Activation of extrasynaptic NMDA receptors induces a PKC-dependent switch in AMPA receptor subtypes in mouse cerebellar stellate cells

The repetitive activation of synaptic glutamate receptors can induce a lasting change in the number or subunit composition of synaptic AMPA receptors (AMPARs). However, NMDA receptors that are present extrasynaptically can also be activated by a burst of presynaptic activity, and thus may be involved in the induction of synaptic plasticity. Here we show that the physiological-like activation of extrasynaptic NMDARs induces a lasting change in the synaptic current, by changing the subunit composition of AMPARs at the parallel fibre-to-cerebellar stellate cell synapse. This extrasynaptic NMDAR-induced switch in synaptic AMPARs from GluR2-lacking (Ca²⁺-permeable) to GluR2-containing (Ca²⁺-impermeable) receptors requires the activation of protein kinase C (PKC). These results indicate that the activation of extrasynaptic NMDARs by glutamate spillover is an important mechanism that detects the pattern of afferent activity and subsequently exerts a remote regulation of AMPAR subtypes at the synapse via a PKC-dependent pathway.     

Light triggers expression of philanthotoxin-insensitive Ca2+-permeable AMPA receptors in the developing rat retina

Ca²+-permeable AMPA receptors (AMPARs) are expressed throughout the adult CNS but yet their role in development is poorly understood. In the developing retina, most investigations have focused on Ca²⁺ influx through NMDARs in promoting synapse maturation and not on AMPARs. However, NMDARs are absent from many retinal cells suggesting that other Ca²⁺-permeable glutamate receptors may be important to consider. Here we show that inhibitory horizontal and AII amacrine cells lack NMDARs but express Ca²⁺-permeable AMPARs. Before eye-opening, AMPARs were fully blocked by philanthotoxin (PhTX), a selective antagonist of Ca²⁺-permeable AMPARs. After eye-opening, however, a subpopulation of Ca²⁺-permeable AMPARs were unexpectedly PhTX resistant. Furthermore, Joro spider toxin (JSTX) and IEM-1460 also failed to antagonize, demonstrating that this novel pharmacology is shared by several AMPAR channel blockers. Interestingly, PhTX-insensitive AMPARs failed to express in retinae from dark-reared animals demonstrating that light entering the eye triggers their expression. Eye-opening coincides with the consolidation of inhibitory cell connections suggesting that the developmental switch to a Ca²⁺-permeable AMPAR with novel pharmacology may be critical to synapse maturation in the mammalian retina.     

Impaired synaptic scaling in mouse hippocampal neurones expressing NMDA receptors with reduced calcium permeability

NMDA receptors (NMDARs) play a crucial role for the acquisition of functional AMPARs during Hebbian synaptic plasticity at cortical and hippocampal synapses over a short timescale of seconds to minutes. In contrast, homeostatic synaptic plasticity can occur over longer timescales of hours to days. The induction mechanisms of this activity-dependent synaptic scaling are poorly understood but are assumed to be independent of NMDAR signalling in the cortex. Here we investigated in the hippocampus a potential role of NMDAR-mediated Ca²⁺ influx for synaptic scaling of AMPA currents by genetic means. The Ca²⁺ permeability of NMDARs was reduced by selective postnatal expression in principal neurones of mouse forebrain half of the NR1 subunits with an amino acid substitution at the critical channel site (N598R). This genetic manipulation did not reduce the total charge transfer via NMDARs in nucleated patches (somatic) and at synaptic sites. In contrast, the current amplitude and the charge carried through AMPARs were substantially reduced at somatic and synaptic sites in juvenile and adult mutants, indicating persistent downscaling of AMPA responses. Smaller and less frequent AMPA miniature currents in the mutant demonstrated a postsynaptic locus of this down-regulation. Afferent innervation and release probability were unchanged at CA3-to-CA1 synapses of mutants, as judged from input-output and minimal stimulation experiments. Our results indicate that NMDAR-mediated Ca²⁺ signalling is important for synaptic scaling of AMPA currents in the hippocampus in vivo .     

