Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons

Long-term potentiation (LTP) of excitatory transmission in the hippocampus likely contributes to learning and memory. The mechanisms underlying LTP at these synapses are not well understood, although phosphorylation and redistribution of AMPA receptors may be responsible for this form of synaptic plasticity. We show here that miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons reliably demonstrate LTP when postsynaptic NMDA receptors are briefly stimulated with glycine. LTP of these synapses is accompanied by a rapid insertion of native AMPA receptors and by increased clustering of AMPA receptors at the surface of dendritic membranes. Both LTP and glycine-facilitated AMPA receptor insertion are blocked by intracellular tetanus toxin (TeTx), providing evidence that AMPA receptors are inserted into excitatory synapses via a SNARE-dependent exocytosis during LTP.     

Comprehensive behavioural study of GluR4 knockout mice: implication in cognitive function

Fast excitatory transmission in the mammalian central nervous system is mediated by AMPA‐type glutamate receptors. The tetrameric AMPA receptor complexes are composed of four subunits, GluR1–4. The GluR4 subunit is highly expressed in the cerebellum and the early postnatal hippocampus and is thought to be involved in synaptic plasticity and the development of functional neural circuitry through the recruitment of other AMPA receptor subunits. Previously, we reported an association of the human GluR4 gene (GRIA4) with schizophrenia. To examine the role of the GluR4 subunit in the higher brain function, we generated GluR4 knockout mice and conducted electrophysiological and behavioural analyses. The mutant mice showed normal long‐term potentiation (LTP) in the CA1 region of the hippocampus. The GluR4 knockout mice showed mildly improved spatial working memory in the T‐maze test. Although the retention of spatial reference memory was intact in the mutant mice, the acquisition of spatial reference memory was impaired in the Barnes circular maze test. The GluR4 knockout mice showed impaired prepulse inhibition. These results suggest the involvement of the GluR4 subunit in cognitive function.     

Neurabin contributes to hippocampal long-term potentiation and contextual fear memory

Neurabin is a scaffolding protein that interacts with actin and protein phosphatase-1. Highly enriched in the dendritic spine, neurabin is important for spine morphogenesis and synaptic formation. However, less is known about the role of neurabin in hippocampal plasticity and its possible effect on behavioral functions. Using neurabin knockout (KO) mice, here we studied the function of neurabin in hippocampal synaptic transmission, plasticity and behavioral memory. We demonstrated that neurabin KO mice showed a deficit in contextual fear memory but not auditory fear memory. Whole-cell patch clamp recordings in the hippocampal CA1 neurons showed that long-term potentiation (LTP) was significantly reduced, whereas long-term depression (LTD) was unaltered in neurabin KO mice. Moreover, increased AMPA receptor but not NMDA receptor-mediated synaptic transmission was found in neurabin KO mice, and is accompanied by decreased phosphorylation of GluR1 at the PKA site (Ser845) but no change at the CaMKII/PKC site (Ser831). Pre-conditioning with LTD induction rescued the following LTP in neurabin KO mice, suggesting the loss of LTP may be due to the saturated synaptic transmission. Our results indicate that neurabin regulates contextual fear memory and LTP in hippocampal CA1 pyramidal neurons.     

GRASP-1: a neuronal RasGEF associated with the AMPA receptor/GRIP complex

The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, we have identified several GRIP-associated proteins (GRASPs) that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduced the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 was rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity.     

AMPA receptor trafficking at excitatory synapses

Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.     

Temporal dynamics of NMDA receptor-induced changes in spine morphology and AMPA receptor recruitment to spines

Recent studies have shown that the activation of NMDA receptors can induce rapid changes in dendritic morphology and synaptic recruitment of AMPA receptors in dendritic spines. Here, we analyze the time course of NMDA receptor-induced changes in dendrite morphology and recruitment of AMPA receptors to synapses in cultured neurons. Activation of NMDA receptors causes a rapid transient increase in the size of preexisting spines and then the gradual formation of new dendritic protrusions and spines. NMDA receptor activation also induced GFP-tagged AMPA receptors to cluster in dendrites and to be inserted into the surface of dendritic spines. These results indicate that NMDA receptor activation induces several phases of dendritic plasticity, initial expansion of dendritic spines, followed by the de novo formation of spines and AMPA receptor dendritic clustering and surface expression on spines. Each of these forms of plasticity may have significant effects on the efficacy of synaptic transmission.     

