Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory

Plasticity of the nervous system is dependent on mechanisms that regulate the strength of synaptic transmission. Excitatory synapses in the brain undergo long-term potentiation (LTP) and long-term depression (LTD), cellular models of learning and memory. Protein phosphorylation is required for the induction of many forms of synaptic plasticity, including LTP and LTD. However, the critical kinase substrates that mediate plasticity have not been identified. We previously reported that phosphorylation of the GluR1 subunit of AMPA receptors, which mediate rapid excitatory transmission in the brain, is modulated during LTP and LTD. To test if GluR1 phosphorylation is necessary for plasticity and learning and memory, we generated mice with knockin mutations in the GluR1 phosphorylation sites. The phosphomutant mice show deficits in LTD and LTP and have memory defects in spatial learning tasks. These results demonstrate that phosphorylation of GluR1 is critical for LTD and LTP expression and the retention of memories.     

Time-dependent postsynaptic AMPA GluR1 receptor recruitment in the cingulate synaptic potentiation

The anterior cingulate cortex (ACC) is critical for brain functions including learning, memory, fear and pain. Long-term synaptic potentiation (LTP), a cellular model for learning and memory, has been reported in the ACC neurons. Unlike LTP in the hippocampus and amygdala, two key structures for memory and fear, little is known about the synaptic mechanism for the expression of LTP in the ACC. Here we use whole-cell patch clamp recordings to demonstrate that cingulate LTP requires the functional recruitment of GluR1 AMPA receptors; and such events are rapid and completed within 5-10 min after LTP induction. Our results demonstrate that the GluR1 subunit is essential for synaptic plasticity in the ACC and may play critical roles under physiological and pathological conditions.     

Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins

Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. Recently, it was shown that AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ domain-containing proteins GRIP/ABP and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuated LTD induction by glutamate/depolarization pairing, as did antibodies directed against the PDZ domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ domain-containing proteins.     

Synaptic transmission and plasticity in the absence of AMPA glutamate receptor GluR2 and GluR3

The AMPA glutamate receptor (AMPAR) subunits GluR2 and GluR3 are thought to be important for synaptic targeting/stabilization of AMPARs and the expression of hippocampal long-term depression (LTD). In order to address this hypothesis genetically, we generated and analyzed knockout mice deficient in the expression of both GluR2 and GluR3. We show here that the double knockout mice are severely impaired in basal synaptic transmission, demonstrating that GluR2/3 are essential to maintain adequate synaptic transmission in vivo. However, these mutant mice are competent in establishing several forms of long-lasting synaptic changes in the CA1 region of the hippocampus, including LTD, long-term potentiation (LTP), depotentiation, and dedepression, indicating the presence of GluR2/3-independent mechanisms of LTD expression and suggesting that AMPA receptor GluR1 alone is capable of various forms of synaptic plasticity.     

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.     

Tyrosine phosphorylation of GluR2 is required for insulin-stimulated AMPA receptor endocytosis and LTD

The alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors is subject to functionally distinct constitutive and regulated clathrin-dependent endocytosis, contributing to various forms of synaptic plasticity. In HEK293 cells transiently expressing GluR1 or GluR2 mutants containing domain deletions or point mutations in their intracellular carboxyl termini (CT), we found that deletion of the first 10 amino acids (834-843) selectively reduced the rate of constitutive AMPA receptor endocytosis, whereas truncation of the last 15 amino acids of the GluR2 CT, or point mutation of the tyrosine residues in this region, only eliminated the regulated (insulin-stimulated) endocytosis. Moreover, in hippocampal slices, both insulin treatment and low-frequency stimulation (LFS) specifically stimulated tyrosine phosphorylation of the GluR2 subunits of native AMPA receptors, and the enhanced phosphorylation appears necessary for both insulin- and LFS-induced long-term depression of AMPA receptor-mediated excitatory postsynaptic currents. Thus, our results demonstrate that constitutive and regulated AMPA receptor endocytosis requires different sequences within GluR CTs and tyrosine phosphorylation of GluR2 CT is required for the regulated AMPA receptor endocytosis and hence the expression of certain forms of synaptic plasticity.     

Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro

The acute hippocampal slice preparation has been widely used to study the cellular mechanisms underlying activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although protein phosphorylation has a key role in LTP and LTD, little is known about how protein phosphorylation might be altered in hippocampal slices maintained in vitro. To begin to address this issue, we examined the effects of slicing and in vitro maintenance on phosphorylation of six proteins involved in LTP and/or LTD. We found that AMPA receptor (AMPAR) glutamate receptor 1 (GluR1) subunits are persistently dephosphorylated in slices maintained in vitro for up to 8 h. alpha calcium/calmodulin-dependent kinase II (alphaCamKII) was also strongly dephosphorylated during the first 3 h in vitro but thereafter recovered to near control levels. In contrast, phosphorylation of the extracellular signal-regulated kinase ERK2, the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2), and Src family kinases was significantly, but transiently, increased. Electrophysiological experiments revealed that the induction of LTD by low-frequency synaptic stimulation was sensitive to time in vitro. These findings indicate that phosphorylation of proteins involved in N-methyl-D-aspartate (NMDA) receptor-dependent forms of synaptic plasticity is altered in hippocampal slices and suggest that some of these changes can significantly influence the induction of LTD.     

