Evaluation of PhTX-74 as subtype-selective inhibitor of GluA2-containing AMPA receptors.

The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are glutamate-gated cation channels that mediate fast excitatory synaptic transmission in the central nervous system. AMPARs are tetramers formed by homo- or heteromeric assembly of GluA1-4 subunits to produce multiple subtypes with varying biophysical properties. Polyamine toxins such as joro spider toxins, philanthotoxins (PhTXs), and argiotoxins are use-dependent ion channel blockers of AMPARs widely employed as highly potent antagonists of GluA2-lacking receptor subtypes. In addition to this use, recent findings have indicated that a philanthotoxin analog, PhTX-74, can distinguish among GluA2-containing AMPAR subtypes in the presence of the prototypical transmembrane AMPAR regulatory protein γ-2 (or stargazin). Thus, PhTX-74 may be of potential use in studies of the neurobiological role of GluA2-containing subtypes. We have evaluated the pharmacological profile of PhTX-74 and related polyamine toxins at homo- and heteromeric AMPARs in the presence and absence of γ-2. Determination of IC(50) values for inhibition of glutamate-evoked currents from Xenopus oocytes expressing recombinant homo- or heteromeric combinations of GluA1, GluA2, and GluA3 in the presence of γ-2 shows that PhTX-74 inhibits homomeric GluA1 and GluA3 receptors nonselectively, with IC(50) values in the nanomolar range (252-356 nM), and heteromeric GluA1/A2 and GluA2/A3 receptors nonselectively, with IC(50) values in the micromolar range (22 µM). Thus, in contrast to earlier findings, we find that PhTX-74 cannot pharmacologically discriminate between GluA2-containing AMPAR subtypes.

Synaptic AMPA receptor exchange maintains bidirectional plasticity.

Activity-dependent synaptic delivery of GluR1-, GluR2L-, and GluR4-containing AMPA receptors (-Rs) and removal of GluR2-containing AMPA-Rs mediate synaptic potentiation and depression, respectively. The obvious puzzle is how synapses maintain the capacity for bidirectional plasticity if different AMPA-Rs are utilized for potentiation and depression. Here, we show that synaptic AMPA-R exchange is essential for maintaining the capacity for bidirectional plasticity. The exchange process consists of activity-independent synaptic removal of GluR1-, GluR2L-, or GluR4-containing AMPA-Rs and refilling with GluR2-containing AMPA-Rs at hippocampal and cortical synapses in vitro and in intact brains. In GluR1 and GluR2 knockout mice, initiation or completion of synaptic AMPA-R exchange is compromised, respectively. The complementary AMPA-R removal and refilling events in the exchange process ultimately maintain synaptic strength unchanged, but their long rate time constants ( approximately 15-18 hr) render transmission temporarily depressed in the middle of the exchange. These results suggest that the previously hypothesized "slot" proteins, rather than AMPA-Rs, code and maintain transmission efficacy at central synapses.

Agonist occupancy is essential for forward trafficking of AMPA receptors.

Regulated trafficking of AMPA receptors to cell surface and to synapses is an important determinant of neuronal excitability. In the present study, we have addressed the role of agonist binding and desensitization in the early trafficking of glutamate receptor-D (GluR-D) AMPA receptors. Analysis of point-mutated GluR-D receptors, via electrophysiology and immunofluorescence, revealed that agonist-binding activity is essential for efficient delivery to cell surface in transfected cell lines and in neurons. Cotransfection with stargazin could fully rescue the surface expression of nonbinding mutant receptors in cell lines, indicating that stargazin is able to interact with and promote exit of AMPA receptors from endoplasmic reticulum (ER) independently of agonist binding. Secretion of separately expressed ligand-binding domain constructs showed a similar dependency of agonist binding to that observed with full-length GluR-D, supporting the idea that glutamate-induced closure of the binding site cleft is registered by ER quality control as a necessary priming step for transport competence. In contrast to agonist binding, the ability of the receptor to undergo desensitization had only a minor influence on trafficking. Our results are consistent with the hypothesis that AMPA receptors are synthesized as intrinsically unstable molecules, which require glutamate binding for structural stability and for transport-competence.

Input-specific intrasynaptic arrangements of ionotropic glutamate receptors and their impact on postsynaptic responses.

