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1.  Caldendrin–Jacob: A Protein Liaison That Couples NMDA Receptor Signalling to the Nucleus 
PLoS Biology  2008;6(2):e34.
NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-α to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor–induced cellular degeneration.
Author Summary
Long-lasting changes in communication between nerve cells require the regulation of gene expression. The influx of calcium ions into the cell, particularly through membrane protein called NMDA receptors, plays a crucial role in this process by determining the type of gene expression induced. NMDA receptors can exert multiple and very divergent effects within neuronal cells by impacting opposing phenomena such as synaptic plasticity and neuronal degeneration. We identified a protein termed Jacob that appears to play a pivotal role in such processes by entering the nucleus in response to NMDA receptor activation and controlling gene expression that governs cell survival and the stability of synaptic cell contacts. Removal of Jacob from the nucleus protects neurons from NMDA receptor–induced cell death and increases phosphorylation of the transcription factor CREB, whereas the opposite occurs after targeting Jacob exclusively to the nucleus. The work defines a novel pathway of synapse-to-nucleus communication involved in modelling synapto-dendritic input and NMDA receptor–induced cellular degeneration.
A new signaling mechanism from NMDA receptors to the nucleus plays an important role in the phosphorylation of the transcription factor CREB and neuronal cell survival.
PMCID: PMC2253627  PMID: 18303947
2.  Spontaneous neurotransmission signals through store-driven Ca2+ transients to maintain synaptic homeostasis 
eLife  null;4:e09262.
Spontaneous glutamate release-driven NMDA receptor activity exerts a strong influence on synaptic homeostasis. However, the properties of Ca2+ signals that mediate this effect remain unclear. Here, using hippocampal neurons labeled with the fluorescent Ca2+ probes Fluo-4 or GCAMP5, we visualized action potential-independent Ca2+ transients in dendritic regions adjacent to fluorescently labeled presynaptic boutons in physiological levels of extracellular Mg2+. These Ca2+ transients required NMDA receptor activity, and their propensity correlated with acute or genetically induced changes in spontaneous neurotransmitter release. In contrast, they were insensitive to blockers of AMPA receptors, L-type voltage-gated Ca2+ channels, or group I mGluRs. However, inhibition of Ca2+-induced Ca2+ release suppressed these transients and elicited synaptic scaling, a process which required protein translation and eukaryotic elongation factor-2 kinase activity. These results support a critical role for Ca2+-induced Ca2+ release in amplifying NMDA receptor-driven Ca2+ signals at rest for the maintenance of synaptic homeostasis.
eLife digest
Learning and memory is thought to rely on changes in the strength of the connections between nerve cells. When an electrical impulse travelling through a nerve cell reaches one of these connections (called a synapse), it causes the cell to release chemical transmitter molecules. These bind to receptors on the cell on the other side of the synapse. This starts a series of events that ultimately leads to new receptors being inserted into the membrane of this second cell, which strengthens the connection between the two cells.
The receptors involved in this process belong to two groups, called AMPA and NMDA receptors. Both groups are ion channels that regulate the flow of charged particles from one side of a cell's membrane to the other. In resting nerve cells, NMDA receptors are partially blocked by magnesium ions. However, the binding of the transmitter molecules to AMPA receptors causes these receptors to open and allow positively charged sodium ions into the cell. This changes the electrical charge across the cell membrane, which displaces the magnesium ions from the NMDA receptors so that they too open. Calcium ions then enter the cell through the NMDA receptors and activate a signaling cascade that leads to the production of new AMPA receptors.
Nerve cells also release transmitter molecules in the absence of electrical impulses, and evidence suggests that individual cells can use this ‘spontaneous transmitter release’ to adjust the strength of their synapses. When these spontaneous release levels are high, AMPA receptors are removed from the membrane of the nerve after the synapse to make it less sensitive to the transmitter molecules. Conversely, when spontaneous release levels are low, additional AMPA receptors are added to the membrane to increase the sensitivity.
Reese and Kavalali have now identified the mechanism behind this process by showing that spontaneously released transmitter molecules cause small amounts of calcium to enter the second nerve cell through NMDA receptors, even when these receptors are blocked by magnesium ions. This trickle of calcium triggers the release of more calcium from stores inside the cell, which amplifies the signal. The ultimate effect of the flow of calcium into the cell is to block the production of AMPA receptors, and ensure that the synapse does not become any stronger. As confirmation of this mechanism, Reese and Kavalali showed that simulating low levels of spontaneous activity by blocking the so-called ‘calcium-induced calcium release’ has the opposite effect. This led to more AMPA receptors being produced and stronger synapses. Taken together these findings indicate that spontaneous transmitter release exerts an outsized influence on communication between neurons by maintaining adequate levels of AMPA receptors via these ‘amplified’ calcium signals.
PMCID: PMC4534843  PMID: 26208337
spontaneous neurotransmitter release; NMDA receptor signaling; Ca2+ induced Ca2+ release; homeostatic plasticity; mouse; rat
3.  In developing hippocampal neurons, NR2B-containing NMDA receptors can mediate signalling to neuronal survival and synaptic potentiation, as well as neuronal death 
Neuroscience  2008;158(1):334-343.
It has been suggested that NR2B-containing NMDA receptors have a selective tendency to promote pro-death signalling and synaptic depression, compared to the survival promoting, synapse potentiating properties of NR2A-containing NMDA receptors. A preferential localization of NR2A-containing NMDA receptors at the synapse in maturing neurons could thus explain differences in synaptic vs. extrasynaptic NMDA receptor signalling.
We have investigated whether NMDA receptors can mediate signalling to survival, death, and synaptic potentiation, in neurons at a developmental stage prior to significant NR2A expression and subunit-specific differences between synaptic and extrasynaptic NMDA receptors. We show that in developing hippocampal neurons, the progressive reduction in sensitivity of NMDA receptor currents to the NR2B antagonist ifenprodil applies to both synaptic and extrasynaptic locations. However, the reduction is less acute in extrasynaptic currents, indicating that NR2A does partition preferentially, but not exclusively, into synaptic locations at DIV>12. We then studied NMDA receptor signalling at DIV10, when both synaptic and extrasynaptic NMDA receptors are both overwhelmingly and equally NR2B-dominated. To analyse pro-survival signalling we studied the influence of synaptic NMDA receptor activity on staurosporine-induced apoptosis. Blockade of spontaneous NMDAR activity with MK-801, or ifenprodil exacerbated the apoptotic insult. Furthermore, MK-801 and ifenprodil both antagonized neuroprotection promoted by enhancing synaptic activity. Pro-death signalling induced by a toxic dose of NMDA is also blocked by NR2B-specific antagonists. Using a cell culture model of synaptic NMDA receptor-dependent synaptic potentiation, we find that this is mediated exclusively by NR2B-containing NMDARs, as implicated by NR2B-specific antagonists and the use of selective vs. non-selective doses of the NR2A-preferring antagonist NVP-AAM077.