Age-dependent requirement of AKAP150-anchored PKA and GluR2-lacking AMPA receptors in LTP

Association of PKA with the AMPA receptor GluR1 subunit via the A kinase anchor protein AKAP150 is crucial for GluR1 phosphorylation. Mutating the AKAP150 gene to specifically prevent PKA binding reduced PKA within postsynaptic densities (>70%). It abolished hippocampal LTP in 7-12 but not 4-week-old mice. Inhibitors of PKA and of GluR2-lacking AMPA receptors blocked single tetanus LTP in hippocampal slices of 8 but not 4-week-old WT mice. Inhibitors of GluR2-lacking AMPA receptors also prevented LTP in 2 but not 3-week-old mice. Other studies demonstrate that GluR1 homomeric AMPA receptors are the main GluR2-lacking AMPA receptors in adult hippocampus and require PKA for their functional postsynaptic expression during potentiation. AKAP150-anchored PKA might thus critically contribute to LTP in adult hippocampus in part by phosphorylating GluR1 to foster postsynaptic accumulation of homomeric GluR1 AMPA receptors during initial LTP in 8-week-old mice.     

Disruption of AMPA receptor GluR2 clusters following long-term depression induction in cerebellar Purkinje neurons

Cerebellar long-term depression (LTD) is thought to play an important role in certain types of motor learning. However, the molecular mechanisms underlying this event have not been clarified. Here, using cultured Purkinje cells, we show that stimulations inducing cerebellar LTD cause phosphorylation of Ser880 in the intracellular C-terminal domain of the AMPA receptor subunit GluR2. This phosphorylation is accompanied by both a reduction in the affinity of GluR2 to glutamate receptor interacting protein (GRIP), a molecule known to be critical for AMPA receptor clustering, and a significant disruption of postsynaptic GluR2 clusters. Moreover, GluR2 protein released from GRIP is shown to be internalized. These results suggest that the dissociation of postsynaptic GluR2 clusters and subsequent internalization of the receptor protein, initiated by the phosphorylation of Ser880, are the mechanisms underlying the induction of cerebellar LTD.     

Unified postsynaptic mechanism of plasticity in the striatum, neocortex, hippocampus, and cerebellum

The unitary postsynaptic mechanism of plasticity in striatum, neocortex, hippocampus and cerebellum involves the LTP/LTD excitation as result of AMPA and NMDA receptor phosphorylation/dephosphorylation, while the LTP/LTD of inhibition is the result of the GABA receptor phosphorylation/dephosphorylation. It follows from this mechanism that when NMDA channels are closed, the determinant role in receptor phosphorylation is played by the PKG. When the NMDA channels are open, the determinant role in receptor phosphorylation is played by the PKC and CaMKII.     

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?     

Reversal of aging‐related emotional memory deficits by norepinephrine via regulating the stability of surface AMPA receptors

Aging‐related emotional memory deficit is a well‐known complication in Alzheimer's disease and normal aging. However, little is known about its molecular mechanism. To address this issue, we examined the role of norepinephrine (NE) and its relevant drug desipramine in the regulation of hippocampal long‐term potentiation (LTP), surface expression of AMPA receptor, and associative fear memory in rats. We found that there was a defective regulation of NE content and AMPA receptor trafficking during fear conditioning, which were accompanied by impaired emotional memory and LTP in aged rats. Furthermore, we also found that the exogenous upregulation of NE ameliorated the impairment of LTP and emotional memory via enhancing AMPA receptor trafficking in aged rats, and the downregulation of NE impaired LTP in adult rats. Finally, acute treatment with NE or desipramine rescued the impaired emotional memory in aged rats. These results imply a pivotal role for NE in synaptic plasticity and associative fear memory in aging rats and suggest that desipramine is a potential candidate for treating aging‐related emotional memory deficit.     

