Adenylyl cyclase I regulates AMPA receptor trafficking during mouse cortical 'barrel' map development

Cortical map formation requires the accurate targeting, synaptogenesis, elaboration and refinement of thalamocortical afferents. Here we demonstrate the role of Ca2+/calmodulin-activated type-I adenylyl cyclase (AC1) in regulating the strength of thalamocortical synapses through modulation of AMPA receptor (AMPAR) trafficking using barrelless mice, a mutant without AC1 activity or cortical 'barrel' maps. Barrelless synapses are stuck in an immature state that contains few functional AMPARs that are rarely silent (NMDAR-only). Long-term potentiation (LTP) and long-term depression (LTD) at thalamocortical synapses require postsynaptic protein kinase A (PKA) activity and are difficult to induce in barrelless mice, probably due to an inability to properly regulate synaptic AMPAR trafficking. Consistent with this, both the extent of PKA phosphorylation on AMPAR subunit GluR1 and the expression of surface GluR1 are reduced in barrelless neurons. These results suggest that activity-dependent mechanisms operate through an AC1/PKA signaling pathway to target some synapses for consolidation and others for elimination during barrel map formation.     

State-dependent Ras signaling and AMPA receptor trafficking

Synaptic trafficking of AMPA-Rs, controlled by small GTPase Ras signaling, plays a key role in synaptic plasticity. However, how Ras signals synaptic AMPA-R trafficking is unknown. Here we show that low levels of Ras activity stimulate extracellular signal-regulated kinase kinase (MEK)-p42/44 MAPK (extracellular signal-regulated kinase [ERK]) signaling, whereas high levels of Ras activity stimulate additional Pi3 kinase (Pi3K)-protein kinase B (PKB) signaling, each accounting for approximately 50% of the potentiation during long-term potentiation (LTP). Spontaneous neural activity stimulates the Ras-MEK-ERK pathway that drives GluR2L into synapses. In the presence of neuromodulator agonists, neural activity also stimulates the Ras-Pi3K-PKB pathway that drives GluR1 into synapses. Neuromodulator release increases with increases of vigilance. Correspondingly, Ras-MEK-ERK activity in sleeping animals is sufficient to deliver GluR2L into synapses, while additional increased Ras-Pi3K-PKB activity in awake animals delivers GluR1 into synapses. Thus, state-dependent Ras signaling, which specifies downstream MEK-ERK and Pi3K-PKB pathways, differentially control GluR2L- and GluR1-dependent synaptic plasticity.     

AMPA receptor trafficking and synaptic plasticity: major unanswered questions

Dynamic movements of AMPA receptors in and out of the postsynaptic membrane account for, at least in part, the expression of NMDA receptor-dependent changes in synaptic efficacy such as long-term potentiation and long-term depression. Recently some of key molecules and subunit rules involved in AMPA receptor trafficking have been identified. In this update article, we try to highlight what we believe to be the major conceptual problems and unanswered questions in this rapidly moving field of neuroscience.     

MAPK signaling pathways mediate AMPA receptor trafficking in an in vitro model of classical conditioning

The mitogen-activated protein kinase (MAPK) signal transduction pathways have been implicated in underlying mechanisms of synaptic plasticity and learning. However, the differential roles of the MAPK family members extracellular signal-regulated kinase (ERK) and p38 in learning remain to be clarified. Here, an in vitro model of classical conditioning was examined to assess the roles of ERK and p38 MAPK in this form of learning. Previous studies showed that NMDA-mediated trafficking of synaptic glutamate receptor 4 (GluR4)-containing AMPA receptors (AMPARs) underlies conditioning in this preparation and that this is accomplished through GluR4 interactions with the immediate-early gene protein Arc and the actin cytoskeleton. Here, it is shown that attenuation of conditioned responses (CRs) by ERK and p38 MAPK antagonists is associated with significantly reduced synaptic localization of GluR4 subunits. Western blotting reveals that p38 MAPK significantly increases its activation levels during late stages of conditioning during CR expression. In contrast, ERK MAPK activation is enhanced in early conditioning during CR acquisition. The results suggest that MAPKs have a central role in the synaptic delivery of GluR4-containing AMPARs during in vitro classical conditioning.     