Therefore, within a single neuron, NR2B-NMDA receptors are able to mediate both survival and death signalling, as well as model of NMDA receptor-dependent synaptic potentiation. In this instance, subunit differences cannot account for the dichotomous nature of NMDA receptor signalling.
PMCID: PMC2635533  PMID: 18378405
Apoptosis; necrosis; extrasynaptic; neuroprotection; NR2A
4.  Dynamin-dependent NMDAR endocytosis during LTD and its dependence on synaptic state 
BMC Neuroscience  2005;6:48.
The N-methyl-D-aspartate (NMDA)-type glutamate receptor expressed at excitatory glutamatergic synapses is required for learning and memory and is critical for normal brain function. At a cellular level, this receptor plays a pivotal role in triggering and controlling synaptic plasticity. While it has been long recognized that this receptor plays a regulatory role, it was considered by many to be itself immune to synaptic activity-induced plasticity. More recently, we and others have shown that NMDA receptor-mediated synaptic responses can be subject to activity-dependent depression.
Here we show that depression of synaptic transmission mediated by NMDA receptors displays a state-dependence in its plasticity; NMDA receptors are resistant to activity-induced changes at silent and recently-silent synapses. Once synapses transition to the active state however, NMDA receptors become fully 'plastic'. This state-dependence is identical to that shown by the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor. Furthermore, the down-regulation of NMDAR-mediated responses during synaptic depression is prevented by disruption of dynamin-dependent endocytosis.
NMDA receptor-mediated synaptic responses are plastic in a state-dependent manner. Depending on the plasticity state in which a synapse currently resides, NMDA receptors will either be available or unavailable for down-regulation. The mechanism underlying the down-regulation of NMDA receptor-mediated synaptic responses is endocytosis of the NMDA receptor. Other potential mechanisms, such as receptor diffusion along the plane of the membrane, or changes in the activity of the channel are not supported. The mechanisms of AMPA receptor and NMDA receptor endocytosis appear to be tightly coupled, as both are either available or unavailable for endocytosis in the same synaptic states. Endocytosis of NMDA receptors would serve as a potent mechanism for metaplasticity. Such state-dependent regulation of NMDAR endocytosis will provide fundamental control over downstream NMDA receptor-dependent plasticity of neuronal circuitry.
PMCID: PMC1187896  PMID: 16042781
5.  The glutamate story 
British Journal of Pharmacology  2006;147(Suppl 1):S100-S108.
Glutamatergic synaptic transmission in the mammalian central nervous system was slowly established over a period of some 20 years, dating from the 1950s. Realisation that glutamate and like amino acids (collectively known as excitatory amino acids (EAA)) mediated their excitatory actions via multiple receptors preceded establishment of these receptors as synaptic transmitter receptors. EAA receptors were initially classified as N-methyl-D-aspartate (NMDA) and non-NMDA receptors, the latter subdivided into quisqualate (later AMPA) and kainate receptors after agonists that appeared to activate these receptors preferentially, and by their sensitivity to a range of differentially acting antagonists developed progressively during the 1970s. NMDA receptors were definitively shown to be synaptic receptors on spinal neurones by the sensitivity of certain excitatory pathways in the spinal cord to a range of specific NMDA receptor antagonists. Importantly, specific NMDA receptor antagonists appeared to be less effective at synapses in higher centres. In contrast, antagonists that also blocked non-NMDA as well as NMDA receptors were almost universally effective at blocking synaptic excitation within the brain and spinal cord, establishing both the existence and ubiquity of non-NMDA synaptic receptor systems throughout the CNS. In the early 1980s, NMDA receptors were shown to be involved in several central synaptic pathways, acting in concert with non-NMDA receptors under conditions where a protracted excitatory postsynaptic potential was effected in response to intense stimulation of presynaptic fibres. Such activation of NMDA receptors together with non-NMDA receptors led to the phenomenon of long-term potentiation (LTP), associated with lasting changes in synaptic efficacy (synaptic plasticity) and considered to be an important process in memory and learning. During the 1980s, it was shown that certain glutamate receptors in the brain mediated biochemical changes that were not susceptible to NMDA or non-NMDA receptor antagonists. This dichotomy was resolved in the early 1990s by the techniques of molecular biology, which identified two families of glutamate-binding receptor proteins (ionotropic (iGlu) and metabotropic (mGlu) receptors). Development of antagonists binding to specific protein subunits is currently enabling precise identification of discrete iGlu or mGlu receptor subtypes that participate in a range of central synaptic processes, including synaptic plasticity.
PMCID: PMC1760733  PMID: 16402093
L-Glutamate; excitatory amino acids; ionotropic glutamate receptors; metabotropic glutamate receptors; synaptic transmission
6.  Triheteromeric NMDA Receptors at Hippocampal Synapses 
NMDA receptors are composed of two GluN1 (N1) and two GluN2 (N2) subunits. Constituent N2 subunits control the pharmacological and kinetic characteristics of the receptor. NMDA receptors in hippocampal or cortical neurons are often thought of as diheteromeric, i.e., containing only one type of N2 subunit. However, triheteromeric receptors with more than one type of N2 subunit also have been reported and the relative contribution of di- and triheteromeric NMDA receptors at synapses has been difficult to assess. Because wild-type hippocampal principal neurons express N1, N2A and N2B, we used cultured hippocampal principal neurons from N2A and N2B-knockout mice as templates for diheteromeric synaptic receptors. Summation of N1/N2B and N1/N2A excitatory postsynaptic currents could not account for the deactivation kinetics of wild-type excitatory postsynaptic currents (EPSCs) however. To make a quantitative estimate of NMDA receptor subtypes at wild-type synapses, we used the deactivation kinetics, as well as the effects of the competitive antagonist NVP-AAM077. Our results indicate that three types of NMDA receptors contribute to the wild-type EPSC, with at least two-thirds being triheteromeric receptors. Functional isolation of synaptic triheteromeric receptors revealed deactivation kinetics and pharmacology distinct from either diheteromeric receptor subtype. Because of differences in open probability, synaptic triheteromeric receptors outnumbered N1/N2A receptors by 5.8 to 1 and N1/N2B receptors by 3.2 to 1. Our results suggest that triheteromeric NMDA receptors must be either preferentially assembled or preferentially localized at synapses.
PMCID: PMC3755730  PMID: 23699525
7.  Extracellular Ca2+ ions reduce NMDA receptor conductance and gating 
The Journal of General Physiology  2014;144(5):379-392.