Sex Differences in Long-Term Potentiation at Temporoammonic-CA1 Synapses: Potential Implications for Memory Consolidation

Sex differences in spatial memory have long been observed in humans, non-human primates and rodents, but the underlying cellular and molecular mechanisms responsible for these differences remain obscure. In the present study we found that adolescent male rats outperformed female rats in 7 d and 28 d retention probes, but not in learning trials and immediate probes, in the Morris water maze task. Male rats also had larger long-term potentiation (LTP) at hippocampal temproammonic-CA1 (TA-CA1) synapses, which have been implicated to play a key role in place field and memory consolidation, when protocols designed to elicit late-stage LTP (LLTP) were used. Interestingly, the ratio of evoked AMPA/NMDA currents was found to be smaller at TA-CA1 synapses in male rats compared to female rats. Protein biotinylation experiments showed that male rats expressed more surface GluN1 receptors in hippocampal CA1 stratum lacunosum-moleculare (SLM) than female rats, although GluA1 expression was also slightly higher in male rats. Taken together, our results suggest that differences in the expression of AMPA and NMDA receptors may affect LTP expression at TA-CA1 synapses in adolescent male and female rats, and thus possibly contribute to the observed sex difference in spatial memory.     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

Emotion enhances learning via norepinephrine regulation of AMPA-receptor trafficking

Emotion enhances our ability to form vivid memories of even trivial events. Norepinephrine (NE), a neuromodulator released during emotional arousal, plays a central role in the emotional regulation of memory. However, the underlying molecular mechanism remains elusive. Toward this aim, we have examined the role of NE in contextual memory formation and in the synaptic delivery of GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors during long-term potentiation (LTP), a candidate synaptic mechanism for learning. We found that NE, as well as emotional stress, induces phosphorylation of GluR1 at sites critical for its synaptic delivery. Phosphorylation at these sites is necessary and sufficient to lower the threshold for GluR1 synaptic incorporation during LTP. In behavioral experiments, NE can lower the threshold for memory formation in wild-type mice but not in mice carrying mutations in the GluR1 phosphorylation sites. Our results indicate that NE-driven phosphorylation of GluR1 facilitates the synaptic delivery of GluR1-containing AMPARs, lowering the threshold for LTP, thereby providing a molecular mechanism for how emotion enhances learning and memory.     

受体、突触和记忆

大脑中神经元突触间的信号传递是由许多神经递质受体介导的。在过去,Richard L．Huganir实验室一直致力于神经递质受体功能调节的分子机制。而最近,该实验室又聚焦到大脑中一种最主要的兴奋性受体的研究——谷氨酸受体。谷氨酸受体主要可以分为两大类：AMPA受体和NMDA受体。AMPA受体主要介导了快速的兴奋性突触传递;而NMDA受体则在神经可塑性和发育中起到重要作用。实验发现,AMPA受体和NMDA受体都可以被一系列的蛋白激酶磷酸化,而磷酸化的水平则直接影响了这些受体的功能特性,包括通道电导和受体膜定位等。AMPA受体磷酸化的水平同时还在学习和记忆的细胞模型中发生改变,如长时程增强（LTP）和长时程抑制（LTD）。此外,AMPA受体中GluR1亚单位的磷酸化对于各种形式的可塑性以及空间记忆的维持有重要的作用。实验室主要研究突触部位谷氨酸受体在亚细胞水平的定位和聚集的分子机制。最近,一系列可以直接或间接与AMPA和NMDA受体相互作用的蛋白质得以发现,其中包括一个新发现的蛋白家族GRIPs（glutamate receptor interacting proteins）。GRIPs可以直接和AMPA受体的GluR2／3亚单位的C端结合。GRIPs包含7个PDZ结构域,可以介导蛋白与蛋白直接的相互连接,从而把各个AMPA受体交互连接在一起并与其他蛋白相连。另外,GluR2亚单位的c端还可以和兴奋性突触中的蛋白激酶C结合蛋白（PICK1）的PDZ结构域相互作用。另外,GluR2亚单位的C端也可以与一种参与膜融合的蛋白NSF相互作用。这些与AMPA受体相互作用的蛋白质对于受体在膜上的运输以及定位有至关重要的作用。同时,受体与PICK1和GRIP的结合对于小脑运动学习中的LTD有重要作用。总体上说,该实验室发现了一系列可以调节神经递质受体功能的分子机制,这些工作提示受体功能的调节可能是?     