PKC and polyamine modulation of GluR2-deficient AMPA receptors in immature neocortical pyramidal neurons of the rat

AMPA receptors (AMPARs) mediate the bulk of fast synaptic excitation in the CNS. We have recently shown that AMPAR-dependent synaptic transmission in immature neocortical pyramidal neurons is mediated by GluR2-deficient receptors that can be modulated by intra- or extracellular polyamines (PAs). Phosphorylation of AMPARs, e.g. by PKC, can lead to enhanced excitation, and PAs are known to modulate PKC activity. Therefore, PAs and PKC might interact to influence AMPAR function. To test this hypothesis, we made whole cell recordings from immature (P12–14) layer V pyramidal neurons and assayed two measures of PA influence on synaptic AMPAR function – inward rectification and use-dependent unblock (UDU), with the latter assayed by differences in rectification between a pair of EPSCs evoked at short (50 ms) latencies. We have previously shown that EPSCs in immature pyramidal neurons displayed inward rectification, which was enhanced by intracellular spermine, as was UDU. Staurosporin (ST), a PKC inhibitor, reversed the effect of PA on rectification and UDU, suggesting that PKC modulates postsynaptic activation of AMPARs. Similarly, polyamine-dependent rectification of spontaneous EPSCs was reversed by treatment with ST or GFX109203X, a specific PKC inhibitor. Chelating intracellular Ca²⁺ with BAPTA reproduced the effects of ST. In addition, PA immunoreactivity in layer V pyramidal neurons was reduced by PKC inhibition indicating that PKC activity influences PA metabolism. Taken together, these data support the involvement of postsynaptic PKC activation in both the inward rectification and UDU of EPSCs in immature rat cortex, and suggest an important mechanism by which excitatory synaptic transmission can be dynamically modulated by changes in either [Ca²⁺]_i or [PA]_i.     

Glutamatergic synapses in the rat nucleus tractus solitarii develop by direct insertion of calcium-impermeable AMPA receptors and without activation of NMDA receptors

Calcium influxes through ionotropic glutamate receptors (AMPA and NMDA receptors, AMPARs and NMDARs) are considered to be critical for the shaping and refinement of neural circuits during synaptogenesis. Using a combined morphological and electrophysiological approach, we evaluated this hypothesis at the level of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. We confirmed that in the NTS, the first excitatory synapses appeared at embryonic day 18. We next characterized the biophysical properties of NTS AMPARs. Throughout perinatal development, both evoked and miniature EPSCs recorded in the presence of an NMDAR blocker were insensitive to polyamines and had linear current–voltage relationships. This demonstrated that AMPARs at NTS excitatory synapses were calcium-impermeable receptors composed of a majority of GluR₂ subunits. We then investigated the influence of calcium influxes through NMDARs on the development of NTS synaptic transmission. We found that NMDAR expression at synaptic sites did not precede AMPAR expression. Moreover, NMDAR blockade in utero did not prevent the development of AMPAR synaptic currents and the synaptic clustering of GluR₂ subunits. Thus, our data support an alternative model of synaptogenesis that does not depend on calcium influxes through either AMPARs or NMDARs. This model may be particularly relevant to the formation of neural networks devoted to basic behaviours required at birth for survival.     

Regulation of synaptic signalling by postsynaptic, non-glutamate receptor ion channels

Activation of glutamatergic synapses onto pyramidal neurons produces a synaptic depolarization as well as a buildup of intracellular calcium (Ca²⁺). The synaptic depolarization propagates through the dendritic arbor and can be detected at the soma with a recording electrode. Current influx through AMPA-type glutamate receptors (AMPARs) provides the depolarizing drive, and the amplitudes of synaptic potentials are generally thought to reflect the number and properties of these receptors at each synapse. In contrast, synaptically evoked Ca²⁺ transients are limited to the spine containing the active synapse and result primarily from Ca²⁺ influx through NMDA-type glutamate receptors (NMDARs). Here we review recent studies that reveal that both synaptic depolarizations and spine head Ca²⁺ transients are strongly regulated by the activity of postsynaptic, non-glutamate receptor ion channels. In hippocampal pyramidal neurons, voltage- and Ca²⁺-gated ion channels located in dendritic spines open as downstream consequences of glutamate receptor activation and act within a complex signalling loop that feeds back to regulate synaptic signals. Dynamic regulation of these ion channels offers a powerful mechanism of synaptic plasticity that is independent of direct modulation of glutamate receptors.     