NMDA receptor conductance and gating is directly affected by fluctuations in extracellular calcium concentration.
Brief intracellular Ca2+ transients initiate signaling routines that direct cellular activities. Consequently, activation of Ca2+-permeable neurotransmitter-gated channels can both depolarize and initiate remodeling of the postsynaptic cell. In particular, the Ca2+ transient produced by NMDA receptors is essential to normal synaptic physiology, drives the development and plasticity of excitatory central synapses, and also mediates glutamate excitotoxicity. The amplitude and time course of the Ca2+ signal depends on the receptor’s conductance and gating kinetics; these properties are themselves influenced both directly and indirectly by fluctuations in the extracellular Ca2+ concentration. Here, we used electrophysiology and kinetic modeling to delineate the direct effects of extracellular Ca2+ on recombinant GluN1/GluN2A receptor conductance and gating. We report that, in addition to decreasing unitary conductance, Ca2+ also decreased channel open probability primarily by lengthening closed-channel periods. Using one-channel current recordings, we derive a kinetic model for GluN1/GluN2A receptors in physiological Ca2+ concentrations that accurately describes macroscopic channel behaviors. This model represents a practical instrument to probe the mechanisms that control the Ca2+ transients produced by NMDA receptors during both normal and aberrant synaptic signaling.
PMCID: PMC4210427  PMID: 25348411
8.  GluN2B-containing NMDA receptors regulate depression-like behavior and are critical for the rapid antidepressant actions of ketamine 
eLife  null;3:e03581.
A single, low dose of the NMDA receptor antagonist ketamine produces rapid antidepressant actions in treatment-resistant depressed patients. Understanding the cellular mechanisms underlying this will lead to new therapies for treating major depression. NMDARs are heteromultimeric complexes formed through association of two GluN1 and two GluN2 subunits. We show that in vivo deletion of GluN2B, only from principal cortical neurons, mimics and occludes ketamine's actions on depression-like behavior and excitatory synaptic transmission. Furthermore, ketamine-induced increases in mTOR activation and synaptic protein synthesis were mimicked and occluded in 2BΔCtx mice. We show here that cortical GluN2B-containing NMDARs are uniquely activated by ambient glutamate to regulate levels of excitatory synaptic transmission. Together these data predict a novel cellular mechanism that explains ketamine's rapid antidepressant actions. In this model, basal glutamatergic neurotransmission sensed by cortical GluN2B-containing NMDARs regulates excitatory synaptic strength in PFC determining basal levels of depression-like behavior.
eLife digest
Depression is the leading cause of disability worldwide, with hundreds of millions of people living with the condition. The ‘gold standard’ for depression treatment involves a combination of psychotherapy and medication. Unfortunately, current antidepressant medications do not help everyone, waiting lists for psychotherapy are often long, and both normally take a number of weeks of regular treatment before they begin to have an effect. As patients are often at a high risk of suicide, it is crucial that treatments that act more quickly, and that are safe and effective, are developed.
One substance that may fulfill these requirements is a drug called ketamine. Studies have shown that depression symptoms can be reduced within hours by a single low dose of ketamine, and this effect on mood can last for more than a week. However, progress has been hindered by a lack of knowledge about what ketamine actually does inside the brain.
Neurons communicate with one another by releasing chemicals known as neurotransmitters, which transfer information by binding to receptor proteins on the surface of other neurons. Drugs such as ketamine also bind to these receptors. Ketamine works by blocking a specific receptor called the n-methyl d-aspartate (NMDA) receptor, but how this produces antidepressant effects is not fully understood.
The NMDA receptor is actually formed from a combination of individual protein subunits, including one called GluN2B. Now Miller, Yang et al. have created mice that lack receptors containing these GluN2B subunits in neurons in their neocortex, including the prefrontal cortex, a brain region involved in complex mental processes such as decision-making. This allowed Miller, Yang et al. to discover that when the neurotransmitter glutamate binds to GluN2B-containing NMDA receptors, it limits the production of certain proteins that make it easier for signals to be transmitted between neurons. Suppressing the synthesis of these proteins too much may cause depressive effects by reducing communication between the neurons in the prefrontal cortex.
Both mice lacking GluN2B-containing receptors in their cortical neurons and normal mice treated with ketamine showed a reduced amount of depressive-like behavior. This evidence supports Miller, Yang et al.'s theory that by blocking these NMDA receptors, ketamine restricts their activation. This restores normal levels of protein synthesis, improves communication between neurons in the cortex, and reduces depression.
Understanding how ketamine works to alleviate depression is an important step towards developing it into a safe and effective treatment. Further research is also required to determine the conditions that cause overactivation of the GluN2B-containing NMDA receptors.
PMCID: PMC4270067  PMID: 25340958
depression; cortex; synapse; ketamine; electrophysiology; protein synthesis; mouse; rat
9.  Cell type-specific pharmacology of NMDA receptors using masked MK801 
eLife  null;4:e10206.
N-Methyl-D-aspartate receptors (NMDA-Rs) are ion channels that are important for synaptic plasticity, which is involved in learning and drug addiction. We show enzymatic targeting of an NMDA-R antagonist, MK801, to a molecularly defined neuronal population with the cell-type-selectivity of genetic methods and the temporal control of pharmacology. We find that NMDA-Rs on dopamine neurons are necessary for cocaine-induced synaptic potentiation, demonstrating that cell type-specific pharmacology can be used to dissect signaling pathways within complex brain circuits.
eLife digest
Learning is critical to survival for humans and other animals. The learning process is regulated by receptors on the surface of brain cells called N-Methyl-D-aspartate receptors (or NMDA receptors for short). These receptors help to strengthen signals between brain cells, which allows a new concept or action to be learned. However, it has been difficult to pin down how the role of NMDA receptors selectively in specific types of brain cells. While drugs can be used to quickly block NMDA receptors throughout the brain, it is hard to target drugs to a specific cell type. Also, genetic engineering can be used to selectively knock out NMDA receptors in certain types of brain cells, but these techniques are too slow, and can take weeks or even a lifetime to work.
Now, Yang et al. have developed a clever way to combine an NMDA-blocking drug and genetic engineering to study NMDA receptors' responses to cocaine in specific brain cells. This approach involved first creating an inactive form of an NMDA-blocking drug that can only becomes active when it is processed by an enzyme that is normally produced in pigs' livers. Next, living mouse brain cells, including some that were engineered to express the pig enzyme, were exposed to the drug in the laboratory. The drug blocked the NMDA receptors on brain cells that expressed the enzyme, but not the receptors on nearby brain cells that lacked the enzyme. This occurred even though all the cells produced NMDA receptors and all were exposed to the drug.