Targeted in vivo mutations of the AMPA receptor subunit GluR2 and its interacting protein PICK1 eliminate cerebellar long-term depression

Cerebellar long-term depression (LTD) is a major form of synaptic plasticity that is thought to be critical for certain types of motor learning. Phosphorylation of the AMPA receptor subunit GluR2 on serine-880 as well as interaction of GluR2 with PICK1 have been suggested to contribute to the endocytic removal of postsynaptic AMPA receptors during LTD. Here, we show that targeted mutation of PICK1, the GluR2 C-terminal PDZ ligand, or the GluR2 PKC phosphorylation site eliminates cerebellar LTD in mice. LTD can be rescued in cerebellar cultures from mice lacking PICK1 by transfection of wild-type PICK1 but not by a PDZ mutant or a BAR domain mutant deficient in lipid binding, indicating the importance of these domains in PICK1 function. These results demonstrate that PICK1-GluR2 PDZ-based interactions and GluR2 phosphorylation are required for LTD expression in the cerebellum.     

Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD

Proteins that bind to the cytoplasmic tails of AMPA receptors control receptor trafficking and thus the strength of postsynaptic responses. Here we show that AP2, a clathrin adaptor complex important for endocytosis, associates with a region of GluR2 that overlaps the NSF binding site. Peptides used previously to interfere with NSF binding also antagonize GluR2-AP2 interaction. Using GluR2 mutants and peptide variants that dissociate NSF and AP2 interaction, we find that AP2 is involved specifically in NMDA receptor-induced (but not ligand-dependent) internalization of AMPA receptors, and is essential for hippocampal long-term depression (LTD). NSF function, on the other hand, is needed to maintain synaptic AMPA receptor responses, but is not directly required for NMDA receptor-mediated internalization and LTD.     

Acute BDNF Treatment Upregulates GluR1-SAP97 and GluR2-GRIP1 Interactions: Implications for Sustained AMPA Receptor Expression

Brain-derived neurotrophic factor (BDNF) plays several prominent roles in synaptic plasticity and in learning and memory formation. Reduced BDNF levels and altered BDNF signaling have been reported in several brain diseases and behavioral disorders, which also exhibit reduced levels of AMPAr subunits. BDNF treatment acutely regulates AMPA receptor expression and function, including synaptic AMPAr subunit trafficking, and implicates several well defined signaling molecules that are required to elicit long term potentiation and depression (LTP and LTD, respectively). Long term encoding of synaptic events, as in long term memory formation, requires AMPAr stabilization and maintenance. However, factors regulating AMPAr stabilization in neuronal cell membranes and synaptic sites are not well characterized. In this study, we examine the effects of acute BDNF treatment on levels of AMPAr-associated scaffolding proteins and on AMPAr subunit-scaffolding protein interactions. We also examine the effects of BDNF-dependent enhanced interactions between AMPAr subunits with their specific scaffolding proteins on the accumulation of both types of proteins. Our results show that acute BDNF treatment upregulates the interactions between AMPAr subunits (GluR1 and GluR2) with their scaffold proteins SAP97 and GRIP1, respectively, leading to prolonged increased accumulation of both categories of proteins, albeit with distinct mechanisms for GluR1 and GluR2. Our findings reveal a new role for BDNF in the long term maintenance of AMPA receptor subunits and associated scaffolding proteins at synapses and further support the role of BDNF as a key regulator of synaptic consolidation. These results have potential implications for recent findings implicating BDNF and AMPAr subunits in various brain diseases and behavioral disorders.     

Regulation of the Phosphorylation State of the AMPA Receptor GluR1 Subunit in the Postsynaptic Density

1. Changes in the phosphorylation state of AMPA-type glutamate receptors are thought to underlie activity-dependent synaptic modification. It has been established that the GluR1 subunit is phosphorylated on two distinct sites, Ser-831 and Ser-845, by CaMKII and by PKA, respectively, and that phosphorylation by either kinase correlates with an increase in the AMPA receptor-mediated current. GluR1 is concentrated in postsynaptic densities and it is expected that this particular receptor pool is involved in synaptic modification. The present study describes the regulation of the phosphorylation state of GluR1 in isolated postsynaptic densities.2. Addition of Ca²⁺/calmodulin to the postsynaptic density fraction promotes phosphorylation of GluR1, and under these conditions, dephosphorylation is prevented by the inclusion of phosphatase type 1 inhibitors, microcystin-LR and Inhibitor-1. CaMKII and phosphatase type 1 are also found to be enriched in the PSD fraction compared to the parent fractions.3. On the other hand, the addition of cAMP, either by itself or with exogenous PKA, does not change the phosphorylation state of GluR1. Prior incubation of PSDs under dephosphorylating conditions results in only a small PKA-mediated phosphorylation of GluR1.4. These results support the hypothesis that PSDs contain the molecular machinery to promote the phosphorylation as well as the dephosphorylation of GluR1 on Ser-831, while Ser-845, the site phosphorylated by PKA, appears to be mostly occluded. Thus, it is possible that a large pool of PSD-associated GluR1 is regulated through modification of the phosphorylation state of the Ser-831 site only.     