Differential activity-dependent regulation of the lateral mobilities of AMPA and NMDA receptors

The basis for differences in activity-dependent trafficking of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) remains unclear. Using single-molecule tracking, we found different lateral mobilities for AMPARs and NMDARs: changes in neuronal activity modified AMPAR but not NMDAR mobility, whereas protein kinase C activation modified both. Differences in mobility were mainly detected for extrasynaptic AMPARs, suggesting that receptor diffusion between synaptic and extrasynaptic domains is involved in plasticity processes.     

LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1.

In the CA1 region of the rat hippocampus, long-term potentiation (LTP) requires the activation of NMDA receptors (NMDARs) and leads to an enhancement of AMPA receptor (AMPAR) function. In neonatal hippocampus, this increase in synaptic strength seems to be mediated by delivery of AMPARs to the synapse. Here we studied changes in surface expression of native AMPA and NMDA receptors following induction of LTP in the adult rat brain. In contrast to early postnatal rats, we find that LTP in the adult rat does not alter membrane association of AMPARs. Instead, LTP leads to rapid surface expression of NMDARs in a PKC- and Src-family-dependent manner. The present study suggests a developmental shift in the LTP-dependent trafficking of AMPA receptors. Moreover, our results indicate that insertion of NMDA receptors may be a key step in regulating synaptic plasticity.     

GluN2B subunits of the NMDA receptor contribute to the AMPA receptor internalization during long-term depression in the lateral amygdala of juvenile rats

The lateral nucleus of the amygdala (LA) is a critical structure involved in fear conditioning. We recently showed that regulated exocytosis and endocytosis of postsynaptic A-amino-3-hydroxy-5-methylisoxazole-4-propionate subtype of glutamate receptors (AMPARs) are involved in the expression of N-methyl-D-aspartate subtype glutamate receptors (NMDARs) dependent long-term potentiation (LTP) and long-term depression (LTD) in coronal slices of the LA. However, the molecular mechanisms of this effect remain unclear. In the present study, we investigated the role of distinct NMDAR subtypes in the endocytosis of AMPARs during LTD expression at the synapses of the thalamic inputs to the LA neurons. Here we show that the NMDARs antagonist DL-2-amino-5-phosphonovalerate (D-APV) blocked the induction of LTD and thus prevented endocytosis of surface AMPARs, indicating that NMDAR activation enhanced the internalization of AMPARs in LTD expression. Furthermore, the selective blocking of GluN2B-containing NMDARs completely abolished the NMDAR-induced AMPAR endocytosis, whereas preferential inhibition of GluN2A-containing NMDARs did not block the NMDAR-induced AMPAR endocytosis during LTD expression. These results suggest that there exist a preferred NMDAR subtype for AMPAR internalization and activation of GluN2B-containing NMDARs represent the predominate pathway triggered during the early stages of this NMDAR-induced endocytosis of AMPARs during LTD in the thalamic inputs to the LA of juvenile rats.     

AMPA receptor subunit GluR1 (GluA1) serine-845 site is involved in synaptic depression but not in spine shrinkage associated with chemical long-term depression

The structure of dendritic spines is highly plastic and can be modified by neuronal activity. In addition, there is evidence that spine head size correlates with the synaptic α-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor (AMPAR) content, which suggests that they may be coregulated. Although there is evidence that there are overlapping mechanisms for structural and functional plasticity, the extent of the overlap needs further investigation. Specifically, it is unknown whether AMPAR levels determine spine size or whether both are regulated via parallel pathways. We studied the correlation between spine structural plasticity and long-term synaptic plasticity following chemical-induced long-term depression (chemLTD). In particular, we examined whether the regulation of AMPARs, which is implicated in LTD, is critical for spine morphological plasticity. We used mutant mice specifically lacking the serine-845 site on the type 1 glutamate receptor (GluR1, or GluA1) subunit of AMPARs (mutants). These mice specifically lack N-methyl-D-aspartate (NMDA) receptor (NMDAR)-dependent LTD and NMDAR activation-induced AMPAR endocytosis. We found that chemLTD causes a rapid and persistent shrinkage in spine head volume of hippocampal CA1 pyramidal neurons in wild types similar to that reported in other studies using low-frequency stimulation (LFS)-induced LTD. Surprisingly, we found that although S845A mutant mice display impaired chemLTD, the shrinkage of spine head volume occurred to a similar magnitude to that observed in wild types. Our results suggest that there is dissociation in the molecular mechanisms underlying functional LTD and spine shrinkage and that GluR1-S845 regulation is not necessary for spine morphological plasticity.     