NMDA receptors have been known to play an important role in cocaine addiction for more than 20 years. Drugs like cocaine can co-opt the normally healthy learning process involving NMDA receptors and lead to a maladaptive form of learning that is commonly called addiction. Cocaine strengthens signals between brain cells causing the behaviors associated with using cocaine to become deeply ingrained and difficult to change. Yang et al. used cell type-specific targeting of a drug that blocks NMDA receptors to observe what happened in cocaine-exposed brain cells with, or without, working NMDA receptors.
As expected, the experiments showed that cocaine didn't strengthen brain signals in cells without working NMDA receptors. Specifically, the experiments showed that NMDA receptors on a type of brain cell that release a pleasure-inducing chemical called dopamine are necessary for cocaine–induced synaptic plasticity. The combination technique developed by Yang et al. will likely be used by other scientists to further study the role of NMDA receptors in specific brain cells during addiction and normal brain activity.
PMCID: PMC4594264  PMID: 26359633
NMDA receptor; cell type-specific pharmacology; dopamine; cocaine; synaptic plasticity; mouse
10.  The Effects of NMDA Subunit Composition on Calcium Influx and Spike Timing-Dependent Plasticity in Striatal Medium Spiny Neurons 
PLoS Computational Biology  2012;8(4):e1002493.
Calcium through NMDA receptors (NMDARs) is necessary for the long-term potentiation (LTP) of synaptic strength; however, NMDARs differ in several properties that can influence the amount of calcium influx into the spine. These properties, such as sensitivity to magnesium block and conductance decay kinetics, change the receptor's response to spike timing dependent plasticity (STDP) protocols, and thereby shape synaptic integration and information processing. This study investigates the role of GluN2 subunit differences on spine calcium concentration during several STDP protocols in a model of a striatal medium spiny projection neuron (MSPN). The multi-compartment, multi-channel model exhibits firing frequency, spike width, and latency to first spike similar to current clamp data from mouse dorsal striatum MSPN. We find that NMDAR-mediated calcium is dependent on GluN2 subunit type, action potential timing, duration of somatic depolarization, and number of action potentials. Furthermore, the model demonstrates that in MSPNs, GluN2A and GluN2B control which STDP intervals allow for substantial calcium elevation in spines. The model predicts that blocking GluN2B subunits would modulate the range of intervals that cause long term potentiation. We confirmed this prediction experimentally, demonstrating that blocking GluN2B in the striatum, narrows the range of STDP intervals that cause long term potentiation. This ability of the GluN2 subunit to modulate the shape of the STDP curve could underlie the role that GluN2 subunits play in learning and development.
Author Summary
The striatum of the basal ganglia plays a key role in fluent motor control; pathology in this structure causes the motor symptoms of Parkinson's Disease and Huntington's Chorea. A putative cellular mechanism underlying learning of motor control is synaptic plasticity, which is an activity dependent change in synaptic strength. A known mediator of synaptic potentiation is calcium influx through the NMDA-type glutamate receptor. The NMDA receptor is sensitive to the timing of neuronal activity, allowing calcium influx only when glutamate release and a post-synaptic depolarization coincide temporally. The NMDA receptor is comprised of specific subunits that modify its sensitivity to neuronal activity and these subunits are altered in animal models of Parkinson's disease. Here we use a multi-compartmental model of a striatal neuron to investigate the effect of different NMDA subunits on calcium influx through the NMDA receptor. Simulations show that the subunit composition changes the temporal intervals that allow coincidence detection and strong calcium influx. Our experiments manipulating the dominate subunit in brain slices show that the subunit effect on calcium influx predicted by our computational model is mirrored by a change in the amount of potentiation that occurs in our experimental preparation.
PMCID: PMC3334887  PMID: 22536151
11.  Computational Investigation of the Changing Patterns of Subtype Specific NMDA Receptor Activation during Physiological Glutamatergic Neurotransmission 
PLoS Computational Biology  2011;7(6):e1002106.
NMDA receptors (NMDARs) are the major mediator of the postsynaptic response during synaptic neurotransmission. The diversity of roles for NMDARs in influencing synaptic plasticity and neuronal survival is often linked to selective activation of multiple NMDAR subtypes (NR1/NR2A-NMDARs, NR1/NR2B-NMDARs, and triheteromeric NR1/NR2A/NR2B-NMDARs). However, the lack of available pharmacological tools to block specific NMDAR populations leads to debates on the potential role for each NMDAR subtype in physiological signaling, including different models of synaptic plasticity. Here, we developed a computational model of glutamatergic signaling at a prototypical dendritic spine to examine the patterns of NMDAR subtype activation at temporal and spatial resolutions that are difficult to obtain experimentally. We demonstrate that NMDAR subtypes have different dynamic ranges of activation, with NR1/NR2A-NMDAR activation sensitive at univesicular glutamate release conditions, and NR2B containing NMDARs contributing at conditions of multivesicular release. We further show that NR1/NR2A-NMDAR signaling dominates in conditions simulating long-term depression (LTD), while the contribution of NR2B containing NMDAR significantly increases for stimulation frequencies that approximate long-term potentiation (LTP). Finally, we show that NR1/NR2A-NMDAR content significantly enhances response magnitude and fidelity at single synapses during chemical LTP and spike timed dependent plasticity induction, pointing out an important developmental switch in synaptic maturation. Together, our model suggests that NMDAR subtypes are differentially activated during different types of physiological glutamatergic signaling, enhancing the ability for individual spines to produce unique responses to these different inputs.
Author Summary
Release of glutamate from one neuron onto glutamate receptors on adjacent neurons serves as the primary basis for neuronal communication. Further, different types of glutamate signals produce unique responses within the neuronal network, providing the ability for glutamate receptors to discriminate between alternative types of signaling. The NMDA receptor (NMDAR) is a glutamate receptor that mediates a variety of physiological functions, including the molecular basis for learning and memory. These receptors exist as a variety of subtypes, and this molecular heterogeneity is used to explain the diversity in signaling initiated by NMDARs. However, the lack of reliable experimental tools to control the activation of each subtype has led to debate over the subtype specific roles of the NMDAR. We have developed a stochastic model of glutamate receptor activation at a single synapse and find that NMDAR subtypes detect different types of glutamate signals. Moreover, the presence of multiple populations of NMDAR subtypes on a given neuron allows for differential patterns of NMDAR activation in response to varied glutamate inputs. This model demonstrates how NMDAR subtypes enable effective and reliable communication within neuronal networks and can be used as a tool to examine specific roles of NMDAR subtypes in neuronal function.
PMCID: PMC3127809  PMID: 21738464
12.  Neto1 Is a Novel CUB-Domain NMDA Receptor–Interacting Protein Required for Synaptic Plasticity and Learning 
PLoS Biology  2009;7(2):e1000041.
