Extrasynaptic AMPA Receptors Research Articles

KIBRA regulates activity-induced AMPA receptor expression and synaptic plasticity in an age-dependent manner.

A growing body of human literature implicates KIBRA in memory and neurodevelopmental disorders. Memory and the cellular substrates supporting adaptive cognition change across development. Using an inducible KIBRA knockout mouse, we demonstrate that adult-onset deletion of KIBRA in forebrain neurons impairs long-term spatial memory and long-term potentiation (LTP). These LTP deficits correlate with adult-selective decreases in extrasynaptic AMPA receptors under basal conditions, and we identify a role for KIBRA in LTP-induced AMPAR upregulation. In contrast, juvenile-onset deletion of KIBRA in forebrain neurons did not affect LTP and had minimal effects on basal AMPAR expression. LTP did not increase AMPAR protein expression in juvenile WT mice, providing a potential explanation for juvenile resilience to KIBRA deletion. These data suggest that KIBRA serves a unique role in adult hippocampal function through regulation of basal and activity-dependent AMPAR proteostasis that supports synaptic plasticity.

Correction: Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.

Correction: Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.

Glial EAAT2 regulation of extracellular nTS glutamate critically controls neuronal activity and cardiorespiratory reflexes.

Excitatory amino acid transporter 2 (EAAT2) is present on astrocytes in the nucleus tractus solitarii (nTS), an important nucleus in cardiorespiratory control. Its specific role in influencing nTS neuronal activity and thereby basal and reflex cardiorespiratory function is unknown. The specific role of nTS EAAT2 was determined via whole animal and brainstem slice patch clamp experiments. Astrocytic EAAT2 buffers basal glutamate activation of AMPA-type glutamate receptors and therefore decreases baseline excitability of nTS neurons. EAAT2 modulates cardiorespiratory control and tempers excitatory cardiorespiratory responses to activation of the peripheral chemoreflex. This study supports the concept that nTS astrocyte transporters influence sympathetic nervous system activity and cardiorespiratory reflex function in health and disease. Glutamatergic signalling is critical in the nucleus tractus solitarii (nTS) for cardiorespiratory homeostasis and initiation of sensory reflexes, including the chemoreflex activated during hypoxia. Maintenance of nTS glutamate concentration occurs in part through astrocytic excitatory amino acid transporters (EAATs). We previously established the importance of EAATs in the nTS by demonstrating their inhibition produced neuronal excitation to alter basal cardiorespiratory function. Since EAAT2 is the most expressed EAAT in the nTS, this study specifically determined EAAT2's role in nTS astrocytes, its influence on neuronal and synaptic properties, and ultimately on basal and reflex cardiorespiratory function. The EAAT2-specific antagonist dihydrokainate (DHK) was microinjected into the anaesthetized rat nTS or applied to rat nTS slices. DHK produced depressor, bradycardic and sympathoinhibitory responses and reduced neural respiration in the intact rat, mimicking responses to glutamate excitation. DHK also enhanced responses to glutamate microinjection. DHK elevated extracellular nTS glutamate concentration, depolarized neurons and enhanced spontaneous EPSCs. EAAT2 block also augmented action potential discharge in chemosensitive nTS neurons. Glial recordings confirmed EAAT2 is functional on nTS astrocytes. Neuronal excitation and cardiorespiratory effects following EAAT2 inhibition were due to activation of putative extrasynaptic AMPA receptors as their antagonism blocked DHK responses in the intact rat nTS and the slice. The DHK-induced elevation of extracellular glutamate and neuronal excitation augmented chemoreflex-mediated pressor, sympathoexcitatory and minute neural ventilation responses in the rat. These data shed new light on the important role astrocytic EAAT2 plays on buffering nTS excitation and overall cardiorespiratory function.

Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.

Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 min or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5-10% of bQD-AMPARs were in PSDs and 90-95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD 'slots', our findings suggest that AMPARs rapidly enter stable 'nanodomains' in PSDs with lifetime >15 min, and do not accumulate in extrasynaptic membranes.

Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP.

