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Explosives create shockwaves that cause blast-induced neurotrauma, one of the most common types of traumatic brain injury (TBI) linked to military service. Blast-induced TBIs are often associated with reduced cognitive and behavioral functions due to a variety of factors. To study the direct effects of military explosive blasts on brain tissue, we removed systemic factors by utilizing rat hippocampal slice cultures. The long-term slice cultures were briefly sealed air-tight in serum-free medium, lowered into a 37°C water-filled tank, and small 1.7-gram assemblies of cyclotrimethylene trinitramine (RDX) were detonated 15cm outside the tank, creating a distinct shockwave recorded at the culture plate position. Compared to control mock-treated groups of slices that received equal submerge time, 1-3 blast impacts caused a dose-dependent reduction in the AMPA receptor subunit GluR1. While only a small reduction was found in hippocampal slices exposed to a single RDX blast and harvested 1-2days later, slices that received two consecutive RDX blasts 4min apart exhibited a 26-40% reduction in GluR1, and the receptor subunit was further reduced by 64-72% after three consecutive blasts. Such loss correlated with increased levels of HDAC2, a histone deacetylase implicated in stress-induced reduction of glutamatergic transmission. No evidence of synaptic marker recovery was found at 72h post-blast. The presynaptic marker synaptophysin was found to have similar susceptibility as GluR1 to the multiple explosive detonations. In contrast to the synaptic protein reductions, actin levels were unchanged, spectrin breakdown was not detected, and Fluoro-Jade B staining found no indication of degenerating neurons in slices exposed to three RDX blasts, suggesting that small, sub-lethal explosives are capable of producing selective alterations to synaptic integrity. Together, these results indicate that blast waves from military explosive cause signs of synaptic compromise without producing severe neurodegeneration, perhaps explaining the cognitive and behavioral changes in those blast-induced TBI sufferers that have no detectable neuropathology., (Copyright © 2016 Elsevier Inc. All rights reserved.)

AMPAkines are positive modulators of AMPA receptors, and previous work has shown that these compounds can facilitate synaptic plasticity and improve learning and memory in both animals and humans; thus, their role in the treatment of cognitive impairment is worthy of investigation. In this study, we have utilized an organotypic slice model in which chloroquine-induced lysosomal dysfunction produces many of the pathogenic attributes of Alzheimer's disease. Our previous work demonstrated that synaptic AMPA receptor function is impaired in hippocampal slice cultures exhibiting lysosomal dysfunction leading to protein accumulation. The present study investigated the effect of the AMPAkine CX516 on AMPAR-mediated synaptic transmission as well as the CX516 induced modification of single channel AMPA receptor properties in this organotypic slice-culture model. In whole cell recordings from CA1 pyramidal neurons in chloroquine-treated slices we observed a significant decrease in AMPAR-mediated mEPSC frequency and amplitude indicating synaptic dysfunction. Following application of CX516, these parameters returned to nearly normal levels. Similarly, we report chloroquine-induced impairment of AMPAR single channel properties (decreased probability of opening and mean open time), and significant recovery of these properties following CX516 administration. These results suggest that AMPA receptors may be potential pharmaceutical targets for the treatment of neurodegenerative diseases, and highlights AMPAkines, in particular, as possible therapeutic agents.

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.

The early processes that lead to synaptic dysfunction during aging are not clearly understood. Dysregulation of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors may cause age-related cognitive decline. Using hippocampal slice cultures exhibiting lysosomal dysfunction, an early marker of brain aging that is linked to protein accumulation, we identified alterations to AMPA and NMDA receptor-mediated synaptic currents. The miniature and spontaneous excitatory postsynaptic currents that were examined after 3, 6, and 9 days of lysosomal disruption showed progressive changes in amplitude, frequency, and rise and decay kinetics. To investigate whether modifications in specific channel properties of single synaptic receptors contributed to changes in the amplitude and time course of synaptic currents, we examined the single channel properties of synaptic AMPA and NMDA receptors. The channel open probability and the mean open times showed decreases in both receptor populations, whereas the closed times were increased without any change in the channel conductance. The Western blot analysis revealed a progressive decline in synaptic markers including glutamate receptor subunits. These results indicate that lysosomal dysfunction leads to progressive functional perturbation of AMPA and NMDA receptors in this slice model of protein accumulation, suggesting that age-related cognitive decline could result from altered glutamate receptor function before reductions in synaptic density.