The N-methyl-D-aspartate receptor (NMDAR), a major excitatory ligand-gated ion channel in the central nervous system (CNS), is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1), a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP) at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans.
Author Summary
The fundamental unit for information processing in the brain is the synapse, a highly specialized site of communication between the brain's multitude of individual neurons. The strength of the communication at each synapse changes in response to neuronal activity—a process called synaptic plasticity—allowing networks of neurons to adapt and learn. How synaptic plasticity occurs is a major question in neurobiology. A central player in synaptic plasticity is an assembly of synaptic proteins called the NMDA receptor complex. Here, we discovered that the protein Neto1 is a component of the NMDA receptor complex. Neto1-deficient mice had a dramatic decrease in the number of NMDA receptors at synapses and consequently, synaptic plasticity and learning were impaired. By indirectly enhancing the function of the residual NMDA receptors in Neto1-deficient mice with a small molecule, we restored synaptic plasticity and learning to normal levels. Our findings establish the principle that inherited abnormalities of synaptic plasticity and learning due to NMDA receptor dysfunction can be pharmacologically corrected. Our discoveries also suggest that synaptic proteins that share a molecular signature, called the CUB domain, with Neto1 may be important components of synaptic receptors across species, because several CUB-domain proteins in worms have also been found to regulate synaptic receptors.
Spatial learning and memory depend on the N-methyl-D-aspartic acid receptor, a synaptic ion channel regulated by Neto1. Impaired cognition due to the absence of Neto1 can be rescued pharmacologically, a finding with implications for the therapy of inherited learning defects in humans.
PMCID: PMC2652390  PMID: 19243221
13.  Biphasic Synaptic Ca Influx Arising from Compartmentalized Electrical Signals in Dendritic Spines 
PLoS Biology  2009;7(9):e1000190.
Dendritic spines compartmentalize synaptically-evoked biochemical signals. The authors show that electrical compartmentalization provided by a spine endows the associated synapse with additional modes of calcium signaling by shaping the kinetics of synaptic calcium currents.
Excitatory synapses on mammalian principal neurons are typically formed onto dendritic spines, which consist of a bulbous head separated from the parent dendrite by a thin neck. Although activation of voltage-gated channels in the spine and stimulus-evoked constriction of the spine neck can influence synaptic signals, the contribution of electrical filtering by the spine neck to basal synaptic transmission is largely unknown. Here we use spine and dendrite calcium (Ca) imaging combined with 2-photon laser photolysis of caged glutamate to assess the impact of electrical filtering imposed by the spine morphology on synaptic Ca transients. We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale. Furthermore, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively) that are colocalized on individual spine heads interact to produce two kinetically and mechanistically distinct phases of synaptically evoked Ca influx. Rapid depolarization of the spine triggers a brief and large Ca current whose amplitude is regulated in a graded manner by the number of open AMPARs and whose duration is terminated by the opening of small conductance Ca-activated potassium (SK) channels. A slower phase of Ca influx is independent of AMPAR opening and is determined by the number of open NMDARs and the post-stimulus potential in the spine. Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine. These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.
Author Summary
The vast majority of excitatory synapses in the mammalian central nervous system are made onto dendritic spines, small (< 1 fL) membranous structures stippled along the dendrite. The head of each spine is separated from its parent dendrite by a thin neck – a morphological feature that intuitively suggests it might function to limit the transmission of electrical and biochemical signals. Unfortunately, the extremely small size of spines has made direct measurements of their electrical properties difficult and, therefore, the functional implications of electrical compartmentalization have remained elusive. In this study, we use spatiotemporally controlled stimulation to generate calcium signals within the spine head and/or neighboring dendrite. By comparing these measurements we demonstrate that spines create specialized electrical signaling compartments, which has at least two functional consequences. First, synaptic stimulation, but not similar dendritic depolarization, can trigger the activation of voltage-gated calcium channels within the spine. Second, voltage changes in the spine head arising from compartmentalization shape the time course of synaptically evoked calcium influx such that it is biphasic. Thus, the electrical compartmentalization provided by spines allows for multiple modes of calcium signaling at excitatory synapses.
PMCID: PMC2734993  PMID: 19753104
14.  Metaplasticity at Single Glutamatergic Synapses 
Neuron  2010;66(6):859-870.
Optimal function of neuronal networks requires interplay between rapid forms of Hebbian plasticity and homeostatic mechanisms that adjust the threshold for plasticity, termed metaplasticity. Numerous forms of rapid synapse plasticity have been examined in detail. However, the rules that govern synaptic metaplasticity are much less clear. Here we demonstrate a local subunit-specific switch in NMDA receptors that alternately primes or prevents potentiation at single synapses. Prolonged suppression of neurotransmitter release enhances NMDA receptor currents, increases the number of functional NMDA receptors containing NR2B, and augments calcium transients at single dendritic spines. This local switch in NMDA receptors requires spontaneous glutamate release, but is independent of action potentials. Moreover, single inactivated synapses exhibit a lower induction threshold for both long-term synaptic potentiation and plasticity-induced spine growth. Thus, spontaneous glutamate release adjusts plasticity threshold at single synapses by local regulation of NMDA receptors, providing a novel spatially delimited form of synaptic metaplasticity.
PMCID: PMC2911980  PMID: 20620872
15.  D1/5 modulation of synaptic NMDA receptor currents 
Converging evidence suggests that salience-associated modulation of behavior is mediated by the release of monoamines and that monoaminergic activation of D1/5 receptors is required for normal hippocampal-dependent learning and memory. However, it is not understood how D1/5 modulation of hippocampal circuits can affect salience-associated learning and memory. We have observed in CA1 pyramidal neurons that D1/5 receptor activation elicits a bi-directional long-term plasticity of NMDA receptor-mediated synaptic currents with the polarity of plasticity determined by NMDA receptor, NR2A/B subunit composition. This plasticity results in a decrease in the NR2A/NR2B ratio of subunit composition. Synaptic responses mediated by NMDA receptors that include NR2B subunits are potentiated by D1/5 receptor activation, while responses mediated by NMDA receptors that include NR2A subunits are depressed. Furthermore, these bidirectional, subunit-specific effects are mediated by distinctive intracellular signaling mechanisms. As there is a predominance of NMDA receptors composed of NR2A subunits observed in entorhinal-CA1 inputs and a predominance of NMDA receptors composed of NR2B subunits in CA3-CA1 synapses, potentiation of synaptic NMDA currents predominates in the proximal CA3-CA1 synapses, while depression of synaptic NMDA currents predominates in the distal entorhinal-CA1 synapses. Finally, all of these effects are reproduced by the release of endogenous monoamines through activation of D1/5 receptors. Thus, endogenous D1/5 activation can, 1) decrease the NR2A/B ratio of NMDAR subunit composition at glutamatergic synapses, a rejuvenation to a composition similar to developmentally immature synapses, and, 2) in CA1, bias NMDA receptor responsiveness towards the more highly processed tri-synaptic CA3-CA1 circuit and away from the direct entorhinal-CA1 input.