Synaptic incorporation of AMPA receptors during LTP is controlled by a PKC phosphorylation site on GluR1

Incorporation of GluR1-containing AMPA receptors into synapses is essential to several forms of neural plasticity, including long-term potentiation (LTP). Numerous signaling pathways that trigger this process have been identified, but the direct modifications of GluR1 that control its incorporation into synapses are unclear. Here, we show that phosphorylation of GluR1 by PKC at a highly conserved serine 818 residue is increased during LTP and critical for LTP expression. GluR1 is phosphorylated by PKC at this site in vitro and in vivo. In addition, acute phosphorylation at GluR1 S818 by PKC, as well as a phosphomimetic mutation, promotes GluR1 synaptic incorporation. Conversely, preventing GluR1 S818 phosphorylation reduces LTP and blocks PKC-driven synaptic incorporation of GluR1. We conclude that the phosphorylation of GluR1 S818 by PKC is a critical event in the plasticity-driven synaptic incorporation of AMPA receptors.     

NMDA receptor-mediated PIP5K activation to produce PI(4,5)P₂ is essential for AMPA receptor endocytosis during LTD

NMDA receptor activation leads to clathrin-dependent endocytosis of postsynaptic AMPA receptors. Although this process controls long-term depression (LTD) induction in the hippocampus, how it is regulated by neuronal activities is not completely clear. Here, we show that Ca2? influx through the NMDA receptor activates calcineurin and protein phosphatase 1 to dephosphorylate phosphatidylinositol 4-phosphate 5-kinaseγ661 (PIP5Kγ661), the major phosphatidylinositol 4,5-bisphosphate (PI(4,5)P?)-producing enzyme in the brain. Bimolecular fluorescence complementation analysis revealed that the dephosphorylated PIP5Kγ661 became associated with the clathrin adaptor protein complex AP-2 at postsynapses in situ. NMDA-induced AMPA receptor endocytosis and low-frequency stimulation-induced LTD were completely blocked by inhibiting the association between dephosphorylated PIP5Kγ661 and AP-2 and by overexpression of a kinase-dead PIP5Kγ661 mutant in hippocampal neurons. Furthermore, knockdown of PIP5Kγ661 inhibited the NMDA-induced AMPA receptor endocytosis. Therefore, NMDA receptor activation controls AMPA receptor endocytosis during hippocampal LTD by regulating PIP5Kγ661 activity at postsynapses.     

Long-term depression requires postsynaptic AMPA GluR2 receptor in adult mouse cingulate cortex

Synaptic long-term depression (LTD) is thought to be important for various brain functions such as learning, memory, and development. Although anterior cingulated cortex (ACC) has been demonstrated to contribute to learning and memory, no studies has been reported about the synaptic mechanisms for cingulate LTD. Here, we used integrative genetic, pharmacological and electrophysiological approaches to demonstrate that AMPA GluR2, but not GluR3, subunit is critical for cingulate LTD. We found that LTD was absent in adult cingulate slices of GluR2 knockout mice. Furthermore, postsynaptic injections of peptides that inhibit AMPA GluR2-PDZ interactions blocked the induction of LTD. To determine if the requirement for AMPA receptor-PDZ interaction is time-dependent, we injected the same inhibiting peptide into the postsynaptic cells 5 min after the induction of LTD. We found that LTD was not affected by the peptide, providing the first evidence that postsynaptic AMPA GluR2-mediated depression occurs rapidly (within t = 5 min). Genetic deletion of GluR3 did not affect cingulate LTD. Our results provide the first study of cingulate LTD mechanism using whole-cell patch-clamp recording in adult cortical slices and demonstrate that postsynaptic AMPA GluR2 subunit is crucial for synaptic depression in the ACC of adult mice.     

Regulation of AMPA Receptor Trafficking by O-Glycosylation

The present study investigated the role of O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) in AMPA receptor trafficking. Alloxan, an inhibitor of O-GlcNAc transferase, potentiated responses of AMPA receptors composed of the GluR1 subunit expressed in Xenopus oocytes. No potentiating effect of alloxan was obtained with mutant GluR1 (S831A) receptor lacking CaMKII phosphorylation site. Alloxan facilitated basal synaptic transmission to approximately 120% of basal levels and enhanced Schaffer collateral-CA1 long-term potentiation (LTP) in rat hippocampal slices, especially in the late phase of the LTP. Alloxan stimulated translocation of the GluR1 and GluR2 subunit from the cytosol towards the plasma membrane in rat hippocampal slices with the LTP, although it had no effect on subcellular distribution of the NR1 subunit. Taken together, the results of the present study show that alloxan regulates AMPA receptor trafficking by inhibiting O-GlcNAcylation, to modulate hippocampal synaptic transmission and synaptic plasticity.