The brains of Alzheimer's disease (AD) patients have large numbers of plaques that contain amyloid beta (Abeta) peptides which are believed to play a pivotal role in AD pathology. Several lines of evidence have established the inhibitory role of Abeta peptides on hippocampal memory encoding, a process that relies heavily on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function. In this study the modulatory effects of the two major Abeta peptides, Abeta(1-40) and Abeta(1-42), on synaptic AMPA receptor function was investigated utilizing the whole cell patch clamp technique and analyses of single channel properties of synaptic AMPA receptors. Bath application of Abeta(1-42) but not Abeta(1-40) reduced both the amplitude and frequency of AMPA receptor mediated excitatory postsynaptic currents in hippocampal CA1 pyramidal neurons by approximately 60% and approximately 45%, respectively, in hippocampal CA1 pyramidal neurons. Furthermore, experiments with single synaptic AMPA receptors reconstituted in artificial lipid bilayers showed that Abeta(1-42) reduced the channel open probability by approximately 42% and channel open time by approximately 65% and increased the close times by several fold. Abeta(1-40), however, did not show such inhibitory effects on single channel properties. Application of the reverse sequence peptide Abeta(42-1) also did not alter the mEPSC or single channel properties. These results suggest that Abeta(1-42) but not Abeta(1-40) closely interacts with and exhibits inhibitory effects on synaptic AMPA receptors and may contribute to the memory impairment observed in AD.

Previous work showed that the glycosaminoglycan (GAG) dextran sulfate (500 kDa) altered the binding and channel properties of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. The current study compared the effects of dextran sulfate with another GAG, fucoidan (100-180 kDa), to determine whether GAG-mediated changes in high-affinity binding of AMPA receptors have a concomitant influence on specific channel properties. Dextran sulfate was more potent in inhibiting high-affinity AMPA binding to solubilized receptors (EC(50) of 7 nM) compared to fucoidan (EC(50) of 124 nM). Similarly, dextran sulfate was more potent in modulating the channel properties of purified and reconstituted AMPA receptors. Dextran sulfate, at 1 mug/ml (2 nM), produced a three to fourfold increase in open channel probability and a threefold increase in mean burst duration of channel activity elicited by 283 nM AMPA. The mean open time was increased by two to threefold and closed times were decreased by two to eightfold. Fucoidan produced similar effects at a concentration many times higher than that of dextran sulfate. Dextran sulfate and fucoidan had no effect on the single channel conductance or the ability of a specific antagonist to block AMPA channels. The effects of GAGs on multichannel patches showed an interactive channel gating behavior resulting in macroscopic currents with long lived open channel life times. These findings suggest that GAG components of proteoglycans can interact with and alter the binding affinity of AMPA receptors and modulate their functional properties.

Synaptic glutamate receptors play a prominent role in the excitatory neurotransmission in the vertebrate central nervous system. Although elucidation of the functional properties of glutamate receptors using electrophysiologic analyses has yielded important information, methodological and technological limitations have prevented direct measurement of single channel properties of synaptic receptors. Here, we have isolated murine mossy fiber synaptosomes and reconstituted them into small artificial lipid bilayers to characterize the single-channel properties of synaptic alpha amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. The reconstituted synaptosomal receptors were activated by nanomolar concentrations of AMPA and blocked by a potent AMPA receptor antagonist. The synaptosomal AMPA receptors exhibited channel conductances of 14-56 pS and linear current-voltage relationship. The open and closed dwell time distributions of single channel currents were best described by three exponentials. These channels frequently exhibited burst behavior with long burst duration of approx 60 ms. Experiments with multichannel recordings revealed that steady state probabilities could not be fitted using a binomial distribution, indicating a cooperative channel gating behavior that would account for larger membrane currents. Our findings suggest that isolation, reconstitution into lipid bilayers, and subsequent single channel analysis of synaptosomal receptors is a useful method for investigation of synaptic AMPA receptors.