Dendritic hold and read: a gated mechanism for short term information storage and retrieval

Two contrasting theories have been proposed to explain the mechanistic basis of short term memory. One theory posits that short term memory is represented by persistent neural activity supported by reverberating feedback networks. An alternate, more recent theory posits that short term memory can be supported by feedforward networks. While feedback driven memory can be implemented by well described mechanisms of synaptic plasticity, little is known of possible molecular and cellular mechanisms that can implement feedforward driven memory. Here we report such a mechanism in which the memory trace exists in the form of glutamate-bound but Mg(2+)-blocked NMDA receptors on the thin terminal dendrites of CA1 pyramidal neurons. Because glutamate dissociates from subsets of NMDA receptors very slowly, excitatory synaptic transmission can leave a silent residual trace that outlasts the electrical activity by hundreds of milliseconds. Read-out of the memory trace is possible if a critical level of these bound-but-blocked receptors accumulates on a dendritic branch that will allow these quasi-stable receptors to sustain a regenerative depolarization when triggered by an independent gating signal. This process is referred to here as dendritic hold and read (DHR). Because the read-out of the input is not dependent on repetition of the input and information flows in a single-pass manner, DHR can potentially support a feedforward memory architecture.     

Multiple forms of long-term potentiation and multiple regulatory sites of N-methyl-D-aspartate receptors: Role of the redox site

Long-term potentiation (LTP) is a form of synaptic plasticity thought to be involved in learning and memory. Althrough extensively studied, mainly in the CA1 region of the hippocampus, the mechanisms underlying the induction and expression of LTP are poorly elucidated. This is probably due to the fact that LTP is not a unique process and indeed recent studies have shown that several forms of LTP could be generated depending on the experimental conditions. Furthermore, LTP is generally associated with a long-lasting increase of the synaptic efficacy of AMPA receptors but an increasing number of data also suggested that NMDA receptors could be potentiated as well. NMDA receptor responses are modulated by a large number of extracellular and intracellular events, providing additional possibilities for the generation of LTP. The role of these different modulatory sites of the NMDA receptor and their relation with LTP are reviewed with a particular attention to the redox site which seems to be a selective target to distinguish between AMPA and NMDA-LTP. © 1995 John Wiley & Sons, Inc.     

Ion Channel Gradients in the Apical Tuft Region of CA1 Pyramidal Neurons

Dendritic ion channels play a critical role in shaping synaptic input and are fundamentally important for synaptic integration and plasticity. In the hippocampal region CA1, somato-dendritic gradients of AMPA receptors and the hyperpolarization-activated cation conductance (I_h) counteract the effects of dendritic filtering on the amplitude, time-course, and temporal integration of distal Schaffer collateral (SC) synaptic inputs within stratum radiatum (SR). While ion channel gradients in CA1 distal apical trunk dendrites within SR have been well characterized, little is known about the patterns of ion channel expression in the distal apical tuft dendrites within stratum lacunosum moleculare (SLM) that receive distinct input from the entorhinal cortex via perforant path (PP) axons. Here, we measured local ion channels densities within these distal apical tuft dendrites to determine if the somato-dendritic gradients of I_h and AMPA receptors extend into distal tuft dendrites. We also determined the densities of voltage-gated sodium channels and NMDA receptors. We found that the densities of AMPA receptors, I_h, and voltage-gated sodium channels are similar in tuft dendrites in SLM when compared with distal apical dendrites in SR, while the ratio of NMDA receptors to AMPA receptors increases in tuft dendrites relative to distal apical dendrites within SR. These data indicate that the somato-dendritic gradients of I_h and AMPA receptors in apical dendrites do not extend into the distal tuft, and the relative densities of voltage-gated sodium channels and NMDA receptors are poised to support nonlinear integration of correlated SC and PP input.     

AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss

Beta amyloid (Abeta), a peptide generated from the amyloid precursor protein (APP) by neurons, is widely believed to underlie the pathophysiology of Alzheimer's disease. Recent studies indicate that this peptide can drive loss of surface AMPA and NMDA type glutamate receptors. We now show that Abeta employs signaling pathways of long-term depression (LTD) to drive endocytosis of synaptic AMPA receptors. Synaptic removal of AMPA receptors is necessary and sufficient to produce loss of dendritic spines and synaptic NMDA responses. Our studies indicate the central role played by AMPA receptor trafficking in Abeta-induced modification of synaptic structure and function.     

Perinatal Exposure to Bisphenol-A Impairs Spatial Memory through Upregulation of Neurexin1 and Neuroligin3 Expression in Male Mouse Brain

Bisphenol-A (BPA), a well known endocrine disruptor, impairs learning and memory in rodents. However, the underlying molecular mechanism of BPA induced impairment in learning and memory is not well known. As synaptic plasticity is the cellular basis of memory, the present study investigated the effect of perinatal exposure to BPA on the expression of synaptic proteins neurexin1 (Nrxn1) and neuroligin3 (Nlgn3), dendritic spine density and spatial memory in postnatal male mice. The pregnant mice were orally administered BPA (50 µg/kgbw/d) from gestation day (GD) 7 to postnatal day (PND) 21 and sesame oil was used as a vehicle control. In Morris water maze (MWM) test, BPA extended the escape latency time to locate the hidden platform in 8 weeks male mice. RT-PCR and Immunoblotting results showed significant upregulation of Nrxn1 and Nlgn3 expression in both cerebral cortex and hippocampus of 3 and 8 weeks male mice. This was further substantiated by in-situ hybridization and immunofluorescence techniques. BPA also significantly increased the density of dendritic spines in both regions, as analyzed by rapid Golgi staining. Thus our data suggest that perinatal exposure to BPA impairs spatial memory through upregulation of expression of synaptic proteins Nrxn1 and Nlgn3 and increased dendritic spine density in cerebral cortex and hippocampus of postnatal male mice.     

Distinct quantal features of AMPA and NMDA synaptic currents in hippocampal neurons: implication of glutamate spillover and receptor saturation

Excitatory postsynaptic currents (EPSCs) were studied in the CA1 pyramidal cells of rat hippocampal slices. Components mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and by N-methyl-D-aspartate (NMDA) receptors were separated pharmacologically. Quantal parameters of AMPA and NMDA receptor-mediated EPSCs were obtained using both maximal likelihood and autocorrelation techniques. Enhancement of transmitter release with 4-aminopyridine caused a significant increase in quantal size of NMDA EPSC. This was accompanied by a slowing of the EPSC decay. The maximal number of quanta in the NMDA current was unchanged, while the probability of quantal event dramatically enhanced. In contrast, neither the quantal size nor the kinetics of AMPA EPSC was altered by 4-aminopyridine, while the maximal number of quanta increased. These changes in the quantal parameters are consistent with a transition to multivesicular release of the neurotransmitter. Spillover of excessive glutamate on the nonsynaptic areas of dendritic spines causes an increase in the quantal size of NMDA synaptic current. The difference in quantal behavior of AMPA and NMDA EPSCs implies that different mechanisms underlie their quantization: the additive response of nonsaturated AMPA receptors contrasts with the variable involvement of saturated intrasynaptic and nonsaturated extrasynaptic NMDA receptors.     

Roles of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling pathway in activity-dependent dendritic protein synthesis in hippocampal neurons

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by alphaCaMKII 5' and 3' untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous alphaCaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.     