PMCID: PMC2684496  PMID: 19279248
D1 [D-1]; NMDA receptor; Synaptic plasticity; Ca1; Hippocampal function; Amphetamine; Hippocampus; Current; glutamate
16.  Structural Plasticity Can Produce Metaplasticity 
PLoS ONE  2009;4(11):e8062.
Synaptic plasticity underlies many aspect of learning memory and development. The properties of synaptic plasticity can change as a function of previous plasticity and previous activation of synapses, a phenomenon called metaplasticity. Synaptic plasticity not only changes the functional connectivity between neurons but in some cases produces a structural change in synaptic spines; a change thought to form a basis for this observed plasticity. Here we examine to what extent structural plasticity of spines can be a cause for metaplasticity. This study is motivated by the observation that structural changes in spines are likely to affect the calcium dynamics in spines. Since calcium dynamics determine the sign and magnitude of synaptic plasticity, it is likely that structural plasticity will alter the properties of synaptic plasticity.
Methodology/Principal Findings
In this study we address the question how spine geometry and alterations of N-methyl-D-aspartic acid (NMDA) receptors conductance may affect plasticity. Based on a simplified model of the spine in combination with a calcium-dependent plasticity rule, we demonstrated that after the induction phase of plasticity a shift of the long term potentiation (LTP) or long term depression (LTD) threshold takes place. This induces a refractory period for further LTP induction and promotes depotentiation as observed experimentally. That resembles the BCM metaplasticity rule but specific for the individual synapse. In the second phase, alteration of the NMDA response may bring the synapse to a state such that further synaptic weight alterations are feasible. We show that if the enhancement of the NMDA response is proportional to the area of the post synaptic density (PSD) the plasticity curves most likely return to the initial state.
Using simulations of calcium dynamics in synaptic spines, coupled with a biophysically motivated calcium-dependent plasticity rule, we find under what conditions structural plasticity can form the basis of synapse specific metaplasticity.
PMCID: PMC2779489  PMID: 19956610
17.  A Model of NMDA Receptor-Mediated Activity in Dendrites of Hippocampal CA1 Pyramidal Neurons 
Journal of neurophysiology  1992;68(6):2248-2259.
The role of synaptic activation of NMDA (N-methyl-d-aspartate) receptor-mediated conductances on CA1 hippocampal pyramidal cells in short-term excitability changes was studied with the use of a computational model. Model parameters were based on experimental recordings from dendrites and somata and previous hippocampal simulations. Representation of CA1 neurons included NMDA and non-NMDA excitatory dendritic synapses, dendritic and somatic inhibition, five intrinsic membrane conductances, and provision for activity-dependent intracellular and extracellular ion concentration changes.The model simulated somatic and dendritic potentials recorded experimentally. The characteristic CA1 spike afterdepolarization was a consequence of the longitudinal spread of dendritic charge, reactivation of slow Ca2+-dependent K+ conductances, slow synaptic processes (NMDA-dependent depolarizing and γ-aminobutyric acid–mediated hyperpolarizing currents) and was sensitive to extracellular potassium accumulation. Calcium currents were found to be less important in generating the spike afterdepolarization.Repetitive activity was influenced by the cumulative activation of the NMDA-mediated synaptic conductances, the frequency-dependent depression of inhibitory synaptic responses, and a shift in the potassium reversal potential. NMDA receptor activation produced a transient potentiation of the excitatory postsynaptic potential (EPSP). The frequency dependence of EPSP potentiation was similar to the experimental data, reaching a maximal value near 10 Hz.Although the present model did not have compartments for dendritic spines, Ca2+ accumulation was simulated in a restricted space near the intracellular surface of the dendritic membrane. The simulations demonstrated that the Ca2+ component of the NMDA-operated synaptic current can be a significant factor in increasing the Ca2+ concentration at submembrane regions, even in the absence of Ca2+ spikes.Elevation of the extracellular K+ concentration enhanced the dendritic synaptic response during repetitive activity and led to an increase in intracellular Ca2+ levels. This increase in dendritic excitability was partly mediated by NMDA receptor-mediated conductances.Blockade of Ca2+-sensitive K+ conductances in the dendrites increased the size of EPSPs leading to a facilitation of dendritic and somatic spike activity and increased [Ca2+]i. NMDA receptor-mediated conductances appeared as an amplifying component in this mechanism, activated by the relatively depolarized membrane potential.The results suggest that dendritic NMDA receptors, by virtue of their voltage-dependency, can interact with a number of voltage-sensitive conductances to increase the dendritic excitatory response during periods of repetitive synaptic activation. These findings support experimental results that implicate NMDA receptor-mediated conductances in the short-term response plasticity of the CA1 hippocampal pyramidal neuron.
PMCID: PMC2605954  PMID: 1337105
18.  NMDA Receptors in GABAergic Synapses during Postnatal Development 
PLoS ONE  2012;7(5):e37753.
GABA (gamma-aminobutyric-acid), the main inhibitory neurotransmitter in the adult brain, exerts depolarizing (excitatory) actions during development and this GABAergic depolarization cooperates with NMDARs (N-methyl-D-aspartate receptors) to drive spontaneous synchronous activity (SSA) that is fundamentally important for developing neuronal networks. Although GABAergic depolarization is known to assist in the activation of NMDARs during development, the subcellular localization of NMDARs relative to GABAergic synapses is still unknown. Here, we investigated the subcellular distribution of NMDARs in association with GABAergic synapses at the developmental stage when SSA is most prominent in mice. Using multiple immunofluorescent labeling and confocal laser-scanning microscopy in the developing mouse hippocampus, we found that NMDARs were associated with both glutamatergic and GABAergic synapses at postnatal day 6–7 and we observed a direct colocalization of GABAA- and NMDA-receptor labeling in GABAergic synapses. Electron microscopy of pre-embedding immunogold-immunoperoxidase reactions confirmed that GluN1, GluN2A and GluN2B NMDAR subunits were all expressed in glutamatergic and GABAergic synapses postsynaptically. Finally, quantitative post-embedding immunogold labeling revealed that the density of NMDARs was 3 times higher in glutamatergic than in GABAergic synapses. Since GABAergic synapses were larger, there was little difference in the total number of NMDA receptors in the two types of synapses. In addition, receptor density in synapses was substantially higher than extrasynaptically. These data can provide the neuroanatomical basis of a new interpretation of previous physiological data regarding the GABAAR-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABAAR-mediated depolarization assists NMDAR activation right inside GABAergic synapses and this effective spatial cooperation of receptors and local change of membrane potential will reach developing glutamatergic synapses with a higher probability and efficiency even further away on the dendrites. This additional level of cooperation that operates within the depolarizing GABAergic synapse, may also allow its own modification triggered by Ca2+-influx through the NMDA receptors.