Differential roles of GRIP1a and GRIP1b in AMPA receptor trafficking

Regulated trafficking controls AMPA receptor (AMPAR) number at the postsynaptic membrane to modify the efficiency of synaptic transmission. The PDZ proteins GRIP1 and the related ABP-L/GRIP2 bind AMPAR subunit GluA2, and have been proposed to play a role in AMPAR trafficking associated with Long Term Depression (LTD) of synaptic transmission. Both GRIP1 and ABP-L/GRIP2 exist in different splice isoforms, including alternative 18 amino acid domains at the extreme N-terminus, which determine whether the protein can be palmitoylated. The implications of this differential splicing for AMPAR trafficking is unknown. Here, we use surface biotinylation and quantitative Western blotting to show that the N-terminal splice variants GRIP1a and GRIP1b have differential effects in NMDA-induced AMPAR internalization in cultured hippocampal neurons. GRIP1a inhibits, but GRIP1b enhances this trafficking event. We further demonstrate that GRIP1a and GRIP1b have dramatically different subcellular distributions in cultured neurons and exhibit NMDA-dependent colocalisation with early endosomes. We propose that GRIP1 palmitoylation modulates NMDA-induced AMPAR internalisation by differential regulation of the early endosomal system.     

New role of delta2-glutamate receptors in AMPA receptor trafficking and cerebellar function

Previous gene knockout studies have shown that the orphan glutamate receptor delta2 (GluRdelta2) is critically involved in synaptogenesis between parallel fibers and Purkinje cells during development. However, the precise function of GluRdelta2 and whether it is functional in the mature cerebellum remain unclear. To address these issues, we developed an antibody specific for the putative ligand-binding region of GluRdelta2, and application of this antibody to cultured Purkinje cells induced AMPA receptor endocytosis, attenuated synaptic transmission and abrogated long-term depression. Moreover, injection of this antibody into the subarachnoidal supracerebellar space of adult mice caused transient cerebellar dysfunction, such as ataxic gait and poor performance in the rotorod test. These results indicate that GluRdelta2 is involved in AMPA receptor trafficking and cerebellar function in adult mice.     

Transmembrane AMPA receptor regulatory proteins and AMPA receptor function in the cerebellum

Heterogeneity among AMPA receptor (AMPAR) subtypes is thought to be one of the key postsynaptic factors giving rise to diversity in excitatory synaptic signaling in the CNS. Recently, compelling evidence has emerged that ancillary AMPAR subunits—the so-called transmembrane AMPA receptor regulatory proteins (TARPs)—also play a vital role in influencing the variety of postsynaptic signaling. This TARP family of molecules controls both trafficking and functional properties of AMPARs at most, if not all, excitatory central synapses. Furthermore, individual TARPs differ in their effects on the biophysical and pharmacological properties of AMPARs. The critical importance of TARPs in synaptic transmission was first revealed in experiments on cerebellar granule cells from stargazer mice. These lack the prototypic TARP stargazin, present in granule cells from wild-type animals, and consequently lack synaptic transmission at the mossy fibre-to-granule cell synapse. Subsequent work has identified many other members of the stargazin family which act as functional TARPs. It has also provided valuable information about specific TARPs present in many central neurons. Because much of the initial work on TARPs was carried out on stargazer granule cells, the important functional properties of TARPs present throughout the cerebellum have received particular attention. Here we discuss some of these recent findings in relation to the main TARPs and the AMPAR subunits identified in cerebellar neurons and glia.     

Fc receptor signaling and trafficking: a connection for antigen processing

Summary: Most hematopoietic cells express a wide variety of receptors for tbe Fc portion of immunoglobulins (FcR) belonging to the immun-noreceptor family. FcRs are multichain complexes composed of ligand-binding a chains, which determine Ig binding, and signal tranduction subunits, bearing a conserved immunoreceptor tyrosine-based activation motif (ITAM). Besides signaling, most FcyRs also efficiently internalize antigen-antibodies complexes and thus induce efficient processing of antigens into peptides presented by major histocompatibility complex (MHC) class II molecules. Importantly, ITAMs and cyiosolic effectors of cell signaling also determine FcyR's endocytic transport and antigen presentation capacities.     