Cross talk between synaptic receptors mediates NMDA-induced suppression of inhibition

Past research has shown that calcium influx through NMDA receptors (NMDARs) depresses GABA(A) currents. We examined upstream triggers of this suppression, including involvement of target synaptic GABA(A) receptors and the NMDARs triggering suppression. In hippocampal neurons, conditioning with 20 μM NMDA for 20 s caused 50% suppression of GABA responses. The suppression was delayed by ≈ 60 s following NMDA application and persisted for at least 5 min following conditioning. Pharmacology experiments suggested a shift in both the sensitivity to GABA and a loss of functional receptors. NMDA conditioning strongly suppressed inhibitory postsynaptic currents and speeded decay kinetics. Synaptic NMDAR conditioning was necessary to suppress GABA current in pyramidal neurons; extrasynaptic NMDAR activation did not suppress, even when matched to synaptic activation. We found no evidence that specific synaptic NMDAR subunits mediate depression of GABA responses. Although physical colocalization of glutamate and GABA(A) receptors is mostly likely in extrasynaptic regions, our evidence suggests that NMDAR-induced suppression of GABA responsiveness prominently affects precise, moment-to-moment signaling from synaptic receptors to synaptic receptors.     

Subunit interaction with PICK and GRIP controls Ca2+ permeability of AMPARs at cerebellar synapses

At many excitatory central synapses, activity produces a lasting change in the synaptic response by modifying postsynaptic AMPA receptors (AMPARs). Although much is known about proteins involved in the trafficking of Ca2+-impermeable (GluR2-containing) AMPARs, little is known about protein partners that regulate subunit trafficking and plasticity of Ca2+-permeable (GluR2-lacking) AMPARs. At cerebellar parallel fiber-stellate cell synapses, activity triggers a novel type of plasticity: Ca2+ influx through GluR2-lacking synaptic AMPARs drives incorporation of GluR2-containing AMPARs, generating rapid, lasting changes in excitatory postsynaptic current properties. Here we examine how glutamate receptor interacting protein (GRIP, also known as AMPAR binding protein or ABP) and protein interacting with C-kinase-1 (PICK) regulate subunit trafficking and plasticity. We find that repetitive synaptic activity triggers loss of synaptic GluR2-lacking AMPARs by selectively disrupting their interaction with GRIP and that PICK drives activity-dependent delivery of GluR2-containing receptors. This dynamic regulation of AMPARs provides a feedback mechanism for controlling Ca2+ permeability of synaptic receptors.     

AMPA receptor-dependent, light-evoked D-serine release acts on retinal ganglion cell NMDA receptors

NMDA receptor (NMDAR) activation requires coincident binding of the excitatory neurotransmitter glutamate and a coagonist, either glycine or d-serine. Changes in NMDAR currents during neural transmission are typically attributed to glutamate release against a steady background of coagonist, excluding the possibility of coagonist release. AMPA receptor (AMPAR) stimulation evokes d-serine release, but it is unknown whether this is a physiological phenomenon capable of influencing synaptic responses. In this study, we utilized the intact retina to determine whether light-evoked synaptic activity in retinal ganglion cells (RGCs) is shaped by a dynamic pool of coagonist. The application of AMPAR antagonist abolished light-evoked NMDAR currents, which were rescued by adding coagonist to the bath. When NMDA was globally applied to RGCs via bath or picospritzing, the coagonist occupancy was also dependent on AMPARs but to a lesser extent than that observed during light responses, suggesting a difference in extrasynaptic coagonist regulation. By saturating the glutamate binding site of NMDARs, we were able to detect released coagonist reaching RGCs during light-evoked responses. Mutant mice lacking the d-serine-synthesizing enzyme serine racemase were deficient in coagonist release. Coagonist release in wild-type retinas was notably greater in ON than in OFF responses and depended on AMPARs. These findings suggest activity-dependent modulation of coagonist availability, particularly d-serine, and may add an extra dimension to NMDAR coincidence detection in the retina.     

