Protein kinase C promotes N-methyl-D-aspartate (NMDA) receptor trafficking by indirectly triggering calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and long-term potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.     

Activation of metabotropic glutamate receptors in conjunction with postsynaptic depolarization triggers a long-term depression of the N-methyl-D-aspartate receptor-mediated synaptic potential in the rat hippocampus

The mechanism responsible for long-term depression (LTD) of pharmacologically isolated N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic potential (EPSP_NMDA) was studied. Intracellular recordings were made from CA1 cells of rat hippocampal slices in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (10 µM) and picrotoxin (50 µM), which block non-NMDA and GABA_A receptors, respectively. Intracellular injections of depolarizing pulses (500 ms, 0.3–0.7 nA) at 1 Hz for 5 min in the absence of synaptic stimulation caused a persistent increase in the amplitude of EPSP_NMDA. However, coupling postsynaptic depolarization with synaptic activity induced LTD. The EPSP_NMDA LTD could be blocked byL-2-amino-3-phosphonopropionic acid (50 µM) or (RS)--methyl-4-carboxyphenylglycine (200 µM), specific antagonists for metabotropic glutamate receptors (mGluR). Furthermore, application oftrans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 50 µM), a specific mGluR agonist, in conjunction with postsynaptic depolarizing elicited LTD. In contrast, the mGluR agonists quisqualate or t-ACPD when given alone produced a sustained enhancement of EPSP_NMDA. Finally, coupled depolarization did not evoke LTD in slices pretreated with the protein kinase C (PKC) inhibitor calphostin c (60 nM). The present results demonstrate that activation of mGluR is necessary for the induction of LTD of EPSP_NMDA and suggest that NMDA receptors are subject to bidirectional regulation by mGluR. Furthermore, the induction of LTD is likely to involve the stimulation of PKC.     

Bidirectional plasticity gated by hyperpolarization controls the gain of postsynaptic firing responses at central vestibular nerve synapses

Linking synaptic plasticity with behavioral learning requires understanding how synaptic efficacy influences postsynaptic firing in neurons whose role in behavior is understood. Here, we examine plasticity at a candidate site of motor learning: vestibular nerve synapses onto neurons that mediate reflexive movements. Pairing nerve activity with changes in postsynaptic voltage induced bidirectional synaptic plasticity in vestibular nucleus projection neurons: long-term potentiation relied on calcium-permeable AMPA receptors and postsynaptic hyperpolarization, whereas long-term depression relied on NMDA receptors and postsynaptic depolarization. Remarkably, both forms of plasticity uniformly scaled synaptic currents evoked by pulse trains, and these changes in synaptic efficacy were translated into linear increases or decreases in postsynaptic firing responses. Synapses onto local inhibitory neurons were also plastic but expressed only long-term depression. Bidirectional, linear gain control of vestibular nerve synapses onto projection neurons provides a plausible mechanism for motor learning underlying adaptation of vestibular reflexes.     

Zinc reverses glycine-dependent inactivation of NMDARs in cultured rat hippocampal neurons

In the presence of glutamate and co-agonists, e.g., glycine, the N-methyl-D-aspartate receptor (NMDAR) plays an important role in physiological and pathophysiological brain processes. Previous studies indicate glycine could inhibit NMDAR responses induced by high concentration of NMDA in hippocampal neurons. The mechanism underlying this inhibitory impact, however, has been unclear. In this study, the whole-cell patch-clamp recording and Ca²⁺ imaging with Fluo-3/AM under laser scanning confocal microscope were used to analyze the possible involvement of NMDAR subunits in this effect. We found that the peak current of NMDARs and Ca²⁺ influx induced by high concentration of NMDA were reduced by treatment of glycine (0.03?C10 ??mol L^?1) in a dose-dependent manner, and that the glycine-dependent inhibition of NMDAR responses, which were induced at 300 ??mol L^?1 NMDA, was reversed by ZnCl₂ through the blocking of the NR2A subunit of NMDARs, but was less influenced by ifenprodil, a NR2B inhibitor. Our results suggest that the glycine-dependent inactivation of NMDARs is potentially modulated by the regulatory subunit NR2A.     

CaMKII translocation requires local NMDA receptor-mediated Ca2+ signaling

Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca(2+) affecting several downstream signaling pathways that involve Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca(2+) responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIalpha translocation was observed. In cells solely expressing Ca(2+)-impermeable NMDARs containing NR1(N598R)-mutant subunits, prolonged NMDA application elevated internal Ca(2+) to the same degree as in wild-type controls, yet failed to translocate CaMKIIalpha. Brief local NMDA application evoked smaller Ca(2+) transients in dendritic spines of mutant compared to wild-type cells. CaMKIIalpha mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1(N598R) cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca(2+) entry directly through NMDARs, rather than other Ca(2+) sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event.     