PMCID: PMC3360635  PMID: 22662211
19.  NR2 subunits and NMDA receptors on lamina II inhibitory and excitatory interneurons of the mouse dorsal horn 
Molecular Pain  2010;6:26.
NMDA receptors expressed by spinal cord neurons in the superficial dorsal horn are involved in the development of chronic pain associated with inflammation and nerve injury. The superficial dorsal horn has a complex and still poorly understood circuitry that is mainly populated by inhibitory and excitatory interneurons. Little is known about how NMDA receptor subunit composition, and therefore pharmacology and voltage dependence, varies with neuronal cell type. NMDA receptors are typically composed of two NR1 subunits and two of four NR2 subunits, NR2A-2D. We took advantage of the differences in Mg2+ sensitivity of the NMDA receptor subtypes together with subtype preferring antagonists to identify the NR2 subunit composition of NMDA receptors expressed on lamina II inhibitory and excitatory interneurons. To distinguish between excitatory and inhibitory interneurons, we used transgenic mice expressing enhanced green fluorescent protein driven by the GAD67 promoter.
Analysis of conductance ratio and selective antagonists showed that lamina II GABAergic interneurons express both the NR2A/B containing Mg2+ sensitive receptors and the NR2C/D containing NMDA receptors with less Mg2+ sensitivity. In contrast, excitatory lamina II interneurons express primarily NR2A/B containing receptors. Despite this clear difference in NMDA receptor subunit expression in the two neuronal populations, focally stimulated synaptic input is mediated exclusively by NR2A and 2B containing receptors in both neuronal populations.
Stronger expression of NMDA receptors with NR2C/D subunits by inhibitory interneurons compared to excitatory interneurons may provide a mechanism to selectively increase activity of inhibitory neurons during intense excitatory drive that can provide inhibitory feedback.
PMCID: PMC2879240  PMID: 20459616
20.  Effects of Ethanol on Phosphorylation Site Mutants of Recombinant NMDA Receptors 
Alcohol (Fayetteville, N.Y.)  2010;45(4):373-380.
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by the neurotransmitter glutamate. These channels are highly expressed by brain neurons and are critically involved in excitatory synaptic transmission. Results from previous studies show that both native and recombinant NMDA receptors are inhibited by ethanol at concentrations associated with signs of behavioral impairment and intoxication. Given the important role that NMDA receptors play in synaptic transmission and brain function, it is important to understand the factors that regulate the ethanol inhibition of these receptors. One dynamic mechanism for regulating ethanol action may be via phosphorylation of NMDA subunits by serine-threonine and tyrosine kinases. Both NR1 and NR2 subunits contain multiple sites of phosphorylation and in the NR1 subunit, most of these are contained within the C1 domain, a carboxy-terminal cassette that is subject to alternative splicing. While results from our previous studies suggest that single phosphorylation sites do not greatly affect ethanol sensitivity of NMDA receptors, it is likely that in vivo, these subunits are phosphorylated at multiple sites by different kinases. In the present study, we constructed a series of NMDA receptor mutants at serine (S) or threonine (T) residues proposed to be sites of phosphorylation by PKA and various isoforms of PKC. Ethanol (100 mM) inhibited currents from wild-type NR1/2A and NR1/2B receptors expressed in HEK293 cells by approximately 25% and 30% respectively. This inhibition was not different in single site mutants expressing alanine (A) or aspartate/glutamate (D/E) at positions T879, S896 or T900. The mutant NR1(S890D) showed greater ethanol inhibition than NR1(890A) containing receptors although this was only observed when it was combined with the NR2A subunit. Ethanol inhibition was not altered by aspartate substitution at four serines (positions 889, 890, 896, 897) or when T879D was added to the four serine-substituted mutant. Ethanol inhibition was increased when T900E was added to the five serine/threonine substituted mutant but again this was selective for NR2A containing receptors. Together with previously published data, these findings suggest that modification of putative phosphorylation sites could contribute to the overall acute ethanol sensitivity of recombinant NMDA receptors. Supported by R37 AA009986.
PMCID: PMC3095724  PMID: 21163614
PKA; PKC; phosphorylation; electrophysiology; alcohol
21.  Ethanol Inhibition of Recombinant NMDA Receptors Is Not Altered by Co-Expression of CaMKII-α or CaMKII-β 
Alcohol (Fayetteville, N.Y.)  2008;42(5):425-432.
Previous studies have shown that the N-methyl-D-aspartate (NMDA) receptor is an important target for the actions of ethanol in the brain. NMDA receptors are glutamate-activated ion channels that are highly expressed in neurons. They are activated during periods of significant glutamatergic synaptic activity and are an important source of the signaling molecule calcium in the post-synaptic spine. Alterations in the function of NMDA receptors by drugs or disease are associated with deficits in motor, sensory and cognitive processes of the brain. Acutely, ethanol inhibits ion flow through NMDA receptors while sustained exposure to ethanol can induce compensatory changes in the density and localization of the receptor. Defining factors that govern the acute ethanol sensitivity of NMDA receptors is an important step in how an individual responds to ethanol. In the present study, we investigated the effect of calcium-calmodulin dependent protein kinase II (CaMKII) on the ethanol sensitivity of recombinant NMDA receptors. CaMKII is a major constituent of the post-synaptic density and is critically involved in various forms of learning and memory. NMDA receptor subunits were transiently expressed in human embryonic kidney 293 cells (HEK 293) along with CaMKII-α or CaMKII-β tagged with the green fluorescent protein (GFP). Whole cell currents were elicited by brief exposures to glutamate and were measured using patchclamp electrophysiology. Neither CaMKII-α or CaMKII-β had any significant effect on the ethanol inhibition of NR1/2A or NR1/2B receptors. Ethanol inhibition was also unaltered by deletion of CaMKII binding domains in NR1 or NR2 subunits or by phospho-site mutants that mimic or occlude CaMKII phosphorylation. Chronic treatment of cortical neurons with ethanol had no significant effect on the expression of CaMKII-α or CaMKII-β. The results of this study suggest that CaMKII is not involved in regulating the acute ethanol sensitivity of NMDA receptors.