Kainate receptors and the induction of mossy fibre long-term potentiation

There is intense interest in understanding the molecular mechanisms involved in long-term potentiation (LTP) in the hippocampus. Significant progress in our understanding of LTP has followed from studies of glutamate receptors, of which there are four main subtypes (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), mGlu and kainate). This article summarizes the evidence that the kainate subtype of glutamate receptor is an important trigger for the induction of LTP at mossy fibre synapses in the CA3 region of the hippocampus. The pharmacology of the first selective kainate receptor antagonists, in particular the GLU(K5) subunit selective antagonist LY382884, is described. LY382884 selectively blocks the induction of mossy fibre LTP, in response to a variety of different high-frequency stimulation protocols. This antagonist also inhibits the pronounced synaptic facilitation of mossy fibre transmission that occurs during high-frequency stimulation. These effects are attributed to the presence of presynaptic GLU(K5)-subunit-containing kainate receptors at mossy fibre synapses. Differences in kainate receptor-dependent synaptic facilitation of AMPA and NMDA receptor-mediated synaptic transmission are described. These data are discussed in the context of earlier reports that glutamate receptors are not involved in mossy fibre LTP and more recent experiments using kainate receptor knockout mice, that argue for the involvement of GLU(K6) but not GLU(K5) kainate receptor subunits. We conclude that activation of presynaptic GLU(K5)-containing kainate receptors is an important trigger for the induction of mossy fibre LTP in the hippocampus.     

A computer model of unitary responses from associational/commissural and perforant path synapses in hippocampal CA3 pyramidal cells

Despite the central position of CA3 pyramidal cells in the hippocampal circuit, the experimental investigation of their synaptic properties has been limited. Recent slice experiments from adult rats characterized AMPA and NMDA receptor unitary synaptic responses in CA3b pyramidal cells. Here, excitatory synaptic activation is modeled to infer biophysical parameters, aid analysis interpretation, explore mechanisms, and formulate predictions by contrasting simulated somatic recordings with experimental data. Reconstructed CA3b pyramidal cells from the public repository NeuroMorpho.Org were used to allow for cell-specific morphological variation. For each cell, synaptic responses were simulated for perforant pathway and associational/commissural synapses. Means and variability for peak amplitude, time-to-peak, and half-height width in these responses were compared with equivalent statistics from experimental recordings. Synaptic responses mediated by AMPA receptors are best fit with properties typical of previously characterized glutamatergic receptors where perforant path synapses have conductances twice that of associational/commissural synapses (0.9 vs. 0.5 nS) and more rapid peak times (1.0 vs. 3.3 ms). Reanalysis of passive-cell experimental traces using the model shows no evidence of a CA1-like increase of associational/commissural AMPA receptor conductance with increasing distance from the soma. Synaptic responses mediated by NMDA receptors are best fit with rapid kinetics, suggestive of NR2A subunits as expected in mature animals. Predictions were made for passive-cell current clamp recordings, combined AMPA and NMDA receptor responses, and local dendritic depolarization in response to unitary stimulations. Models of synaptic responses in active cells suggest altered axial resistivity and the presence of synaptically activated potassium channels in spines.     

Rapid and Transient Learning-Associated Increase in NMDA NR1 Subunit in the Rat Hippocampus

Several lines of evidence indicate that glutamate NMDA receptors are critically involved in long-term potentiation (LTP) and in certain forms of learning. It was previously demonstrated that memory formation of an inhibitory avoidance task in chick is specifically associated with an increase in the density of NMDA receptor in selected brain regions. Here we report on the effect of a one trial inhibitory avoidance training in rats, a hippocampal-dependent learning task, on the levels of different subunits of the glutamate NMDA receptor in synaptic plasma membranes (SPM) isolated from the hippocampus. Training rats on a one trial inhibitory avoidance task results in a rapid, transient and selective increase (+33 %, p < 0.05) in NMDA NR1 subunit expression in hippocampal SPM of rats sacrificed 30 min posttraining. No changes were observed at 0 or 120 min after training or in shocked animals in comparison to naive control rats. In addition, no training-associated increase in the levels of NMDA NR2A and NR2B or AMPA GluR 2/3 subunits was observed at any timepoint tested. In conclusion, the present findings support the hypothesis that alterations in expression of synaptic NMDA NR1 subunits in the hippocampus are specifically associated with memory formation of an inhibitory avoidance task and strongly suggest that hippocampal NMDA receptors are crucially involved in the neural mechanisms underlying certain forms of learning.These authors contributed equally to this work