D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period

N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC) at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP) at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.     

Differential Regulation of GABAB Receptor Trafficking by Different Modes of N-methyl-d-aspartate (NMDA) Receptor Signaling

Inhibitory GABA_B receptors (GABA_BRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABA_BRs are heterodimers of GABA_B1 and GABA_B2 subunits and here we show that the trafficking and surface expression of GABA_BRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABA_BR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABA_BRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABA_B1 but decreases GABA_B2 surface expression. The increase in surface GABA_B1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABA_B2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABA_BR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival.     

Regulation of Long-Term Plasticity Induction by the Channel and C-Terminal Domains of GluN2 Subunits

Conventional long-term potentiation (LTP) and long-term depression (LTD) are induced by different patterns of synaptic stimulation, but both forms of synaptic modification require calcium influx through NMDA receptors (NMDARs). A prevailing model (the “calcium hypothesis”) suggests that high postsynaptic calcium elevation results in LTP, whereas moderate elevations give rise to LTD. Recently, additional evidence has come to suggest that differential activation of NMDAR subunits also factors in determining which type of plasticity is induced. While the growing amount of data suggest that activation of NMDARs containing specific GluN2 subunits plays an important role in the induction of plasticity, it remains less clear which subunit is tied to which form of plasticity. Additionally, it remains to be determined which properties of the subunits confer upon them the ability to differentially induce long-term plasticity. This review highlights recent studies suggesting differential roles for the subunits, as well as findings that begin to shed light on how two similar subunits may be linked to the induction of opposing forms of plasticity.     

Control of excitatory synaptic transmission by C-terminal Src kinase

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca(2+), followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca(2+) into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus.     

Glutamate-mediated extrasynaptic inhibition: direct coupling of NMDA receptors to Ca(2+)-activated K+ channels

NMDA receptors (NMDARs) typically contribute to excitatory synaptic transmission in the CNS. While Ca(2+) influx through NMDARs plays a critical role in synaptic plasticity, direct actions of NMDAR-mediated Ca(2+) influx on neuronal excitability have not been well established. Here we show that Ca(2+) influx through NMDARs is directly coupled to activation of BK-type Ca(2+)-activated K+ channels in outside-out membrane patches from rat olfactory bulb granule cells. Repetitive stimulation of glutamatergic synapses in olfactory bulb slices evokes a slow inhibitory postsynaptic current (IPSC) in granule cells that requires both NMDARs and BK channels. The slow IPSC is enhanced by glutamate uptake blockers, suggesting that extrasynaptic NMDARs underlie the response. These findings reveal a novel inhibitory action of extrasynaptic NMDARs in the brain.     

Allosteric Regulation of the N-Methyl-d-Aspartate Receptor-Linked Ion Channel Complex and Effects of Ethanol in Ethanol-Withdrawal Seizure-Prone and -Resistant Mice

Abstract: The effects of ethanol, glycine, and spermidine on the specific binding of [³H]MK-801 were characterized in Triton-treated membranes prepared from the hippocampus and cortex of ethanol-withdrawal seizure-prone (WSP) and -resistant (WSR) mice. Glycine, an allosteric agonist at the NMDA receptor-linked ion channel complex, caused an increase in specific [³H]MK-801 binding to hippocampal membrane preparations. There were no significant differences in EC₅₀ values between the selected lines for the effect of glycine (WSP, 391.7 ± 48.4 nM; WSR, 313.4 ± 77 nM) in the presence of 10 µM NMDA or in the maximal response to the agonist (WSP, 1.75 ± 0.26 pmol/mg of protein; WSR, 1.67 ± 0.22 pmol/mg of protein). The EC₅₀ values for the spermidine-induced increase in [³H]MK-801 binding in membranes from hippocampus in the absence (WSP, 11.7 ± 0.83 µM; WSR, 9.98 ± 1.29 µM) or in the presence of 10 µM glycine and 10 µM NMDA (WSP, 2.1 ± 0.35 µM; WSR, 2.37 ± 0.42 µM) also did not differ. Similar results were obtained in cortical membranes. Saturation isotherms indicated that there was no difference in the density of [³H]MK-801 binding sites, or in their affinity for the radioligand, between the mouse lines. In addition, administration of ethanol by inhalation (24 h) to WSP and WSR mice did not cause an increase in the density of [³H]MK-801 binding sites, and there was no difference in the density or affinity of binding sites between the mouse lines. Withdrawal from ethanol (6 h), which causes an increase in the severity of handling-induced convulsions in WSP mice, also did not alter the binding site density or affinity for radioligand. The results suggest that the characteristics of the NMDA receptor-linked ion channel complex in the tissue preparations described here do not differ in WSP and WSR mice. Thus, genetic differences in seizure susceptibility during ethanol withdrawal can be dissociated from the total density of hippocampal or cortex NMDA receptors under activating conditions.     