Strengthening of synaptic connections by NMDA-receptor-dependent long-term potentiation (LTP) shapes neural circuits and mediates learning and memory. During NMDA-receptor-dependent LTP induction, Ca2+-influx stimulates recruitment of synaptic AMPA-receptors, thereby strengthening synapses. How Ca2+ induces AMPA-receptor recruitment, however, remains unclear. Here we show that, in pyramidal neurons of the hippocampal CA1-region, blocking postsynaptic expression of both synaptotagmin-1 and synaptotagmin-7, but not of synaptotagmin-1 or synaptotagmin-7 alone, abolished LTP. LTP was rescued by wild-type but not by Ca2+-binding-deficient mutant synaptotagmin-7. Blocking postsynaptic synaptotagmin-1/7 expression did not impair basal synaptic transmission, synaptic or extrasynaptic AMPA-receptor levels, or other AMPA-receptor trafficking events. Moreover, expression of dominant-negative mutant synaptotagmin-1 that inhibited Ca2+-dependent presynaptic vesicle exocytosis also blocked Ca2+-dependent postsynaptic AMPA-receptor exocytosis, thereby abolishing LTP. Our results suggest that postsynaptic synaptotagmin-1 and synaptotagmin-7 act as redundant Ca2+-sensors for Ca2+-dependent exocytosis of AMPA-receptors during LTP, thus delineating a simple mechanism for the recruitment of AMPA-receptors that mediates LTP.

Functional properties of extrasynaptic AMPA and NMDA receptors during postnatal hippocampal neurogenesis

In the mammalian hippocampus, new granule cells are continuously generated throughout life. Although it is well known that they rapidly form several thousand new glutamatergic synapses, the underlying mechanisms are not well understood. As extrasynaptic NMDA receptors are believed to support the generation of new spines, we have studied the functional properties of extrasynaptic ionotropic glutamate receptors in newborn granule cells in juvenile rats during and after synaptic integration. Using the fast application of glutamate to outside-out membrane patches, we show that all immature granule cells express functional AMPA and NMDA receptors. The density of AMPA receptors was small in cells starting to receive excitatory synaptic input (∼30 pS μm(-2)) but substantially increased during synaptic integration to finally reach ∼120 pS μm(-2) in fully mature cells. Interestingly, AMPA receptors showed a biphasic change in desensitization time constant which was slowest during synaptic integration and substantially faster before and afterwards. This was paralleled by a change in the non-desensitizing current component which was maximal during synaptic integration and about 50% smaller afterwards. Surprisingly, the NMDA receptor kinetics and density in young cells was already comparable to mature cells (∼10 pS μm(-2)), leading to an enhanced NMDA/AMPA receptor density ratio. Similar to somatic outside-out patches, iontophoretic application of glutamate onto dendrites also revealed an enhanced dendritic NMDA/AMPA ratio in young cells. These data indicate that prolonged AMPA receptor currents in newly generated young granule cells might support the effective activation of extrasynaptic NMDA receptors and therefore constitute a competitive advantage over mature cells for new synapse formation.

Regulation of phosphorylation of synaptic and extrasynaptic GluA1 AMPA receptors in the rat forebrain by amphetamine

The AMPA receptor is regulated by phosphorylation. Two major phosphorylation sites (S831 and S845) are located in the intracellular C-terminal tail of GluA1 subunits. The phosphorylation on these sites controls receptor expression and function and is subject to the regulation by psychostimulants. In this study, we further characterized the regulation of S831 and S845 phosphorylation by amphetamine (AMPH) in the adult rat striatum and medial prefrontal cortex (mPFC) in vivo. We focused on the specific fraction of GluA1/AMPA receptors enriched from synaptic and extrasynaptic membranes, using a pre-validated biochemical fractionation procedure. We found that acute AMPH administration elevated GluA1 S845 phosphorylation in the defined synaptic membrane from the striatum in a dose-dependent manner. AMPH also induced a comparable increase in S845 phosphorylation in the extrasynaptic fraction of striatal GluA1. Similar increases in S845 phosphorylation in both synaptic and extrasynaptic pools were observed in the mPFC. In contrast, S831 phosphorylation was not altered in synaptic and extrasynaptic GluA1 in striatal neurons and synaptic GluA1 in mPFC neurons in response to AMPH, although a moderate increase in S831 phosphorylation was seen in extrasynaptic GluA1 in the mPFC after an AMPH injection at a high dose. Total synaptic and extrasynaptic GluA1 expression remained stable in the two regions after AMPH administration. Our data demonstrate the differential sensitivity of S845 and S831 phosphorylation to dopamine stimulation. S845 is a primary site where phosphorylation of GluA1 is upregulated by AMPH in striatal and mPFC neurons at both synaptic and extrasynaptic compartments.