To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.

TARP redundancy is critical for maintaining AMPA receptor function.

Transmembrane AMPA receptor regulatory proteins (TARPs) are AMPA receptor auxiliary subunits that influence diverse aspects of receptor function. However, the full complement of physiological roles for TARPs in vivo remains poorly understood. Here we find that double knock-out mice lacking TARPs gamma-2 and gamma-3 are profoundly ataxic and fail to thrive. We demonstrate that these TARPs are critical for the synaptic targeting and kinetics of AMPA receptors in cerebellar Golgi cells, but that either alone is sufficient to fully preserve function. By analyzing the few remaining synaptic AMPA receptors in the gamma-2, gamma-3 double knock-out mice, we unexpectedly find that these TARPs specify AMPA receptor subunit composition. This study establishes a new role for TARPs in regulating AMPA receptor assembly and suggests that TARPs are necessary for proper AMPA receptor localization and function in most, if not all, neurons of the CNS.

Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A convergently regulate the synaptic ratio of ionotropic glutamate receptor subclasses.

A current hypothesis proposes that fragile X mental retardation protein (FMRP), an RNA-binding translational regulator, acts downstream of glutamatergic transmission, via metabotropic glutamate receptor (mGluR) G(q)-dependent signaling, to modulate protein synthesis critical for trafficking ionotropic glutamate receptors (iGluRs) at synapses. However, direct evidence linking FMRP and mGluR function with iGluR synaptic expression is limited. In this study, we use the Drosophila fragile X model to test this hypothesis at the well characterized glutamatergic neuromuscular junction (NMJ). Two iGluR classes reside at this synapse, each containing common GluRIIC (III), IID and IIE subunits, and variable GluRIIA (A-class) or GluRIIB (B-class) subunits. In Drosophila fragile X mental retardation 1 (dfmr1) null mutants, A-class GluRs accumulate and B-class GluRs are lost, whereas total GluR levels do not change, resulting in a striking change in GluR subclass ratio at individual synapses. The sole Drosophila mGluR, DmGluRA, is also expressed at the NMJ. In dmGluRA null mutants, both iGluR classes increase, resulting in an increase in total synaptic GluR content at individual synapses. Targeted postsynaptic dmGluRA overexpression causes the exact opposite GluR phenotype to the dfmr1 null, confirming postsynaptic GluR subtype-specific regulation. In dfmr1; dmGluRA double null mutants, there is an additive increase in A-class GluRs, and a similar additive impact on B-class GluRs, toward normal levels in the double mutants. These results show that both dFMRP and DmGluRA differentially regulate the abundance of different GluR subclasses in a convergent mechanism within individual postsynaptic domains.

Stargazin modulates AMPA receptor antagonism.

AMPA receptors mediate the majority of fast synaptic transmission and underlie several forms of synaptic plasticity. AMPARs also have an important role in several neuronal pathologies. Therefore, studying the structure and function of these receptors is important for understanding general mechanisms of synaptic transmission as well as for the development of new therapies. A recent study identified the apparent binding sites for GYKI 53655 (GYKI) and CP-465,022 (CP) at the interface between the glutamate binding core and the transmembrane domains. The emerging role of transmembrane AMPA receptor regulatory proteins (TARPs) in AMPAR function raises the possibility that the antagonism of these receptors is also affected by TARPs such as stargazin. Here we compare the antagonism of the competitive antagonist CNQX and the negative allosteric modulators GYKI, and CP in the absence and presence of stargazin. We found that stargazin decreases the apparent affinity of GluR1 for CNQX, which is most likely explained by a partial agonistic effect of CNQX. In contrast, stargazin increases the affinity for GYKI, and has only a small effect on CP binding. Because inhibition of recently described GYKI insensitive receptors is restored by co-expression with stargazin, our data suggest that the identified residues do not constitute the full GYKI binding site. We could show that the ectodomain of stargazin controls the change in agonist sensitivity. Mutations in the identified binding regions for GYKI and CP dramatically reduced surface expression. Our data provides further evidence that TARPs alter the conformation of pore-forming subunits and thereby affects antagonist interaction.

Isoform-specific early trafficking of AMPA receptor flip and flop variants.

