, extracellular signals, and electrical activity. Despite an increasing understanding of dendrite development, it is unclear if mechanisms that sculpt specific dendrite patterns are an integral part of those that control dendrite growth and branching, or if independent mechanisms can regulate these two aspects of dendrite development(Espinosa, 2009).
N-methyl-D-aspartate-type glutamate receptors (NMDARs) play a central role in activity-dependent regulation of dendrite development. NMDARs function mainly as heterotetramers of two obligate NR1 subunits and a combination of two NR2 subunits (A-D). Each NMDAR subunit combination confers distinct functional properties, including the regulation of unitary conductance, binding affinity, and gating and desensitization kinetics. For example, compared to NR2A-containing receptors, NR2B-containing NMDARs have a 3- to 4-fold slower decay time course of NMDAR-mediated excitatory postsynaptic currents, resulting in a larger Ca2+ influx. In the mammalian brain, the NR1 mRNA is found ubiquitously, whereas the NR2 subunits are differentially expressed, both temporally and spatially. At embryonic stages, the NR2B subunit is expressed in the entire brain, while the NR2D subunit is expressed selectively in the diencephalon, the mesencephalon, and the spinal cord. From the time of birth to adulthood, expression of NR2B becomes restricted to the forebrain and NR2D expression peaks 1 week after birth but is then strongly reduced. During this time, the expression levels increase for NR2A in the forebrain and for NR2C in the cerebellum. The expression pattern of NR2B suggests that it plays a more important role during development than do other NR2 subunits. Indeed, NR2B knockout mice die shortly after birth, similar to NR1 knockout mice, but knockout mice for NR2A, 2C, or 2D are fully viable (Espinosa, 2009).
Pharmacological agents that block NMDARs have been used to study their function in dendrite development. In the Xenopus retinotectal system, NMDAR antagonists inhibit dendritic arborization of tectal neurons during development (Rajan, 1998) or in response to visual stimulation (Sin, 2002). NMDAR function in dendrite development has also been examined in knockout mice. In the cortex-specific NR1 knockout, individual layer 4 stellate cells lose oriented arborization and grow exuberant dendrites and spines (Datwani, 2002). NMDARs are also necessary for dendritic spine formation induced by sensory activity and long-term potentiation (Engert, 1999; Maletic-Savati, 1999). Cortex-specific NR1 knockout results in reduced spine densities (Ultanir, 2007). Although these studies have revealed important functions for NMDARs in multiple aspects of dendrite development, it is unclear to what extent the observed defects are caused by the cell-autonomous perturbation of NMDAR function. These experiments cannot exclude secondary consequences of perturbing the NMDAR in other neurons in the circuit. In the Xenopus retinotectal system, NMDAR blockade also affects the arborization of the retinal ganglion cell axon termini (Cline, 1990; Ruthazer, 2003), which may indirectly perturb tectal cell dendrite development. In cortex-specific NR1 knockout mice, although thalamocortical axons are genetically unperturbed, their terminal arborization patterns are grossly altered in response to NR1 knockout in cortical cells (Lee, 2005), and barrels do not form properly (Datwani, 2002). It is therefore difficult to determine if the unoriented dendrites of layer 4 stellate neurons reflect the cell-autonomous requirement for NMDAR or if they are a secondary consequence of the general pattern formation defects in the barrel cortex. Recently, genetic perturbations of NR2A and NR2B subunits have been reported using overexpression and morpholino-mediated knockdown in single Xenopus tectal cells. Compared to overexpression, knockdown of NR2B has minor consequences on dendrite development (Espinosa, 2009).
This study used the mosaic analysis with double markers (MADM) system to knock out NR2B in isolated single neurons to assess the cell-autonomous function of NR2B in dendrite development. MADM permits simultaneous gene inactivation and distinct labeling of homozygous mutant cells and their wild-type siblings in the same animal through Cre/LoxP-mediated interchromosomal mitotic recombination events. Moreover, infrequent recombination generates isolated single knockout cells, allowing unambiguous assessment of cell-autonomous function of genes. This study found that in two types of neurons analyzed, dGCs and bSCs, NR2B is dispensable for general dendrite growth and branching but is required for dendrite patterning critical for information processing. This study also indicates that molecular mechanisms that regulate activity-dependent dendrite patterning are separable from those that control general dendrite growth and branching (Espinosa, 2009).
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