Impaired Regulation of Synaptic Strength in Hippocampal Neurons from GluR1-Deficient Mice

Neurons of the central nervous system (CNS) exhibit a variety of forms of synaptic plasticity, including associative long-term potentiation and depression (LTP/D), homeostatic activity-dependent scaling and distance-dependent scaling. Regulation of synaptic neurotransmitter receptors is currently thought to be a common mechanism amongst many of these forms of plasticity. In fact, glutamate receptor 1 (GluR1 or GluRA) subunits containing L-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors have been shown to be required for several forms of hippocampal LTP and a particular hippocampal-dependent learning task. Because of this importance in associative plasticity, we sought to examine the role of these receptors in other forms of synaptic plasticity in the hippocampus. To do so, we recorded from the apical dendrites of hippocampal CA1 pyramidal neurons in mice lacking the GluR1 subunit (GluR1 −/−). Here we report data from outside-out patches that indicate GluR1-containing receptors are essential to the extrasynaptic population of AMPA receptors, as this pool was nearly empty in the GluR1 −/− mice. Additionally, these receptors appear to be a significant component of the synaptic glutamate receptor pool because the amplitude of spontaneous synaptic currents recorded at the site of input and synaptic AMPA receptor currents evoked by focal glutamate uncaging were both substantially reduced in these mice. Interestingly, the impact on synaptic weight was greatest at distant synapses such that the normal distance-dependent synaptic scaling used by these cells to counter dendritic attenuation was lacking in GluR1 −/− mice. Together the data suggest that the highly regulated movement of GluR1-containing AMPA receptors between extrasynaptic and synaptic receptor pools is critically involved in establishing two functionally diverse forms of synaptic plasticity: LTP and distance-dependent scaling.     

Non-ionotropic cross-talk between AMPA and NMDA receptors in rodent hippocampal neurones

Many fast excitatory synapses in the hippocampus are enriched with both AMPARs (α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors) and NMDARs ( N -methyl- D -aspartate receptors). Their proximity allows them to be activated simultaneously by the same neurotransmitter, L -glutamate. Activation of AMPARs leads to influx of sodium and calcium ions, which can increase or decrease NMDAR activity through sodium concentration-dependent cascades or a calcium-calmodulin-dependent inactivation process, respectively. Here we provide evidence that the activation of AMPARs inhibits NMDARs through a non-ionotropic mechanism. NMDA-induced current in isolated rat CA1 hippocampal cells and nucleated patches of cultured mouse hippocampal neurones decreased when AMPARs were activated. Conversely, when AMPARs were blocked, the NMDA component of glutamate-induced current increased. The inhibitory action of AMPAR activation on NMDAR-mediated current depends upon the open state of AMPA channels and rapidly diminishes after deactivation of AMPARs. The inhibitory action was independent of membrane voltage, univalent cation fluxes and calcium influx. The AMPA-NMDA cross-inhibition also occurred in evoked synaptic current in CA1 neurones from intact mouse hippocampal slices. This cross-talk may play a role in preventing overexcitation during bursting activities in the hippocampus.     

Postfusional regulation of cleft glutamate concentration during LTP at 'silent synapses'

'Silent synapses' show responses from high-affinity NMDA receptors (NMDARs) but not low-affinity AMPA receptors (AMPARs), but gain AMPAR responses upon long-term potentiation (LTP). Using the rapidly reversible NMDAR antagonist l-AP5 to assess cleft glutamate concentration ([glu]cleft), we found that it peaked at <170 microM at silent neonatal synapses, but greatly increased after potentiation. Cyclothiazide (CTZ), a potentiator of AMPAR, revealed slowly rising AMPA EPSCs at silent synapses; LTP shortened their rise times. Thus, LTP at silent synapses increased rate-of-rise and peak amplitude of [glu]cleft. Release probability reported by NMDARs remained unchanged during LTP, implying that [glu]cleft increases arose from immediately presynaptic terminals. Our data suggest that changes in the dynamics of fusion-pore opening contribute to LTP.     

Capsaicin-sensitive neurogenic sensory vasodilatation in the dura mater of the rat