Flip and flop splice variants of AMPA receptor subunits are expressed in distinct but partly overlapping patterns and impart different desensitization kinetics to cognate receptor channels. In the absence of specific antibodies, isoform-specific differences in trafficking or localization of native flip and flop subunits remain uncharacterized. We report that in several transfected cell lines, transport of homomeric glutamate receptor (GluR)-D(flop) receptors is largely blocked at the endoplasmic reticulum (ER) exit, whereas GluR-D(flip) undergoes complex glycosylation and reaches the plasma membrane at >10x higher levels than GluR-D(flop), as determined by immunofluorescence, patch-clamp recordings and biochemical assays. The transport difference between flip and flop is independent of activity, is primarily determined by amino acid residue 780 (Leu in flop, Val in flip), and is manifested even in the secretion of the soluble ligand-binding domain, suggesting it is independent of oligomerization. Coexpression with stargazin or with the flip isoform rescues the surface expression of GluR-D(flop) near to the level exhibited by GluR-D(flip). Our results demonstrate that the extracellular flip/flop region, via interactions with ER luminal splice form-specific protein(s), plays a hitherto unappreciated and important role in AMPA-receptor trafficking.

Protein-protein coupling/uncoupling enables dopamine D2 receptor regulation of AMPA receptor-mediated excitotoxicity.

here is considerable evidence that dopamine D2 receptors can modulate AMPA receptor-mediated neurotoxicity. However, the molecular mechanism underlying this process remains essentially unclear. Here we report that D2 receptors inhibit AMPA-mediated neurotoxicity through two pathways: the activation of phosphoinositide-3 kinase (PI-3K) and downregulation of AMPA receptor plasma membrane expression, both involving a series of protein-protein coupling/uncoupling events. Agonist stimulation of D2 receptors promotes the formation of the direct protein-protein interaction between the third intracellular loop of the D2 receptor and the ATPase N-ethylmaleimide-sensitive factor (NSF) while uncoupling the NSF interaction with the carboxyl tail (CT) of the glutamate receptor GluR2 subunit of AMPA receptors. Previous studies have shown that full-length NSF directly couples to the GluR2CT and facilitates AMPA receptor plasma membrane expression. Furthermore, the CT region of GluR2 subunit is also responsible for several other intracellular protein couplings, including p85 subunit of PI-3K. Therefore, the direct coupling of D2-NSF and concomitant decrease in the NSF-GluR2 interaction results in a decrease of AMPA receptor membrane expression and an increase in the interaction between GluR2 and the p85 and subsequent activation of PI-3K. Disruption of the D2-NSF interaction abolished the ability of D2 receptor to attenuate AMPA-mediated neurotoxicity by blocking the D2 activation-induced changes in PI-3K activity and AMPA receptor plasma membrane expression. Furthermore, the D2-NSF-GluR2-p85 interactions are also responsible for the D2 inhibition of ischemia-induced cell death. These data may provide a new avenue to identify specific targets for therapeutics to modulate glutamate receptor-governed diseases, such as stroke.

Regulation of editing and expression of glutamate alpha-amino-propionic-acid (AMPA)/kainate receptors by antidepressant drugs.

BACKGROUND: Several reports have shown that the glutamatergic system is involved in both the pathogenesis of affective and stress-related disorders and in the action of antidepressant drugs. In particular, antidepressant treatment was shown to modulate expression and function of ionotropic glutamate receptors, to inhibit glutamate release and to restore synaptic plasticity impaired by stress. METHODS: We analyzed the mRNA expression and RNA editing of alpha-amino-propionic-acid (AMPA) and kainate (KA) receptor subunits, in the pre-frontal/frontal cortex (P/FC) and hippocampus (HI) of rats chronically treated with three different drugs: the selective serotonin (5-HT) reuptake inhibitor fluoxetine, the selective noradrenaline (NA) reuptake inhibitor reboxetine and the tricyclic antidepressant desipramine. RESULTS: Our data showed that fluoxetine and desipramine exerted moderate but selective effects on glutamate receptor expression and editing, while reboxetine appeared to be the drug that affects glutamate receptors (GluR) most. The most consistent effect, observed with pronoradrenergic drugs (desipramine and reboxetine), was a decrease of GluR3 expression both in P/FC and HI. Interestingly, in HI, the same drugs also decreased the editing levels of either the flip (desipramine) or flop (reboxetine) form of GluR3. CONCLUSIONS: Overall, these results point to specific and regionally discrete changes in the expression and editing level of glutamate receptors and, in particular, to a selective reduction of conductance for GluR3-containing receptors following treatment with antidepressant drugs. These data support the hypothesis that changes in glutamate neurotransmission are involved in the therapeutic effects induced by these drugs.