Voltage-gated sodium channel organization in neurons: Protein interactions and trafficking pathways

In neurons, voltage-gated sodium (Nav) channels underlie the generation and propagation of the action potential. The proper targeting and concentration of Nav channels at the axon initial segment (AIS) and at the nodes of Ranvier are therefore vital for neuronal function. In AIS and nodes, Nav channels are part of specific supra-molecular complexes that include accessory proteins, adhesion proteins and cytoskeletal adaptors. Multiple approaches, from biochemical characterization of protein–protein interactions to functional studies using mutant mice, have addressed the mechanisms of Nav channel targeting to AIS and nodes. This review summarizes our current knowledge of both the intrinsic determinants and the role of partner proteins in Nav targeting. A few fundamental trafficking mechanisms, such as selective endocytosis and diffusion/retention, have been characterized. However, a lot of exciting questions are still open, such as the mechanism of differentiated Nav subtype localization and targeting, and the possible interplay between electrogenesis properties and Nav concentration at the AIS and the nodes.     

Neurotransmitter receptor interactions with G-proteins: a critical link in receptor response mechanisms

The interaction of membrane bound receptors with guanine nucleotide binding regulatory proteins is necessary for the transduction of many extracellular signals across the cell membrane. Age-related impairments of this interaction illustrate that the efficiency of receptor effector coupling is complex, and is an additional factor for consideration in the future study of receptor mediated functions.     

The colocalization of CGRP receptor and AMPA receptor in the spinal dorsal horn neuron of rat: a morphological and electrophysiological study

Both the calcitonin gene-related peptide (CGRP) receptor and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor are involved in the transmission of sensory information from primary afferent to the spinal cord. The present study found that there was a colocalization of CGRP receptor and AMPA receptor in a single spinal dorsal horn neuron in rat determined by double immunofluorescence labeling image methods. Furthermore, our results showed that the evoked discharge frequency of the wide dynamic range (WDR) neuron, one type of the dorsal horn neurons, increased significantly after micro-iontophoretic delivery of CGRP or AMPA alone tested by extracellular recording, indicating a functional colocalization of CGRP receptor and AMPA receptor in a single spinal dorsal horn neuron. The results of the present study found a morphological and functional colocalization of the CGRP receptor and AMPA receptor in a single dorsal horn neuron that involved in the transmission and modulation of sensory information from primary afferent to the spinal cord in rats.     

Glutamate AMPA receptor subunit 1 gene (GRIA1) and DSM-IV-TR schizophrenia: a pilot case-control association study in an Italian sample.

Glutamatergic dysfunction is one of the major hypotheses for the pathogenesis of schizophrenia. The GRIA1 gene encodes for one (GluR1) of the four (GluR1-4) ionotropic AMPA receptor subunits. GRIA1 is a good candidate gene for susceptibility to schizophrenia since it maps in 5q33, a region where the presence of susceptibility loci has been suggested by independent genome-wide scans and because its expression has been found to be decreased in the brain of some schizophrenia patients. We present data from a case-control association study on the Italian population with eight polymorphisms spanning the whole GRIA1 gene. Single-locus analysis revealed a significantly different allele distribution in cases and in controls of two SNPs (rs707176, 0.41 vs. 0.31, P = 0.009; rs2963944, 0.41 vs. 0.30, P = 0.007), and one microsatellite (rs10631988, allele 9: 0.40 vs. 0.29, P = 0.004). Haplotype analysis showed an increased frequency of a specific haplotype for these markers (C09CC, 0.39 vs. 0.28, P = 0.009). Therefore our data indicate that GRIA1 may be involved in susceptibility to DSM-IV-TR schizophrenia.     

Parvalbumin-containing interneurons in rat hippocampus have an AMPA receptor profile suggestive of vulnerability to excitotoxicity

-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors mediate excitatory neurotransmission in the central nervous system, and contain combinations of four subunits (GluR1-4). We developed a GluR3-specific monoclonal antibody and quantified the cellular distribution of GluR3 in rat hippocampus. GluR3 immunoreactivity was detected in all pyramidal neurons and most interneurons. In addition, we found a subset of parvalbumin (PV)-containing interneurons in the hippocampus and neocortex that was notable for its intense GluR3 immunoreactivity and lack of GluR2 immunoreactivity. Such an expression pattern of AMPA receptor subunits is likely to make these interneurons selectively vulnerable to excitotoxicity.     