PSD-95 is one of the most abundant proteins found in the postsynaptic density of excitatory synapses. However, the precise functional role played by PSD-95 in regulating synaptic transmission and plasticity remains undefined. To address this issue, we have overexpressed PSD-95 in cortical pyramidal neurons in organotypic brain slices using particle-mediated gene transfer and assessed the consequences on synaptic transmission and plasticity. The AMPA receptor/NMDA receptor (AMPAR/NMDAR) ratio of evoked EPSCs recorded at +40 mV was greater in PSD-95-transfected pyramidal neurons than in controls. This difference could not be accounted for by a change in rectification of AMPAR-mediated synaptic currents since the current-voltage curves obtained in controls and in PSD-95-transfected neurons were indistinguishable. However, the amplitude of AMPAR-mediated evoked EPSCs was larger in PSD-95-transfected neurons compared to matched controls. Paired-pulse ratio analysis suggested that overexpression of PSD-95 did not alter presynaptic release probability. Transfection of PSD-95 was further accompanied by an increase in the frequency, but not amplitude, of AMPAR-mediated mEPSCs. Together, these results indicate that transfection of PSD-95 increased AMPAR-mediated synaptic transmission. Furthermore, they suggest that this phenomenon reflects an increased number of synapses expressing AMPARs rather than an increased number or function of these receptors at individual synapses. We tested the consequences of these changes on synaptic plasticity and found that PSD-95 transfection greatly enhanced the probability of observing long-term depression. These results thus identify a physiological role for PSD-95 and demonstrate that this protein can play a decisive role in controlling synaptic strength and activity-dependent synaptic plasticity.     

The density of AMPA receptors activated by a transmitter quantum at the climbing fibre-Purkinje cell synapse in immature rats

We aimed to estimate the number of AMPA receptors (AMPARs) bound by the quantal transmitter packet, their single-channel conductance and their density in the postsynaptic membrane at cerebellar Purkinje cell synapses. The synaptic and extrasynaptic AMPARs were examined in Purkinje cells in 2- to 4-day-old rats, when they receive synaptic inputs solely from climbing fibres (CFs). Evoked CF EPSCs and whole-cell AMPA currents displayed roughly linear current-voltage relationships, consistent with the presence of GluR2 subunits in synaptic and extrasynaptic AMPARs. The mean quantal size, estimated from the miniature EPSCs (MEPSCs), was ∼300 pS. Peak-scaled non-stationary fluctuation analysis of spontaneous EPSCs and MEPSCs gave a weighted-mean synaptic channel conductance of ∼5 pS (∼7 pS when corrected for filtering). By applying non-stationary fluctuation analysis to extrasynaptic currents activated by brief glutamate pulses (5 m m ), we also obtained a small single-channel conductance estimate for extrasynaptic AMPARs (∼11 pS). This approach allowed us to obtain a maximum open probability ( P _o,max) value for the extrasynaptic receptors ( P _o,max= 0.72). Directly resolved extrasynaptic channel openings in the continued presence of glutamate exhibited clear multiple-conductance levels. The mean area of the postsynaptic density (PSD) of these synapses was 0.074 μm², measured by reconstructing electron-microscopic (EM) serial sections. Postembedding immunogold labelling by anti-GluR2/3 antibody revealed that AMPARs are localised in PSDs. From these data and by simulating error factors, we estimate that at least 66 AMPARs are bound by a quantal transmitter packet at CF-Purkinje cell synapses, and the receptors are packed at a minimum density of ∼900 μm⁻² in the postsynaptic membrane.     

Switch to Ca2+-permeable AMPA and reduced NR2B NMDA receptor-mediated neurotransmission at dorsal horn nociceptive synapses during inflammatory pain in the rat

Glutamate receptor response properties of nociceptive synapses on neurokinin 1 receptor positive (NK1R⁺) lamina I neurons were determined 3 days after induction of chronic peripheral inflammation with Freund's Complete Adjuvant (CFA). A significant increase in the AMPAR/NMDAR ratio was found during inflammation, which was associated with a significant reduction in the quantal amplitude of NMDAR-mediated synaptic currents. A significant shortening of the quantal AMPA current decay, a greater inward rectification of the AMPAR-mediated eEPSC amplitude and an increased sensitivity to the Ca²⁺-permeable AMPAR channel blocker 1-naphthylacetyl spermine (NAS) was also observed, indicating an increase in the contribution of Ca²⁺-permeable AMPARs at this synapse during inflammation. Furthermore the reduced effectiveness of the NR2B-specific antagonist CP-101,606 on NMDAR-mediated eEPSCs together with a decrease in Mg²⁺ sensitivity suggests a down regulation of the highly Mg²⁺-sensitive and high conductance NR2B subunit at this synapse. These changes in glutamatergic receptor function during inflammation support the selective effectiveness of Ca²⁺-permeable AMPAR antagonists in inflammatory pain models and may underlie the reported ineffectiveness of NR2B antagonists in spinal antinociception.