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.     

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.     

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.     

The function of GluR1 and GluR2 in cerebellar and hippocampal LTP and LTD is regulated by interplay of phosphorylation and O‐GlcNAc modification

Long‐term potentiation (LTP) and long‐term depression (LTD) are the current models of synaptic plasticity and widely believed to explain how different kinds of memory are stored in different brain regions. Induction of LTP and LTD in different regions of brain undoubtedly involve trafficking of AMPA receptor to and from synapses. Hippocampal LTP involves phosphorylation of GluR1 subunit of AMPA receptor and its delivery to synapse whereas; LTD is the result of dephosphorylation and endocytosis of GluR1 containing AMPA receptor. Conversely the cerebellar LTD is maintained by the phosphorylation of GluR2 which promotes receptor endocytosis while dephosphorylation of GluR2 triggers receptor expression at the cell surface and results in LTP. The interplay of phosphorylation and O‐GlcNAc modification is known as functional switch in many neuronal proteins. In this study it is hypothesized that a same phenomenon underlies as LTD and LTP switching, by predicting the potential of different Ser/Thr residues for phosphorylation, O‐GlcNAc modification and their possible interplay. We suggest the involvement of O‐GlcNAc modification of dephosphorylated GluR1 in maintaining the hippocampal LTD and that of dephosphorylated GluR2 in cerebral LTP. J. Cell. Biochem. 109: 585–597, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

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.     

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.     

D1 dopamine receptor stimulation increases GluR1 surface expression in nucleus accumbens neurons

The goal of this study was to understand how dopamine receptors, which are activated during psychostimulant administration, might influence glutamate-dependent forms of synaptic plasticity that are increasingly recognized as important to drug addiction. Regulation of the surface expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR1 plays a critical role in long-term potentiation, a well-characterized form of synaptic plasticity. Primary cultures of rat nucleus accumbens neurons were used to examine whether dopamine receptor stimulation influences cell surface expression of GluR1, detected using antibody to the extracellular portion of GluR1 and fluorescence microscopy. Surface GluR1 labeling on processes of medium spiny neurons and interneurons was increased by brief (5-15 min) incubation with a D1 agonist (1 microm SKF 81297). This effect was attenuated by the D1 receptor antagonist SCH 23390 (10 microm) and reproduced by the adenylyl cyclase activator forskolin (10 microm). Labeling was decreased by glutamate (10-50 microm, 15 min). These results are the first to demonstrate modulation of AMPA receptor surface expression by a non-glutamatergic G protein-coupled receptor. Normally, this may enable ongoing regulation of AMPA receptor transmission in response to changes in the activity of dopamine projections to the nucleus accumbens. When dopamine receptors are over-stimulated during chronic drug administration, this regulation may be disrupted, leading to inappropriate plasticity in neuronal circuits governing motivation and reward.     

Expression of cerebellar long-term depression requires postsynaptic clathrin-mediated endocytosis

Cerebellar long-term depression (LTD) is a cellular model system of information storage that may underlie certain forms of motor learning. While cerebellar LTD is expressed as a selective modification of postsynaptic AMPA receptors, this might involve changes in receptor number/distribution, unitary conductance, kinetics, or glutamate affinity. The observation that GluR2-containing synaptic AMPA receptors could be internalized by regulated clathrin-mediated endocytosis suggested that this process could underlie LTD expression. To test this hypothesis, we postsynaptically applied dynamin and amphiphysin peptides that interfere with the clathrin endocytotic complex and found that they blocked LTD expression in cultured Purkinje neurons. In addition, induction of LTD and attenuation of AMPA responses by stimulation of clathrin-mediated endocytosis occluded each other. These findings suggest that the expression of cerebellar LTD requires clathrin-mediated internalization of postsynaptic AMPA receptors.     

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?     

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.     