In this work, we investigated the involvement of calpains in the neurotoxicity induced by short-term exposure to kainate (KA) in non-desensitizing conditions of AMPA receptor activation (cyclothiazide present, CTZ), in cultured rat hippocampal neurons. The calpain inhibitor MDL28170 had a protective effect in cultures treated with KA plus CTZ (p < 0.01), preventing the decrease in MTT reduction caused by exposure to KA (p < 0.001). Caspase inhibition by ZVAD-fmk was not neuroprotective against the toxic effect of KA. At 1 h after treatment, we could already observe significantly increased calpain activity, which was prevented by MDL 28170 and NBQX. Western blot analysis of calpain substrates, GluR1, neuronal nitric oxide synthase (nNOS) and nonerythroid spectrin (fodrin), showed a time-dependent and MDL 28170-sensitive proteolysis of these proteins. This effect was due to calpains, but not caspases, since ZVAD-fmk was ineffective in preventing proteolytic events. Breakdown products of fodrin (BDPs) were detected as early as 15 min after exposure to KA. Overall, these results show early activation of calpains following activation of AMPA receptors as well as compromise of neuronal survival, likely due to proteolytic events that affect proteins involved in neuronal signaling.

Previous work found evidence that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors interact with and are functionally regulated by the glycosaminoglycan heparin. The present study tested whether dextran species affect ligand binding, channel kinetics, and calcium permeability of AMPA receptors. Dextran sulfate of 500 kDa markedly reduced high affinity [3H]AMPA binding in solubilized hippocampal membranes. In isolated receptors reconstituted in a lipid bilayer, the same dextran sulfate prolonged the lifetime of open states exhibited by AMPA-induced channel fluctuations. The large polysaccharide further changed the single channel kinetics by increasing the open channel probability five- to sixfold. Such modulation of channel activity corresponded with enhanced levels of calcium influx as shown in hippocampal neurons loaded with Fluo3AM dye. With an exposure time of <1 min, AMPA produced a dose-dependent increase in intracellular calcium that was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX). Dextran sulfate, at the same concentration range that modified ligand binding (EC50 of 5-10 nM), enhanced the AMPA-induced calcium influx by as much as 60%. The enhanced influx was blocked by CNQX, although unchanged by the N-methyl-D-aspartate (NMDA) receptor antagonist AP5. Confocal microscopy showed that the increase in calcium occurred in neuronal cell bodies and their processes. Interestingly, smaller 5-8-kDa dextran sulfate and a non-sulfated dextran of 500 kDa had little or no effect on the binding, channel, and calcium permeability properties. Together, these findings suggest that synaptic polysaccharide species modulate hippocampal AMPA receptors in a sulfate- and size-dependent manner., (Copyright 2003 Wiley-Liss, Inc.)

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic receptors have been linked to survival signaling, especially when the receptors are allosterically modulated by members of the Ampakine family. While increased glutamatergic communication through AMPA receptors has been shown to protect against toxic conditions that target hippocampal subfield CA1, protection in other subfields has not been shown. Accordingly, positive modulation of AMPA receptors by Ampakine compounds CX727 and CX516 was tested for effects on trimethyltin (TMT) neurotoxicity in rat hippocampal slice cultures. TMT was applied for 4 h followed by a rapid washout and antagonistic quenching of AMPA and N-methyl-D-aspartate (NMDA) receptors. After a 24-h period, the TMT-exposed slices exhibited increased levels of calpain-mediated spectrin breakdown as well as synaptic deterioration. TMT selectively targeted CA3 pyramidal neurons and dentate gyrus (DG) granule cells as evidenced by degeneration and neuronal loss. The cytoskeletal and synaptic damage was reduced when Ampakine modulation was initiated during the postinsult period. Furthermore, the extent of protection was comparable to that produced by the NMDA receptor antagonist AP5. The above results were substantiated by histological experiments, revealing that Ampakine treatment prevented TMT-induced cell loss in CA3 and DG. These results indicate that AMPA receptor signals are part of cellular repair responses following exposure to an environmental toxin.

In the brain, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate glutamatergic neurotransmission and, when intensely activated, can induce excitotoxic cell death. In addition to their ionotropic properties, however, AMPA receptors have been functionally coupled to a variety of signal transduction events involving Src-family kinases, G-proteins, and the mitogen-activated protein kinase (MAPK). In the present study, we tested whether AMPA receptors are linked to appropriate signaling events in order to prevent neuronal injury and/or enhance recovery. AMPA stimulation in hippocampal slice cultures caused the selective activation of MAPK through the upstream activator MAPK kinase (MEK). Inhibition of either component of the AMPA receptor--MAPK pathway potentiated cellular damage due to serum deprivation, suggesting that this pathway facilitates compensatory signals in response to injury. Correspondingly, positive modulation of AMPA receptors with the Ampakine 1-(quinoxalin-6-ylcarbonyl)piperidine (CX516) enhanced MAPK activation and reduced the extent of synaptic and neuronal degeneration resulting from excitotoxic episodes. CX516 was neuroprotective when infused into slices either before or after the insult. The Ampakine derivative also elicited neuroprotection in an in vivo model of excitotoxicity as evidenced by reduction in lesion size and preservation of two different types of neurons. Interestingly, the AMPA receptor--MAPK pathway selectively protects against excitotoxicity since enhancing the pathway did not protect against the nonexcitotoxic, slow pathology initiated by lysosomal dysfunction. The results indicate that glutamatergic communication is important for cellular maintenance and that AMPA receptors activate survival signals to counterpoise their own excitotoxic potential.