PKCα Is Required for Inflammation-Induced Trafficking of Extrasynaptic AMPA Receptors in Tonically Firing Lamina II Dorsal Horn Neurons During the Maintenance of Persistent Inflammatory Pain

Persistent inflammation promotes internalization of synaptic GluR2-containing, Ca2+-impermeable AMPA receptors (AMPARs) and insertion of GluR1-containing, Ca2+-permeable AMPARs at extrasynaptic sites in dorsal horn neurons. Previously we have shown that internalization of synaptic GluR2-containing AMPARs requires activation of spinal cord protein kinase C alpha (PKCα), but molecular mechanisms that underlie altered trafficking of extrasynaptic AMPARs are unclear. Here, using antisense (AS) oligodeoxynucleotides (ODN) that specifically knock down PKCα, we found that a decrease in dorsal horn PKCα expression prevents complete Freund's adjuvant (CFA)-induced increase in functional expression of extrasynaptic Ca2+-permeable AMPARs in substantia gelatinosa (SG) neurons of the rat spinal cord. Augmented AMPA-induced currents and associated [Ca2+]i transients were abolished, and the current rectification 1 day post-CFA was reversed. These changes were observed specifically in SG neurons characterized by intrinsic tonic firing properties, but not in those that exhibited strong adaptation. Finally, dorsal horn PKCα knockdown produced an antinociceptive effect on CFA-induced thermal and mechanical hypersensitivity during the maintenance period of inflammatory pain, indicating a role for PKCα in persistent inflammatory pain maintenance. Our results indicate that inflammation-induced trafficking of extrasynaptic Ca2+-permeable AMPARs in tonically firing SG neurons depends on PKCα, and that this PKCα-dependent trafficking may contribute to persistent inflammatory pain maintenance. PerspectiveThis study shows that PKCα knockdown blocks inflammation-induced upregulation of extrasynaptic Ca2+-permeable AMPARs in dorsal horn neurons and produces an antinociceptive effect during the maintenance period of inflammatory pain. These findings have potential implications for use of PKCα gene-silencing therapy to prevent and/or treat persistent inflammatory pain.

Extrasynaptic AMPA receptors in the dorsal horn: Evidence and functional significance

Extrasynaptic AMPA receptors (AMPARs) are widely expressed in the brain, spinal cord and periphery. These receptors are critically involved in activity-dependent synaptic transmission and changes in their functioning are causally linked to multiple neuropathologies in the central nervous system (CNS). However, most studies in this field have been concentrated on elucidating synaptic AMPAR functioning, leaving a possible involvement of an extrasynaptic pool of AMPARs in normal and pathological signaling open for consideration. Here, we review the present evidence for extrasynaptic AMPAR function in the dorsal horn neurons of the spinal cord, linking these receptors to neurotransmission and non-synaptic signaling in this part of the CNS. In addition, we summarize current knowledge about the role of extrasynaptic AMPARs in the development and maintenance of pain states during inflammation. This knowledge potentially suggests the development of alternative therapies to prevent and/or treat inflammatory pain.

Development of inflammation-induced hyperalgesia and allodynia is associated with the upregulation of extrasynaptic AMPA receptors in tonically firing lamina II dorsal horn neurons

Persistent peripheral inflammation changes AMPA receptor (AMPAR) trafficking in dorsal horn neurons by promoting internalization of GluR2-containing, Ca2+-impermeable AMPARs from the synapses and by increasing insertion of GluR1-containing, Ca2+-permeable AMPARs in extrasynaptic plasma membrane. These changes contribute to the maintenance of persistent inflammatory pain. However, much less is known about AMPAR trafficking during development of persistent inflammatory pain and direct studies of extrasynaptic AMPARs functioning during this period are still lacking. Using Complete Freund's adjuvant (CFA)-induced model of long-lasting peripheral inflammation, we showed that remarkable hyperalgesia and allodynia developes in 1–3 h after intraplantar CFA injection. By utilizing patch-clamp recording combined with Ca2+ imaging, we found a significant upregulation of extrasynaptic AMPARs in substantia gelatinosa (SG) neurons of the rat spinal cord 2–3 h after CFA injection. This upregulation was manifested as a robust increase in the amplitude of AMPAR-mediated currents 2–3 h post-CFA. These changes were observed specifically in SG neurons characterized by intrinsic tonic firing properties, but not in those that exhibited strong adaptation. Our results indicate that CFA-induced inflammation increases functional expression of extrasynaptic AMPARs in tonically firing SG neurons during development of pain hypersensitivity and that this increase may contribute to the development of peripheral persistent pain.