PMCID: PMC2629600  PMID: 18562151
electrophysiology; alcohol; ion channel; kinase; phosphorylation
22.  Spontaneous and evoked glutamate release activate two populations of NMDA receptors with limited overlap 
In a synapse, spontaneous and action potential-driven neurotransmitter release are assumed to activate the same set of postsynaptic receptors. Here, we tested this assumption using MK-801, a well-characterized use-dependent blocker of NMDA receptors. NMDA receptor-mediated spontaneous miniature excitatory postsynaptic currents (NMDA-mEPSCs) were substantially decreased by MK-801 within 2-minutes in a use-dependent manner. In contrast, MK-801 application at rest for 10-minutes did not significantly impair the subsequent NMDA receptor-mediated evoked EPSCs (NMDA-eEPSCs). Brief stimulation in the presence of MK-801 significantly depressed evoked NMDA-eEPSCs but only mildly affected the spontaneous NMDA-mEPSCs detected on the same cell. Optical imaging of synaptic vesicle fusion showed that spontaneous and evoked release could occur at the same synapse albeit without correlation between their kinetics. In addition, modeling glutamate diffusion and NMDA receptor activation revealed that postsynaptic densities larger than ~0.2 μm2 can accommodate two populations of NMDA receptors with largely non-overlapping responsiveness. Collectively, these results support the premise that spontaneous and evoked neurotransmission activate distinct sets of NMDA receptors and signal independently to the postsynaptic side.
PMCID: PMC2578837  PMID: 18829973
Synaptic vesicle release; synaptic transmission; synaptic communication; Synapse; NMDA receptor; Synaptic plasticity; neurotransmission
23.  NMDA receptor activation requires remodeling of inter-subunit contacts within ligand-binding heterodimers 
Nature communications  2011;2:498.
Two classes of glutamate-activated channels mediate excitation at central synapses: N-methyl-d-aspartic acid (NMDA) receptors and non-NMDA receptors. Despite substantial structural homology, each class generates signals with characteristic kinetics and mediates distinct synaptic functions. In non-NMDA receptors, the strength of inter-subunit contacts within agonist-binding domains is inversely correlated with functional desensitization. Here we test how the strength of these contacts affects NMDA receptor activation by combining mutagenesis and single-channel current analyses. We show that receptors with covalently linked dimers had dramatically lower activity due to high barriers to opening and unstable open states but had intact desensitization. Based on these observations, we suggest that in NMDA receptors rearrangements at the heterodimer interface represent an early and integral step of the opening sequence but are not required for desensitization. These results demonstrate distinct functional roles in the activation of NMDA and non-NMDA glutamate-gated channels for largely conserved inter-subunit contacts.
PMCID: PMC3899702  PMID: 21988914
24.  Direct pharmacological monitoring of the developmental switch in NMDA receptor subunit composition using TCN 213, a GluN2A-selective, glycine-dependent antagonist 
British Journal of Pharmacology  2012;166(3):924-937.
Developmental switches in NMDA receptor subunit expression have been inferred from studies of GluN2 expression levels, changes in kinetics of glutamatergic synaptic currents and sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists. Here we use TCN 213, a novel GluN2A-selective antagonist to identify the presence of this subunit in functional NMDA receptors in developing cortical neurones.
Two-electrode voltage-clamp (TEVC) recordings were made from Xenopus laevis oocytes to determine the pharmacological activity of TCN 213 at recombinant NMDA receptors. TCN 213 antagonism was studied in cultures of primary cortical neurones, assessing the NMDA receptor dependency of NMDA-induced excitotoxicity and monitoring developmental switches in NMDA receptor subunit composition.
TCN 213 antagonism of GluN1/GluN2A NMDA receptors was dependent on glycine but independent of glutamate concentrations in external recording solutions. Antagonism by TCN 213 was surmountable and gave a Schild plot with unity slope. TCN 213 block of GluN1/GluN2B NMDA receptor-mediated currents was negligible. In cortical neurones, at a early developmental stage predominantly expressing GluN2B-containing NMDA receptors, TCN 213 failed to antagonize NMDA receptor-mediated currents or to prevent GluN2B-dependent, NMDA-induced excitoxicity. In older cultures (DIV 14) or in neurones transfected with GluN2A subunits, TCN 213 antagonized NMDA-evoked currents. Block by TCN 213 of NMDA currents inversely correlated with block by ifenprodil, a selective GluN2B antagonist.
TCN 213 selectively blocked GluN1/GluN2A over GluN1/GluN2B NMDA receptors allowing direct dissection of functional NMDA receptors and pharmacological profiling of developmental changes in native NMDA receptor subunit composition.
PMCID: PMC3417419  PMID: 22022974
NMDA; glutamate; glycine; antagonism; oocyte; two-electrode voltage clamp; electrophysiology; neurotoxicity; development
25.  Direct pharmacological monitoring of the developmental switch in NMDA receptor subunit composition using TCN 213, a GluN2A-selective, glycine-dependent antagonist 
British Journal of Pharmacology  2012;166(3):924-937.
Developmental switches in NMDA receptor subunit expression have been inferred from studies of GluN2 expression levels, changes in kinetics of glutamatergic synaptic currents and sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists. Here we use TCN 213, a novel GluN2A-selective antagonist to identify the presence of this subunit in functional NMDA receptors in developing cortical neurones.
Two-electrode voltage-clamp (TEVC) recordings were made from Xenopus laevis oocytes to determine the pharmacological activity of TCN 213 at recombinant NMDA receptors. TCN 213 antagonism was studied in cultures of primary cortical neurones, assessing the NMDA receptor dependency of NMDA-induced excitotoxicity and monitoring developmental switches in NMDA receptor subunit composition.
TCN 213 antagonism of GluN1/GluN2A NMDA receptors was dependent on glycine but independent of glutamate concentrations in external recording solutions. Antagonism by TCN 213 was surmountable and gave a Schild plot with unity slope. TCN 213 block of GluN1/GluN2B NMDA receptor-mediated currents was negligible. In cortical neurones, at a early developmental stage predominantly expressing GluN2B-containing NMDA receptors, TCN 213 failed to antagonize NMDA receptor-mediated currents or to prevent GluN2B-dependent, NMDA-induced excitoxicity. In older cultures (DIV 14) or in neurones transfected with GluN2A subunits, TCN 213 antagonized NMDA-evoked currents. Block by TCN 213 of NMDA currents inversely correlated with block by ifenprodil, a selective GluN2B antagonist.
TCN 213 selectively blocked GluN1/GluN2A over GluN1/GluN2B NMDA receptors allowing direct dissection of functional NMDA receptors and pharmacological profiling of developmental changes in native NMDA receptor subunit composition.
PMCID: PMC3417419  PMID: 22022974
NMDA; glutamate; glycine; antagonism; oocyte; two-electrode voltage clamp; electrophysiology; neurotoxicity; development

Results 1-25 (1420288)