Single-cell optogenetic excitation drives homeostatic synaptic depression

Homeostatic processes have been proposed to explain the discrepancy between the dynamics of synaptic plasticity and the stability of brain function. Forms of synaptic plasticity such as long-term potentiation alter synaptic activity in a synapse- and cell-specific fashion. Although network-wide excitation triggers compensatory homeostatic changes, it is unknown whether neurons initiate homeostatic synaptic changes in response to cell-autonomous increases in excitation. Here we employ optogenetic tools to cell-autonomously excite CA1 pyramidal neurons and find that a compensatory postsynaptic depression of both AMPAR and NMDAR function results. Elevated calcium influx through L-type calcium channels leads to activation of a pathway involving CaM kinase kinase and CaM kinase 4 that induces synaptic depression of AMPAR and NMDAR responses. The synaptic depression of AMPARs but not of NMDARs requires protein synthesis and the GluA2 AMPAR subunit, indicating that downstream of CaM kinase activation divergent pathways regulate homeostatic AMPAR and NMDAR depression.     

Bidirectional changes in spatial dendritic integration accompanying long-term synaptic modifications

Information processing in the neuron requires spatial summation of synaptic inputs at the dendrite. In CA1 pyramidal neurons of the hippocampus, a brief period of correlated pre- and postsynaptic activity, which induces long-term potentiation (LTP) or long-term depression (LTD), results in a persistent increase or decrease in the linearity of spatial summation, respectively. Such bidirectional modification of the summation property is specific to the modified input and reflects localized dendritic changes involving I(h) channels and NMDA receptors. Thus, correlated pre- and postsynaptic activity alters not only the strength of the activated input but also its dendritic integration with other inputs.     

Neocortical LTD via coincident activation of presynaptic NMDA and cannabinoid receptors

There is a consensus that NMDA receptors (NMDARs) detect coincident pre- and postsynaptic activity during induction of long-term potentiation (LTP), but their role in timing-dependent long-term depression (tLTD) is unclear. We examine tLTD in neocortical layer 5 (L5) pyramidal pairs and find that tLTD is expressed presynaptically, implying retrograde signaling. CB1 agonists produce depression that mimics and occludes tLTD. This agonist-induced LTD requires presynaptic activity and NMDAR activation, but not postsynaptic Ca(2+) influx. Further experiments demonstrate the existence of presynaptic NMDARs that underlie the presynaptic activity dependence. Finally, manipulating cannabinoid breakdown alters the temporal window for tLTD. In conclusion, tLTD requires simultaneous activation of presynaptic NMDA and CB1 receptors. This novel form of coincidence detection may explain the temporal window of tLTD and may also impart synapse specificity to cannabinoid retrograde signaling.     

NMDA Receptor Activation: Two Targets for Two Co-Agonists

Neuronal N-methyl-d-aspartate receptors (NMDARs) play a critical role in synaptic plasticity. Their activation requires not only binding of their ligand glutamate and membrane depolarization but also the presence of a co-agonist, glycine or d-serine. An increasing body of experimental evidence suggests that different populations of NMDARs could be gated by different co-agonists. Here we discuss how the spatial distribution of co-agonist sources and uptake mechanisms, together with diffusional properties of the synaptic environment, could shape NMDAR co-agonist supply and therefore NMDAR-dependent plasticity.     

A Glycine Site Associated with N-Methyl-d-Aspartic Acid Receptors: Characterization and Identification of a New Class of Antagonists

Membranes from rat telencephalon contain a single class of strychnine-insensitive glycine sites. That these sites are associated with N-methyl-D-aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate-type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.     

Modulation of synaptic plasticity by stress hormone associates with plastic alteration of synaptic NMDA receptor in the adult hippocampus

Stress exerts a profound impact on learning and memory, in part, through the actions of adrenal corticosterone (CORT) on synaptic plasticity, a cellular model of learning and memory. Increasing findings suggest that CORT exerts its impact on synaptic plasticity by altering the functional properties of glutamate receptors, which include changes in the motility and function of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype of glutamate receptor (AMPAR) that are responsible for the expression of synaptic plasticity. Here we provide evidence that CORT could also regulate synaptic plasticity by modulating the function of synaptic N-methyl-D-aspartate receptors (NMDARs), which mediate the induction of synaptic plasticity. We found that stress level CORT applied to adult rat hippocampal slices potentiated evoked NMDAR-mediated synaptic responses within 30 min. Surprisingly, following this fast-onset change, we observed a slow-onset (>1 hour after termination of CORT exposure) increase in synaptic expression of GluN2A-containing NMDARs. To investigate the consequences of the distinct fast- and slow-onset modulation of NMDARs for synaptic plasticity, we examined the formation of long-term potentiation (LTP) and long-term depression (LTD) within relevant time windows. Paralleling the increased NMDAR function, both LTP and LTD were facilitated during CORT treatment. However, 1-2 hours after CORT treatment when synaptic expression of GluN2A-containing NMDARs is increased, bidirectional plasticity was no longer facilitated. Our findings reveal the remarkable plasticity of NMDARs in the adult hippocampus in response to CORT. CORT-mediated slow-onset increase in GluN2A in hippocampal synapses could be a homeostatic mechanism to normalize synaptic plasticity following fast-onset stress-induced facilitation.     