Gadolinium reduces AMPA receptor desensitization and deactivation in hippocampal neurons.

The actions of the trivalent cation Gd(3+) on whole cell AMPA receptor-mediated currents were studied in isolated hippocampal neurons, in nucleated or outside-out patches taken from cultured hippocampal neurons, and on miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured hippocampal neurons. Glutamate, AMPA, or kainate was employed to activate AMPA receptors. Applications of relatively low concentrations of Gd(3+) (0.1-10 microM) substantially enhanced steady-state whole cell glutamate and kainate-evoked currents without altering peak currents, suggesting that desensitization was reduced. However, higher concentrations (>30 microM) depressed steady-state currents, indicating an underlying inhibition of channel activity. Lower concentrations of Gd(3+) also increased the potency of peak glutamate-evoked currents without altering that of steady-state currents. An ultrafast perfusion system and nucleated patches were then used to better resolve peak glutamate-evoked currents. Low concentrations of Gd(3+) reduced peak currents, enhanced steady-state currents, and slowed the onset of desensitization, providing further evidence that this cation reduces desensitization. In the presence of cyclothiazide, a compound that blocks desensitization, a low concentration Gd(3+) inhibited both peak and steady-state currents, indicating that Gd(3+) both reduces desensitization and inhibits these currents. Gd(3+) reduced the probability of channel opening at the peak of the currents but did not alter the single channel conductance calculated using nonstationary variance analysis. Recovery from desensitization was enhanced, and glutamate-evoked current activation and deactivation were slowed by Gd(3+). The Gd(3+)-induced reduction in desensitization did not require the presence of the GluR2 subunit as this effect was seen in hippocampal neurons from GluR2 null-mutant mice. Gd(3+) reduced the time course of decay of mEPSCs perhaps as a consequence of its slowing of AMPA receptor deactivation although an increase in the frequency of mEPSCs also suggested enhanced presynaptic release of transmitter. These results demonstrate that Gd(3+) potently reduces AMPA receptor desensitization and mimics a number of the properties of the positive modulators of AMPA receptor desensitization such as cyclothiazide.     

Differential expression of AMPA receptor subunits in dopamine neurons of the rat brain: a double immunocytochemical study

We have examined the distribution of dopamine neurons expressing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits (glutamate receptors 1, 2/3 and 4) in the A8-A15 regions of the rat brain using double immunofluorescence. The distribution of glutamate receptor 1- or 2/3-like immunoreactive neurons completely overlapped that of tyrosine hydroxylase-like immunoreactive neurons in dopamine cell groups in the retrorubral field (A8), the substantia nigra (A9), the ventral tegmental area and the nucleus raphe linealis (A10), and the rostral hypothalamic periventricular nucleus (A14, A15). In the caudal hypothalamic periventricular nucleus (A11), arcuate nucleus (A12) and zona incerta (A13), the distribution was partially overlapping. Neurons double-labeled for tyrosine hydroxylase and glutamate receptor 1 or 2/3 immunoreactivities were, however, exclusively found in certain dopamine cell regions: in areas A14-A15, 85-88% of tyrosine hydroxylase-containing neurons expressed glutamate receptor 1 and 22-25% expressed glutamate receptor 2/3, while in areas A8-A10, 20-43% expressed glutamate receptor 1 and 63-84% expressed glutamate receptor 2/3. In contrast, the double-labeled neurons were hardly detected in the A11-A13 regions. No tyrosine hydroxylase-positive neurons displayed glutamate receptor 4 immunoreactivity, though a partially overlapping distribution of tyrosine hydroxylase- and glutamate receptor 4-immunopositive neurons was also seen in regions A8-10, A11 and A13.The present study has demonstrated the morphological evidence for direct modulation of dopamine neurons via AMPA receptors in rat mesencephalon and hypothalamus. This distribution may provide the basis for a selective dopamine neuron loss in neurodegenerative disorders, such as Parkinson's disease.     