PDZ binding of TARPγ-8 controls synaptic transmission but not synaptic plasticity

Reduction in synaptic transmission and plasticity in mice lacking the hippocampus-enriched AMPAR auxiliary subunit, TARP©-8, could be due to reduction in AMPAR expression or a direct role of ©-8. Here, we generated TARP©-8Δ4 knock-in mice lacking the C-terminal PDZ ligand. We found that synaptic transmission and AMPAR were reduced without changes in extrasynaptic AMPAR expression, but LTP was unaltered. Our findings indicate distinct TARP-dependent mechanisms for synaptic transmission and LTP.

Stargazin (TARP γ-2) Is Required for Compartment-Specific AMPA Receptor Trafficking and Synaptic Plasticity in Cerebellar Stellate Cells

In the cerebellar cortex, parallel fiber-to-stellate cell (PF-SC) synapses exhibit a form of synaptic plasticity manifested as a switch in the subunit composition of synaptic AMPA receptors (AMPARs) from calcium-permeable, GluA2-lacking to calcium-impermeable, GluA2-containing receptors. Here, we examine the role of stargazin (γ-2), canonical member of the transmembrane AMPAR regulatory protein (TARP) family, in the regulation of GluA2-lacking AMPARs and synaptic plasticity in SCs from epileptic and ataxic stargazer mutant mice. We found that AMPAR-mediated synaptic transmission is severely diminished in stargazer SCs, and that the rectification index (RI) of AMPAR current is reduced. Activity-dependent plasticity in the rectification of synaptic AMPARs is also impaired in stargazer SCs. Despite the dramatic loss in synaptic AMPARs, extrasynaptic AMPARs are preserved. We then examined the role of stargazin in regulating the rectification of extrasynaptic AMPARs in nucleated patches and found, in contrast to previous reports, that wild-type extrasynaptic AMPARs have moderate RI values (average RI = 0.38), while those in stargazer SCs are low (average RI = 0.24). The GluA2-lacking AMPAR blocker, philanthotoxin-433 (PhTx-433), was used as an alternative measure of GluA2 content in wild-type and stargazer SCs. Despite the difference in RI, PhTx-433 sensitivity of both synaptic and extrasynaptic AMPARs remains unchanged, suggesting that the dramatic changes in RI and the impairment in synaptic plasticity observed in the stargazer mouse are not the result of a specific impairment in GluA2 trafficking. Together, these data suggest that stargazin regulates compartment-specific AMPAR trafficking, as well as activity-dependent plasticity in synaptic AMPAR rectification at cerebellar PF-SC synapses.

Inflammation alters trafficking of extrasynaptic AMPA receptors in tonically firing lamina II neurons of the rat spinal dorsal horn

Peripheral inflammation alters AMPA receptor (AMPAR) subunit trafficking and increases AMPAR Ca2+ permeability at synapses of spinal dorsal horn neurons. However, it is unclear whether AMPAR trafficking at extrasynaptic sites of these neurons also changes under persistent inflammatory pain conditions. Using patch-clamp recording combined with Ca2+ imaging and cobalt staining, we found that, under normal conditions, an extrasynaptic pool of AMPARs in rat substantia gelatinosa (SG) neurons of spinal dorsal horn predominantly consists of GluR2-containing Ca2+-impermeable receptors. Maintenance of complete Freund’s adjuvant (CFA)-induced inflammation was associated with a marked enhancement of AMPA-induced currents and [Ca2+]i transients in SG neurons, while, as we previously showed, the amplitude of synaptically evoked AMPAR-mediated currents was not changed 24h after CFA. These findings indicate that extrasynaptic AMPARs are upregulated and their Ca2+ permeability increases dramatically. This increase occurred in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibited strong adaptation. This increase was also accompanied by an inward rectification of AMPA-induced currents and enhancement of sensitivity to a highly selective Ca2+-permeable AMPAR blocker, IEM-1460. Electron microcopy and biochemical assays additionally showed an increase in the amount of GluR1 at extrasynaptic membranes in dorsal horn neurons 24h post-CFA. Taken together, our findings indicate that CFA-induced inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca2+-permeable AMPARs in tonically firing excitatory dorsal horn neurons, suggesting that the altered extrasynaptic AMPAR trafficking might participate in the maintenance of persistent inflammatory pain.Persistent peripheral inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca2+-permeable AMPARs within their entire pool specifically in tonically firing SG neurons.