The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity

The AMPA receptor (AMPAR) GluR2 subunit dictates the critical biophysical properties of the receptor, strongly influences receptor assembly and trafficking, and plays pivotal roles in a number of forms of long-term synaptic plasticity. Most neuronal AMPARs contain this critical subunit; however, in certain restricted neuronal populations and under certain physiological or pathological conditions, AMPARs that lack this subunit are expressed. There is a current surge of interest in such GluR2-lacking Ca2+-permeable AMPARs in how they affect the regulation of synaptic transmission. Here, we bring together recent data highlighting the novel and important roles of GluR2 in synaptic function and plasticity.     

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.     

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.     

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.     

NMDA receptor-dependent activation of the small GTPase Rab5 drives the removal of synaptic AMPA receptors during hippocampal LTD

The activity-dependent removal of AMPA receptors from synapses underlies long-term depression in hippocampal excitatory synapses. In this study, we have investigated the role of the small GTPase Rab5 during this process. We propose that Rab5 is a critical link between the signaling cascades triggered by LTD induction and the machinery that executes the activity-dependent removal of AMPA receptors. We have found that Rab5 activation drives the specific internalization of synaptic AMPA receptors in a clathrin-dependent manner and that this activity is required for LTD. Interestingly, Rab5 does not participate in the constitutive cycling of AMPA receptors. Rab5 is able to remove both GluR1 and GluR2 AMPA receptor subunits, leading to GluR1 dephosphorylation. Importantly, NMDA receptor-dependent LTD induction produces a rapid and transient increase of active (GTP bound) Rab5. We propose a model in which synaptic activity leads to Rab5 activation, which in turn drives the removal of AMPA receptors from synapses.     

Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization

Redistribution of postsynaptic AMPA- (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-) subtype glutamate receptors may regulate synaptic strength at glutamatergic synapses, but the mediation of the redistribution is poorly understood. We show that AMPA receptors underwent clathrin-dependent endocytosis, which was accelerated by insulin in a GluR2 subunit-dependent manner. Insulin-stimulated endocytosis rapidly decreased AMPA receptor numbers in the plasma membrane, resulting in long-term depression (LTD) of AMPA receptor-mediated synaptic transmission in hippocampal CA1 neurons. Moreover, insulin-induced LTD and low-frequency stimulation-(LFS-) induced homosynaptic CA1 LTD were found to be mutually occlusive and were both blocked by inhibiting postsynaptic clathrin-mediated endocytosis. Thus, controlling postsynaptic receptor numbers through endocytosis may be an important mechanism underlying synaptic plasticity in the mammalian CNS.     

Synaptic neurotransmission depression in ventral tegmental dopamine neurons and cannabinoid-associated addictive learning

Drug addiction is an association of compulsive drug use with long-term associative learning/memory. Multiple forms of learning/memory are primarily subserved by activity- or experience-dependent synaptic long-term potentiation (LTP) and long-term depression (LTD). Recent studies suggest LTP expression in locally activated glutamate synapses onto dopamine neurons (local Glu-DA synapses) of the midbrain ventral tegmental area (VTA) following a single or chronic exposure to many drugs of abuse, whereas a single exposure to cannabinoid did not significantly affect synaptic plasticity at these synapses. It is unknown whether chronic exposure of cannabis (marijuana or cannabinoids), the most commonly used illicit drug worldwide, induce LTP or LTD at these synapses. More importantly, whether such alterations in VTA synaptic plasticity causatively contribute to drug addictive behavior has not previously been addressed. Here we show in rats that chronic cannabinoid exposure activates VTA cannabinoid CB1 receptors to induce transient neurotransmission depression at VTA local Glu-DA synapses through activation of NMDA receptors and subsequent endocytosis of AMPA receptor GluR2 subunits. A GluR2-derived peptide blocks cannabinoid-induced VTA synaptic depression and conditioned place preference, i.e., learning to associate drug exposure with environmental cues. These data not only provide the first evidence, to our knowledge, that NMDA receptor-dependent synaptic depression at VTA dopamine circuitry requires GluR2 endocytosis, but also suggest an essential contribution of such synaptic depression to cannabinoid-associated addictive learning, in addition to pointing to novel pharmacological strategies for the treatment of cannabis addiction.