Activity-dependent change in AMPA receptor properties in cerebellar stellate cells.

High-frequency synaptic stimulation is thought to cause a rapid and lasting change in the expression of GluR2 subunit-containing AMPA receptors (AMPARs) at synapses in cerebellar stellate cells. We examined whether spontaneous synaptic activity affects the expression of GluR2-containing synaptic AMPARs and whether the change in AMPAR subtypes alters their Ca(2+) permeability and kinetic properties. We used intracellular spermine, which blocks GluR2-lacking receptors at depolarized potentials, to distinguish the presence of GluR2. In most cells, the spontaneous EPSC frequency was low, and evoked EPSCs displayed inwardly rectifying I-V relationships, indicative of the presence of GluR2-lacking AMPARs. However, in cells that displayed a higher rate of spontaneous synaptic activity, EPSCs gave linear I-V plots, suggesting the presence of GluR2-containingAMPARs. This is consistent with the idea that spontaneous synaptic activity increased the expression of GluR2-containing AMPARs at synapses. The Ca(2+) permeability of AMPARs that gave inwardly rectifying currents in outside-out patches from TTX-treated cells was six times higher than in control cells that gave linear or outwardly rectifying I-V plots. However, increased spontaneous synaptic activity did not significantly alter the EPSC decay time. Furthermore, the decay time course ofEPSCs mediated by GluR2-containing receptors was not different from that mediated by a mixed population of receptors at the same synapse. Our results suggest that the level of spontaneous synaptic activity can determine the subunit composition of postsynaptic receptors at this synapse. The activity-induced expression of GluR2-containing receptors significantly reduced the Ca(2+) permeability of AMPARs in stellate cells but did not slow the decay time course of synaptic currents.

New developments in the molecular pharmacology of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and kainate receptors.

Separation of non-N-methyl-D-aspartate subtypes of glutamate receptors, known as alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors, is traced through conventional pharmacology to molecular biology. The physiology and pharmacology of recombinant receptor subtypes (GluR1-7 and KA1-2) are described. Competitive antagonists, e.g., the quinoxalinedione, 2,3-dihyroxy-6-nitro-7-sulphamoyl-benz(F)quinoxaline, and the decahydroisoquinoline, 3S,4aR,6R, 8aR-6-[2-(1(2)H-tetrazol-5-yl)ethyl]-decahydroisoquinolin e-3-carboxylate, have a broad antagonist spectrum, except that the latter is inactive on GluR6. The 2,3-benzodiazepines noncompetitively antagonise the AMPA receptor GluR1-4. Desensitisation of AMPA (GluR1-4) and kainate (GluR5-7 and KA1-2) receptors is blocked by cyclothiazide and concanavalin A, respectively. Polyamine toxins block AMPA receptors not containing GluR2 and unedited kainate receptors (GluR5-6). These data correlate well with results on native neurons characterised by techniques such as in situ hybridisation.

Extracellular protons differentially potentiate the responses of native AMPA receptor subtypes regulating neurotransmitter release.