Novel presynaptic mechanisms for coincidence detection in synaptic plasticity

Long-term plasticity typically relies on postsynaptic NMDA receptors to detect the coincidence of pre- and postsynaptic activity. Recent studies, however, have revealed forms of plasticity that depend on coincidence detection by presynaptic NMDA receptors. In the amygdala, cortical afferent associative presynaptic long-term potentiation (LTP) requires activation of presynaptic NMDA receptors by simultaneous thalamic and cortical afferents. Surprisingly, both types of afferent can also undergo postsynaptically induced NMDA-receptor-dependent LTP. In the neocortex, spike-timing-dependent long-term depression (LTD) requires simultaneous activation of presynaptic NMDA autoreceptors and retrograde signalling by endocannabinoids. In cerebellar LTD, presynaptic NMDA receptor activation suggests that similar presynaptic mechanisms may exist. Recent studies also indicate the existence of presynaptic coincidence detection that is independent of NMDA receptors, suggesting that such mechanisms have a widespread role in plasticity.     

Modulation of NMDA receptors by glycine — introduction to some basic aspects and recent developments

Summary Glycine is a co-agonist at NMDA receptors and it's presence is a prerequisite for channel activation by glutamate or NMDA. Physiological concentrations reduce one form of NMDA receptor-desensitization. Interactions between the glycine_B site and other domains of the NMDA receptor are complex and include the glutamate, Mg⁺ and polyamines sites. Glycine shows different affinities at various NMDA receptor subtypes probably via to allosteric interactions between NMDA2 subunits and the glycine recognition site on the NMDAR1 subunit. There is still some debate whether the glycine_B site is saturatedin vivo but it seems likely that this depends on regional differences in receptor subtype expression, local glycine or D-serene concentrations and the expression of specific glycine transporters.Glycine_B antagonists and partial agonists have been reported to have good therapeutic indices as neuroprotective agents against focal ischaemia and trauma, anti-epileptics, anxiolytics, anti-psychotomimetics and in models of chronic pain. They clearly lack two potentially serious side effects classically associated with NMDA receptor blockade, namely neurodegenerative changes in the cingulate/retrosplenial cortex and psychotomimetic-like effects. This improved therapeutic profile may be partially due to the ability of full glycine_B antagonists to reveal Gycne-sensitive desensitization and possibly also via functional and/or regional NMDA receptor subtype selectivity.     

Preservation of Redox, Polyamine, and Glycine Domains of the N-Methyl-D-Aspartate in Alzheimer's Disease

Abstract: This study used [³H] dizocilpine ([³H] MK-801) binding to the N-methyl-D-aspartate (NMDA) receptor to examine redox, polyamine, and glycine modulatory sites in membranes derived from the superior frontal and the superior temporal cortex of patients with Alzheimer's disease. In control subjects the competitive polyamine site antagonist arcaine inhibited [³H] dizocilpine binding in a dose-dependent fashion and this curve was shifted to the right by the addition of 50 μM spermidine. Arcaine inhibition of binding was more potent in the temporal cortex than in the frontal cortex, in both the absence and presence of 50 μMspermidine. In Alzheimer's disease, arcaine inhibition of [³H] dizocilpine binding (in both the absence and the presence of spermidine) was not different from control in either of the two brain areas examined. The sulfhydryl redox site of the NMDA receptor was assessed using the oxidizing agent 5, 5′-dithio-bis(2-nitrobenzoic acid), which inhibited binding in a dose-dependent fashion. This inhibition was similar in patients with Alzheimer's disease and control subjects. Glycine-stimulated [³H] dizocilpine binding was also unaffected in patients with Alzheimer's disease. However, in the temporal cortex there was a significant age-associated decline in [³H] dizocilpine binding in the presence of 100 μM glutamate (R₈=-0.71) and 100 μM glutamate plus 30 μM glycine (R₈=?0.90). There was also an age-related increase in arcaine IC₅₀ (which reflects an age-related decrease in arcaine affinity) in the frontal cortex, determined both in the absence (R₈= 0.83) and the presence (R₈= 0.79) of spermidine. These data indicate that the NMDA receptor and its modulatory redox, polyamine, and glycine subsites are intact in patients with Alzheimer's disease and that the modulatory activity of polyamine and glycine sites decline with aging.