Ampakines are small benzamide compounds that allosterically produce the positive modulation of AMPA receptors and improve performance on a variety of behavioral tasks. To test if the native synaptic membrane is necessary for the effects of such positive modulators, the mechanism of action of the Ampakine 1-(1,3-benzodioxol-5-ylcarbonyl)-1,2,3,6-tetrahydropyridine (CX509) was investigated in isolated rat brain AMPA receptors reconstituted in lipid bilayers. The drug increased the open time of AMPA-induced single channel current fluctuations with an EC(50) of 4 microM. The action of CX509 was highly selective since it had no effect on the amplitude or close time of channel events. The open time effect had a maximum enhancement of 70-fold and the modulated currents were blocked by CNQX. It is concluded that the synaptic membrane environment is not necessary for Ampakine effects. In fact, CX509 was about 100 times more potent on the reconstituted AMPA receptors than on receptors in their native membrane. These findings indicate that centrally active Ampakines modulate specific kinetic properties of AMPA currents. They also raise the possibility that AMPA receptors are regulated by factors present in situ, thus explaining the more efficient modulatory effects of CX509 when acting on receptors removed from their synaptic location., (Copyright 2001 Wiley-Liss, Inc.)

Glutamate receptors specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have been reported to interact with the highly sulfated glycosaminoglycan, heparin, and to subsequently express lower binding affinity for [3H]AMPA. The present study examined whether heparin also modifies the kinetic properties of single channel activity expressed by isolated AMPA receptors from rat forebrain. Upon application of 280 nM AMPA, the partially purified receptors reconstituted in lipid bilayers expressed bursting channel activity that was inhibited by dinitroquinoxaline-2-3,-dione (DNQX). Treating the receptors with heparin (10 microg/ml) produced no change in conductance but the mean burst length for 280 nM AMPA was nearly doubled. Heparin also prolonged the lifetime of open states of the individual ion channels 3-5-fold, perhaps by causing a decrease in the closing rate constant for channel gating. Heparin had no effect on the lifetime of the closed state or on the amplitude of currents. The single channel open time was voltage-dependent and an increase of applied voltage caused a decrease in the heparin effect on channel open times. While the lifetime of the open channel was increased 3-4 times by heparin at 20 mV, there was no significant change induced at 43 mV. The equivalent electric charge of the channel gate was increased by 40%. The heparin effects were specific as another polysaccharide, dextran, and a monomeric constituent of heparin, glucosamine 2,3-disulfate, failed to have any effect on the receptors. These findings suggest that heparin-containing extracellular matrix components can interact with AMPA receptors and influence their functional properties.

Heparin was found to bind to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and to alter their functional properties. AMPA receptors solubilized in 0.4% Triton X-100 bound to a heparin-agarose column and were eluted by 0.4 M NaCl. Soluble heparin inhibited 10 nM [3H]AMPA binding to detergent-solubilized receptors by 75% (IC50 = 10 micrograms/ml), but had little effect on binding to membrane-associated receptors. The inhibition of [3H]AMPA binding to detergent-solubilized receptors was not observed when binding was measured in the presence of 0.4 M NaCl, and no effect of heparin was observed on binding of the AMPA receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Scatchard analyses of [3H]AMPA binding to solubilized receptors revealed that the inhibition induced by heparin was caused by a decrease in the apparent affinity of a portion of the total binding sites. Studies on AMPA receptors reconstituted in artificial lipid bilayers indicated that 10 micrograms/ml heparin enhanced cooperativity between channels and prolonged the lifetime of the open channel, but did not affect the amplitude of single channel currents. Thus, heparin may be added to the list of compounds known to modulate AMPA receptor function. These data also raise the possibility that heparin-containing proteoglycans, which are known to be concentrated at synaptic junctions, might be able to bind AMPA receptors and influence their functional characteristics.