Learning, AMPA receptor mobility and synaptic plasticity depend on n-cofilin-mediated actin dynamics

Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin-binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n-cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long-term potentiation and long-term depression. Loss of n-cofilin-mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n-cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability.

Ultra-High Resolution Imaging of the Dynamic Nature of Post-Synaptic Molecules

The spatial and temporal regulation in the composition of the postsynaptic membrane of synapses participates in the different forms of synaptic plasticity that trigger the cellular processes of memory formation, consolidation, and retrieval. Neurotransmitter receptors move rapidly in and out of synapses by lateral diffusion. This mobility is crucial to control the number of receptors present at a given synapse. Thus, the equilibrium between the synaptic and extra synaptic AMPA receptor number is crucial in controlling basal transmission and synaptic plasticity. This balance is regulated by the subunit composition of these receptors and by the interaction of intracellular scaffold proteins. However, how the trafficking of receptors and the scaffolding molecules in and out synapse is controlled remains unknown. Here we attempt to determine the relative distribution and trafficking properties of AMPA receptors and various scaffold proteins at unprecedented spatial (< 40nm) and temporal resolution (>50 Hz) using a variety of novel ultra-high resolution fluorescence imaging approaches. We combine Single Particle Tracking (SPT) and Photo Activation Localization Microscopy (PALM) to map trajectories at the level of individual molecules. Here we describe the implementation of a multimodal microscope along with the development of a new dedicated analysis for single molecule segmentation and tracking. Furthermore we will discuss the application of SPT-PALM experiments on living neurons. With this novel approach, we expect to comprehend the motilities of receptors or scaffolding proteins when they traffic between the submicron sized molecular zones of dendritic spines. The combination of this type of detection and analysis will provide the information from thousands of discrete trajectories from a single cell with which it would be possible to appreciate finer details of versatile molecular mechanisms pertinent in the functioning of an excitatory synapse.

AMPA Receptor Subunits Get Their Share of the Pie

Determining the precise subunit composition of AMPARs in situ has been an enduring challenge. In this issue of Neuron, Lu et al. make significant headway into this pesky problem by using a clever single-cell genetic knockout approach. In a Herculean effort, they provide the field with the completeness of a pie chart outlining the contribution of each AMPAR subunit to basal synaptic transmission at CA1 hippocampus synapses--and we get a pie chart for somatic extrasynaptic receptors, too.

Renewed cocaine exposure produces transient alterations in nucleus accumbens AMPA receptor-mediated behavior.

Withdrawal from repeated cocaine is associated with increased synaptic and extrasynaptic AMPA receptor (AMPAR) expression in nucleus accumbens (NAc) neurons and enhanced behavioral sensitivity to AMPAR stimulation. Recent studies found that increased membrane expression of AMPARs is reversed or normalized on cocaine reexposure in withdrawal, but the mechanism for this AMPAR plasticity and the behavioral implications are unknown. Here, we examine the effects of renewed cocaine exposure during withdrawal on enhanced NAc AMPAR sensitivity and investigate the underlying mechanisms. Cocaine reexposure transiently reversed enhanced NAc AMPAR-mediated locomotion 1 d later, while enhancing cocaine-induced locomotion. Reversal in AMPAR sensitivity was prohibited by NAc AMPAR blockade with CNQX during cocaine reexposure and mimicked by intra-NAc infusions of AMPA, suggesting that cocaine-induced glutamate stimulation of NAc AMPARs is necessary for reversing AMPAR responsiveness. Similarly, systemic treatment with the dopamine D(1)-like agonist SKF 81297 [(+/-)-6-chloro-7,8-dihydroxy-l-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide] reversed AMPAR responsiveness in cocaine withdrawal, but the effect was prevented by local NAc AMPAR blockade in the NAc, and not local D(1)-like receptor blockade, suggesting a role for glutamate afferents in the reversal of enhanced AMPAR sensitivity. Together, these findings suggest that cocaine-induced glutamate release in sensitized animals is responsible for dynamic alterations in AMPAR function that contribute to enhanced cocaine sensitivity.