Facilitating actions of an AMPA receptor potentiator upon extinction of contextually conditioned fear response in stressed mice

Extinction of conditioned fear response is thought to be a biological process underlying exposure therapy for anxiety disorders. We have previously reported that an AMPA receptor potentiator, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluorophenoxyacetamide (PEPA), facilitates extinction of fear memory formed through contextual fear conditioning in mice that had never been exposed to experimental stress. On the other hand, recent findings suggest that the fear extinction is impaired in stressed rats or mice. The purpose of the present study was to examine whether PEPA facilitates impaired extinction of fear in stressed mice. For this purpose, mice were applied stress (a 2 h restraint, a 20 min forced swim, and ether inhalation), and contextual fear conditioning was carried out 7 days later. After 1–3 days of conditioning, mice were re-exposed to the context for 6 min, and behavioral freezing response was measured. The time mice spent frozen decreased following every extinction session, and the decrease was remarkably slower in the stressed mice than in control non-stressed mice. PEPA (3, 10, 30 mg/kg body weight) or vehicle was intraperitoneally administered into stressed mice once before the first extinction session. The significant decrease of the freezing response in the extinction sessions was only seen in the 30 mg/kg PEPA-administered stressed mice, compared with vehicle-administered stressed mice. A similar extent of decrease in the freezing response in the extinction sessions was observed in the PEPA-administered (30 mg/kg) and d-cycloserine-administered (30 mg/kg) mice. These results suggest that PEPA facilitates extinction of contextual fear in stressed mice.     

Facilitating actions of an AMPA receptor potentiator upon extinction of contextually conditioned fear response in stressed mice

Mounting evidence supports the association between a polymorphism in the serotonin transporter gene promoter region (5-HTTLPR) and suicidal behaviour. Recently, a novel variant of the 5-HTTLPR L allele was identified. The previously unknown L_G allele produced similar levels of gene expression to the S allele and might have been misclassified as a “high-expression” allele in previous association studies. In this study, we aimed to compare the genotype distribution of the tri-allelic 5-HTTLPR polymorphism in 168 Chinese patients with schizophrenia, including 60 suicide attempters and 108 non-suicide attempters. In our analysis, which used the L_A dominant model, it was found that the L_A allele carriers were significantly more likely to have attempted suicide (p = 0.035). Further analysis showed this association existed only in male patients (p = 0.012). A similar association between the L_A allele and violent suicide attempt was also found (p = 0.028). In addition, logistic regression confirmed our findings that male L_A allele carriers were at a higher risk of suicide, although the lack of a significant association in females may reflect insufficient power due to small sample size. However, no association was found when we examined the traditional bi-allelic 5-HTTLPR. These findings differ from those reported in Caucasian subjects, where no associations have been reported. Different genetic backgrounds may give rise to different allelic distribution, causing differential effects on the expression of endophenotypes of suicide behaviours. Although the potential influence of multiple comparisons might weaken our findings, our study provides preliminary evidence for a potentially gender-specific role of a “high-expression” 5-HTTLPR polymorphism in susceptibility to suicide in Chinese patients with schizophrenia.     

Subunit-specific temporal and spatial patterns of AMPA receptor exocytosis in hippocampal neurons

Using a thrombin cleavage assay in cultured hippocampal neurons, we studied the kinetics, regulation and site of AMPA receptor surface delivery. Surface insertion of the GluR1 subunit occurs slowly in basal conditions and is stimulated by NMDA receptor activation and insulin, whereas GluR2 exocytosis is constitutively rapid. Although both subunits ultimately concentrate in synapses, GluR1 and GluR2 show different spatial patterns of surface accumulation, consistent with GluR1 being inserted initially at extrasynaptic sites and GluR2 being inserted more directly at synapses. The spatiotemporal pattern of surface accumulation is determined by the cytoplasmic tails of GluR subunits, and in heteromeric receptors, GluR1 acts dominantly over GluR2. We propose that GluR1 controls the exocytosis and GluR2/3, the recycling and endocytosis of AMPA receptors.