1. The effects of pH changes on the basal and evoked release of [(3)H]noradrenaline ([(3)H]NA) and [(3)H]5-hydrohytryptamine ([(3)H]5-HT) from hippocampal synaptosomes and of [(3)H]dopamine ([(3)H]DA) and [(3)H]acetylcholine ([(3)H]ACh) from striatal and cortical synaptosomes were investigated in rat brain. 2. Changing pH between 6.4 and 8.0 did not affect the spontaneous release of the four [(3)H]neurotransmitters; alkalinization to pH 8.8 significantly enhanced release. Acidification to pH 6.4 augmented the AMPA-evoked overflows of [(3)H]NA, [(3)H]5-HT and [(3)H]DA, but not that of [(3)H]ACh. In contrast, lowering pH to 6.4 decreased the K(+)-evoked overflows of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 3. AMPA released transmitters in a Ca(2+)-dependent, exocytotic manner since its effects, at pH 7.4 or 6.4, were abolished by omitting external Ca(2+) or by depleting vesicular transmitter stores with bafilomycin A1. AMPA did not evoke carrier-mediated release because the uptake blockers nisoxetine, 6-nitroquipazine, GBR12909 and hemicholinium-3 could not inhibit the AMPA-induced release of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 4. Extraterminal acidification to pH 6.4 prevented the potentiating effect of cyclothiazide on the AMPA-evoked release of [(3)H]NA, [(3)H]DA and [(3)H]5-HT, whereas the proton-insensitive AMPA-evoked release of [(3)H]ACh, previously found to be cyclothiazide-insensitive at pH 7.4 was cyclothiazide-resistant also at pH 6.4. 5. To conclude, the cyclothiazide-sensitive AMPA receptors mediating release of NA, 5-HT and DA, but not the cyclothiazide-insensitive AMPA receptors mediating the release of ACh, become more responsive when external pH is lowered to pathophysiologically relevant values. The results with cyclothiazide suggest that H(+) ions may prevent desensitization of some AMPA receptor subtypes.

Ontogeny of AMPA and NMDA receptor gene expression in the developing sheep white matter and cerebral cortex.

This study examined the hypothesis that the high prevalence of white matter injury in premature infants is associated with increased expression of calcium-permeable forms of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors in pre-myelinating white matter. We characterized expression of subunits of the AMPA, and for reference, the N-methyl-d-aspartate (NMDA), glutamate receptors at 0.5, 0.65, 0.85, and term gestation in the ovine fetal white matter and cerebral cortex. There was a low expression of the critical calcium-impermeable AMPA receptor GluR2 subunit in subcortical white matter both absolutely and relative to other AMPA subunits throughout gestation. In contrast, GluR2 subunit mRNA expression fell in the cerebral cortex with increasing gestation whereas protein expression increased. These findings suggest a vulnerability of subcortical white matter to AMPA receptor-mediated calcium toxicity throughout the second half of gestation. Thus, the hypothesis that AMPA receptor-mediated glutamate toxicity contributes to brain damage in premature infants needs to be revised.

Upregulation of mRNAs coding for AMPA and NMDA receptor subunits and metabotropic glutamate receptors in the dorsal horn of the spinal cord in a rat model of diabetes mellitus.

Recent studies suggest that glutamate plays a pivotal role in the processing of sensory information in the spinal cords of patients with diabetic neuropathy. However, the specific glutamate receptors that that are involved have yet to be determined. We therefore conducted a study to characterize the expression of messenger RNAs (mRNAs) coding for subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and N-methyl-d-aspartate (NMDA) receptors and for metabotropic glutamate receptors (mGluRs) in the dorsal horn of the lumbar segment of the spinal cord in a rat model (streptozotocin [STZ]-induced) of diabetic neuropathy. The levels of mRNAs coding for AMPA receptor subunits, GluR1, GluR2, and GluR3, were significantly increased in all layers (laminae I-V) of the dorsal horn in diabetic (STZ-injected) rats compared to control (vehicle-injected) rats. The hybridization signals for NR2A mRNA and NR2B mRNA were significantly elevated in the deep layer of the dorsal horn of diabetic rats. In diabetic (STZ-induced) rats, the levels of expression of mGluR1 mRNA and mGluR5 mRNA were significantly increased in all layers of the dorsal horn. These results suggest that abnormal expression of multiple glutamate receptors is involved in the development of diabetic neuropathy and that glutamate receptors are promising targets in the treatment of this disorder.

Expression profile of AMPA receptor subunit mRNA in single adult rat brain and spinal cord neurons in situ.

The differential expression of AMPA receptor subunit mRNA was analyzed in the adult rat brain and spinal cord using quantitative RT-PCR with laser capture microdissection. The expression of all four AMPA receptor subunits was demonstrable, with the mRNA expression level for GluR2 being the highest in all the brain areas and neuronal subsets examined. Both the absolute expression level of GluR2 mRNA and its expression relative to the other subunits were the lowest in motoneurons. The unique AMPA receptor expression profile of motoneurons may render them selectively vulnerable to AMPA receptor-mediated excitotoxicity.

Stoichiometries of AMPA receptor subunit mRNAs in rat brain fall into discrete categories.