Polyclonal antibodies against specific carboxy-terminal sequences of known alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR-4) were used to screen regional homogenates and subcellular fractions from rat brain. Affinity purified anti-GluR1 (against amino acids 877-899), anti-GluR2/3 (850-862), and anti-GluR4a and anti-GluR4b (868-881) labeled distinct subunits with the expected molecular weight of approximately 105,000. These antigens were shown to have distinct distributions in the brain. While GluR2/3 epitopes had a distribution profile similar to that of the presynaptic marker synaptophysin, GluR1 was notable for its abundance in the hippocampus and its relatively low density in neocortical areas, and GluR4 was highly enriched in cerebellar tissue. An additional antigen (glutamate receptor-related, GR53) of lower molecular weight (50,000-59,000) was recognized in rat, human, frog, chick and goldfish brain samples by anti-GluR4a as well as by anti-GluR1 at, an antibody that specifically recognizes the extracellular aminoterminal domain of GluR1 (amino acids 163-188). Both antibodies also labeled antigens of approximately 105,000 mol. wt in brain tissue from all species tested. The approximately 53,000 mol. wt antigen was concentrated 10-20-fold in synaptic membranes vs homogenates across rat brain regions. Both the 105,000 and the 53,000 mol. wt proteins were also concentrated in postsynaptic densities, and neither of the two antigens were evident in seven non-brain tissue samples. These data indicate that AMPA receptors have regionally different subunit combinations and that some AMPA receptor composites include proteins other than the conventional 105,000 mol. wt GluR subunits.

Cultured hippocampal slices retain many in vivo features with regard to circuitry, synaptic plasticity, and pathological responsiveness, while remaining accessible to a variety of experimental manipulations. The present study used ligand binding, immunostaining, and in situ hybridization assays to determine the stability of AMPA- and NMDA-type glutamate receptors and other synaptic proteins in slice cultures obtained from 11 day postnatal rats and maintained in culture for at least 4 weeks. Binding of the glutamate receptor ligands [3H]AMPA and [3H]MK-801 exhibited a small and transient decrease immediately after slice preparation, but the binding levels recovered by culture day (CD) 5-10 and remained stable for at least 30 days in culture. Autoradiographic analyses with both ligands revealed labeling of dendritic fields similar to adult tissue. In addition, slices at CD 10-20 expressed a low to high affinity [3H]AMPA binding ratio that was comparable with that in the adult hippocampus (10:1). AMPA receptor subunits GluR1 and GluR2/3 and an NMDA receptor subunit (NMDAR1) exhibited similar postcutting decreases as that exhibited by the ligand binding levels, followed by stable recovery. The GluR4 AMPA receptor subunit was not evident during the first 10 CDs but slowly reached detectable levels thereafter in some slices. Immunocytochemistry and in situ hybridization techniques revealed adult-like labeling of subunit proteins in dendritic processes and their mRNAs in neuronal cell body layers. Long-term maintenance was evident for other synapse-related proteins, including synaptophysin, neural cell adhesion molecule isoforms (NCAMs), and an AMPA receptor related antigen (GR53), as well as for certain structural and cytoskeletal components (e.g., myelin basic protein, spectrin, microtubule-associated proteins). In summary, following an initial and brief depression, many synaptic components were expressed at steady-state levels in long-term hippocampal slices, thus allowing the use of such a culture system for investigations into mechanisms of brain synapses.

Telencephalic membranes from rats of different embryonic (E16, E19) and postnatal (P2, P7, P14, adult) ages were assessed for alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H] AMPA) binding and for immunoreactivity levels of AMPA receptor subunits (GluR1, GluR2/3, and GluR4). In addition, the synaptic markers synaptophysin and NCAM140 (a neural cell adhesion molecule isoform) were examined by immunoblot. The density of [3H]AMPA binding sites increased steadily with advancing age. This increase was due mainly to the development of the large low-affinity component (KD = 400 nM) that dominates the [3H]AMPA binding profile of adult rat brain membranes. As resolved by two-site regression analysis, the high-affinity component (KD = 15 nM) of the [3H]AMPA binding increased by approximately twofold from E16 to adult, whereas the low-affinity component increased by 25-fold. Staining for GluR1 and GluR2/3 increased steadily with increasing age at all time points examined; synaptophysin and NCAM140 exhibited similar ontogenic immunostaining profiles. GluR4 immunoreactivity was first evident at P14 and increased by adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)