Bidirectional redistribution of AMPA but not NMDA receptors after perforant path simulation in the adult rat hippocampus in vivo

Long-term potentiation (LTP) in vitro reveals dynamic regulation of synaptic glutamate receptors. AMPA receptors may be inserted into synapses to increase neurotransmission, whereas NMDA receptors may redistribute within the synapse to alter the probability of subsequent plasticity. To date, the only evidence for these receptor dynamics in the hippocampus is from the studies of dissociated neurons and hippocampal slices taken from young animals. Although synaptic plasticity is induced easily, the extent of AMPA and NMDA receptor mobility after LTP is unknown in the adult, intact hippocampus. To test whether AMPA or NMDAR subunits undergo activity-dependent modifications in adult hippocampal synapses, we induced LTP at perforant path-dentate gyrus (DG) synapses in anesthetized adult rats, using high frequency stimulation (HFS), verified layer-specific Arc induction, and analyzed the distribution of postsynaptic AMPA and NMDAR subunits, using immunogold electron microscopy. The number of synapses with AMPA receptor labeling increased with LTP-inducing HFS in the stimulated region of the dendrite relative to the nonstimulated regions. The opposite trend was noted with low frequency stimulation (LFS). Moreover, HFS increased and LFS decreased the ratio of synaptic to extrasynaptic AMPA receptor labeling in the postsynaptic membrane. In contrast, HFS did not significantly alter NMDAR labeling. Thus, LTP in the adult hippocampus in vivo selectively enhanced AMPA but not NMDAR labeling specifically in synapses undergoing activity-dependent plasticity relative to the remainder of the dendritic tree. The results suggest a mechanism by which rapid adjustments in synaptic strength can occur through localized AMPA receptor mobility and that this process may be competitive across the dendritic tree.

Long‐term depression of neuron to glial signalling in rat cerebellar cortex

Bergmann glial cells enclose synapses throughout the molecular layer of the cerebellum and express extrasynaptic AMPA receptors and glutamate transporters. Accordingly, stimulation of parallel fibres leads to the generation of inward currents in the glia due to AMPA receptor activation and electrogenic uptake of glutamate. Elimination of AMPA receptor Ca(2+) permeability leads to the withdrawal of glial processes and synaptic dysfunction, suggesting that AMPA receptor-mediated Ca(2+) signalling is essential for glial support of the neuronal network. Here we show that glial extrasynaptic currents (ESCs) exhibit activity-dependent plasticity, specifically, long-term depression during repetitive stimulation of parallel fibres at low frequencies (0.033-1 Hz) -- conditions in which Purkinje neuron excitatory postsynaptic currents (EPSCs) remain stable. Both the rate of onset and the magnitude of ESC depression increased with stimulation frequency. Depression was reversible following brief periods of stimulation, but became increasingly persistent as the duration of repetitive stimulation increased. All glial currents -- AMPA receptors, glutamate transporter and a recently discovered slow 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide (NBQX)-sensitive current -- were depressed. Increasing presynaptic release probability by doubling external Ca(2+) concentration did not affect the time course of depression, suggesting that neither decreased release probability nor fatigue of release sites contribute to depression. Inhibition of glutamate uptake caused a dramatic enhancement of the rate of depression, implicating glutamate in the underlying mechanism. The strength of neuron to glial signalling in the cerebellum is therefore dynamically regulated, independently of adjacent synapses, by the frequency of parallel fibre activity.

Photoinactivation of Native AMPA Receptors Reveals Their Real-Time Trafficking

AMPA receptors mediate the majority of the fast excitatory transmission in the central nervous system. Much evidence suggests that the fast trafficking of AMPA receptors into and out of the postsynaptic membrane underlies changes in synaptic strength thought to be necessary for higher cognitive functions such as learning and memory. Despite the abundance of research conducted in this area, a direct, real-time functional assay that measures the trafficking of native AMPA receptors has been lacking. Toward this aim, we use a photoreactive, irreversible antagonist of AMPA receptors, ANQX, to rapidly silence surface AMPA receptors and investigate directly the trafficking of native AMPA receptors in real time. We find that the most dynamic movement of AMPA receptors occurs by lateral movement across the surface of neurons. Fast cycling of surface AMPA receptors with receptors from internal stores does occur but exclusively at extrasynaptic somatic sites. The cycling of synaptic AMPA receptors only occurs on a much longer timescale with complete exchange requiring at least 16 hr. This cycling is not dependent on protein synthesis or action potential driven network activity. These data suggest a revised model of AMPA receptor trafficking wherein a large internal store of AMPA receptors exchanges rapidly with extrasynaptic somatic AMPA receptors, and these newly inserted AMPA receptors then travel laterally along dendrites to reside stably at synapses.