In situ hybridization was used to estimate the relative concentrations of mRNAs encoding different subunits (GluR1-4) of alpha-amino 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors in rat brain and to test the hypothesis that within-region expression profiles reflect a limited number of recurring patterns. Fractional subunit mRNA concentrations were calculated for 33 brain regions, and cluster analysis methods were applied to test for statistically meaningful groupings in the data. Four relatively homogeneous classes were identified and designated as AMPA receptor (AR) categories, numbered according to dominant subunit mRNAs. The AR-1 class (47% GluR1 mRNA) was expressed by structures near the mesodiencephalic border, including basal ganglia-related areas. The AR-2 class (57% GluR2 mRNA) was expressed in cortex and tectum. The AR-1,2 class (31% GluR1, 45% GluR2) was found in the largest number of regions, including such dissimilar cell fields as hippocampus and substantia nigra pars compacta. The AR-2,3 grouping (33% GluR2, 31% GluR3) was associated with the sensory relay and reticular thalamic nuclei. It is suggested that AR-1,2 and AR-2, the most closely related categories in clustering space, are largely telencephalic receptors with the former predominant in the subcortex and the latter in the cortex. The AR-2,3 class is associated with ascending sensory stations, whereas AR-1 appears to include several smaller categories expressed by specialized systems. If the balance of subunit mRNAs is reflected at the protein level, then the present data suggest that forebrain AMPA-type glutamate receptors can be classified into a limited number of recurring types.

The AMPA receptor binding site: focus on agonists and competitive antagonists.

It is generally agreed that (S)-glutamic acid (Glu) receptors are involved in the development of a number of diseases in the central nervous system (CNS), and ligands that interact with these receptors are of significant interest. Selective ligands are indispensable as tools for the elucidation of the physiological role of AMPA receptors and as leads for the development of therapeutic agents. Over the last decade a wide variety of such ligands have been developed and studies on the structure-activity relationships of these compounds have contributed to our understanding of the mechanisms involved in AMPA receptor activation and blockade. Series of selective agonists using the 3-isoxazolol amino acid ibotenic acid (2) as a lead compound have been designed and developed. Other heterocycles, such as the uracil moiety of willardiine (6), have also proved to be highly effective bioisosteres for the distal carboxyl group of Glu. For a number of reasons, the development of competitive antagonists with therapeutic potential has been hampered for example due to the limited solubility of key heterocyclic compounds structurally unrelated to Glu. However, some problems have been overcome, and series of water-soluble, potent and selective quinoxalinediones, indenoimidazones and isatine oximes have now been developed. At the turn of the millennium the crystal structure of GluR2 co-crystallized with different AMPA receptor ligands became available, opening a new era in the design of AMPA receptor ligands on a rational basis.

Nociceptive stimulation induces glutamate receptor down-regulation in the trigeminal nucleus.

The dorsal horn of the subnucleus caudalis of the spinal trigeminal nucleus is a relay of oro-facial pain transmission; increase in subnucleus caudalis neuronal activity in response to tissue injury affects the level of chemical mediators participating in nociceptive processing. We investigated, by means of immunocytochemistry, the expression of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) glutamate receptor subunits in this nucleus in a model of inflammation. Rats injected with formalin in the whisker pad were compared with saline-injected control rats. One and two days after formalin injection, the immunostaining of cell bodies and neuropil of the AMPA receptor subunits GluR1 and GluR2/3 was markedly decreased in the ipsilateral superficial laminae of the subnucleus caudalis compared to the contralateral side. Side differences were not evident in the saline-treated animals. The down-regulation of AMPA GluR1 and GluR2/3 was no longer detectable in the subnucleus caudalis three days after formalin injection. No side difference was detected in the N-methyl-D-aspartate receptor subunit NR2A/B immunoreactivity of the subnucleus caudalis at any time-point in the formalin-injected animals. The modulation of AMPA receptor may be related to the decrease of hyperalgesia evident 1 h after formalin injection, in spite of the increasing perioral inflammation evident later on and characteristic of the formalin model. The present findings point out a selective down-regulation of AMPA receptor subunits in the transduction of trigeminal pain. These data also support the involvement of glutamate receptor subunits in the processing of trigeminal inflammation induced by noxious chemical stimulation.