AMPA Receptor Subunit Composition Research Articles

Modification of AMPA receptor properties following environmental enrichment

Environmental enrichment results in many modifications in the brain such as structural, behavioural, and biochemical changes. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptors for excitatory amino acid glutamate are recently found to be involved in neuronal plasticity. In this study, we examined whether environmental enrichment modified the brain expression of mRNA for subunit composition of AMPA receptors in adult mice using the real-time quantitative PCR method and western blotting. Mice housed in enriched environments showed significantly higher levels of GluR2 and GluR4 subunits in the hippocampus compared to control mice. We concluded that environmental enrichment can change the expression of AMPA receptor subunits and thus might modify the potentials of brain plasticity.

Regulation of synaptic strength and AMPA receptor subunit composition by PICK1.

PICK1 (protein interacting with C kinase-1) regulates the surface expression of the AMPA receptor (AMPAR) GluR2 subunit, however, the functional consequences of this interaction are not well understood. Previous work has suggested that PICK1 promotes the internalization of AMPARs. However, we found that when PICK1 is virally expressed in the CA1 region of hippocampal slices, it causes an increase in AMPAR-mediated EPSC amplitude. This effect is associated with increased AMPAR rectification and sensitivity to polyamine toxin. These effects are blocked by PKC or calcium/calmodulin-dependent protein kinase II inhibitors, indicating that the virally expressed PICK1 signals through an endogenous kinase cascade. In contrast, blockade of interactions with GluR2 at the N-ethylmaleimide-sensitive factor site did not cause a change in subunit composition, suggesting that the effects of PICK1 are not simply a nonspecific consequence of removing AMPARs from the surface. Immunocytochemical and biochemical analyses in dissociated cultured hippocampal neurons show that PICK1 causes a decrease in endogenous GluR2 surface expression but no change in GluR1 surface levels. To address the physiological role of PICK1, we virally expressed C-terminal GluR2 peptides. Blockade of endogenous PICK1 PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain interactions produced opposite effects on synaptic strength and AMPAR rectification to those observed with PICK1 expression. This demonstrates that AMPAR subunit composition is physiologically regulated through a mechanism involving PICK1 PDZ domain interactions. These findings suggest that PICK1 acts to downregulate the GluR2 content of AMPARs at hippocampal CA1 synapses, thereby increasing synaptic strength at resting membrane potentials.

Chronic Prenatal Ethanol Exposure Alters Ionotropic Glutamate Receptor Subunit Protein Levels in the Adult Guinea Pig Cerebral Cortex

Background: The superfamily of glutamate‐gated ion channels mediates fast excitatory synaptic transmission in the central nervous system and is composed of the NMDA, AMPA, and kainate receptors. Binding studies have shown that chronic prenatal and/or neonatal ethanol exposure produces persistent effects on the numbers of some of these channels. However, whether or not this chronic ethanol exposure produces long‐lasting effects on the expression of specific ionotropic receptor subunits remains an open question.Methods: Timed pregnant Dunkin‐Hartley strain guinea pigs received oral administration of one of the following regimens between gestational days 2 and 67: (1) 4 g of ethanol per kilogram of maternal body weight per day with ad libitum access to pellet food and water (ethanol group), (2) isocaloric sucrose‐ and pair‐feeding with ad libitum access to water (sucrose group), or (3) isovolumetric water with ad libitum access to food and water (water group). The maternal blood ethanol concentration produced by the ethanol regimen was 71 ± 12 mM. Adult offspring were killed on postnatal day 61, and cerebral cortical tissue was analyzed for ionotropic glutamate receptor subunit expression by Western immunoblotting.Results: There was a statistically significant decrease in NR2B subunit protein expression and an increase in GluR2/3 subunit protein expression in the ethanol group. Expression of NR1, NR2A, NR2C, GluR1, GluR6/7, and KA2 subunit proteins was not affected.Conclusions: These results demonstrate that chronic prenatal ethanol exposure produces long‐lasting effects on the subunit composition of NMDA and AMPA receptors in the cerebral cortex of the adult guinea pig.

Correlation of AMPA Receptor Subunit Composition with Synaptic Input in the Mammalian Cochlear Nuclei

The composition of AMPA receptors in patches excised from somata and dendrites of six cell types in the mammalian cochlear nuclei was probed and compared electrophysiologically and pharmacologically with the rapid application of glutamate. Cells excited predominantly by auditory nerve fibers had AMPA receptors with exceptionally rapid gating (submillisecond deactivation and desensitization time constants). The nonlinear current-voltage relationship in the presence of spermine showed that few of these receptors had GluR2 subunits, and the insensitivity of desensitization to cyclothiazide indicated that they contained mostly flop splice variants. At synapses made by parallel fibers, AMPA receptors were slowly gating (time constants of deactivation and desensitization >1 msec) and contained higher levels of GluR2 and flip isoforms. However, receptors at auditory nerve synapses on cells that also receive parallel fiber input, the fusiform cells, had intermediate properties with respect to kinetics and contained GluR2 and flip isoforms. Given the diverse biophysical properties, patterns of innervation, patterns of electrical activity, and targets of each cell type in vivo, these data indicate that the kinetics and permeation properties of AMPA receptors are linked to factors associated with synaptic connectivity.

Differentiation of corticothalamic and collateral thalamocortical synapses on mouse reticular nucleus neurons by EPSC amplitude and AMPA receptor subunit composition

AMPA receptors mediate fast synaptic transmission at collateral synapses of corticothalamic and thalamocortical axons in the thalamic reticular nucleus (RTN). These synapses are important in generating synchrony in the thalamocortical network. Whole cell recording in the mouse thalamocortical slice preparation was combined with high-resolution immunoelectron microscopy to characterize AMPA-mediated conductances at the two synapses and to correlate these with differential expression of GluR3 and GluR4 subunits. Thalamocortical collateral (TC) synapses had larger mean EPSC amplitudes (1914±1814 pS) than corticothalamic (CT) synapses (400±257 pS), and rise and decay times of TC EPSCs were faster and less variable than CT EPSCs, probably reflecting proximal and dispersed locations of TC and CT terminals, respectively, on RTN cells. In situ hybridization and immunocytochemical studies revealed that GluR1 and GluR2 subunits are not expressed in the RTN and GluR4 subunits are expressed at higher levels than GluR3 subunits. Immunoelectron microscopy revealed gold particles representing GluR3 or GluR4 subunits concentrated at single postsynaptic densities (PSD) characteristic of CT synapses and at the two to seven split PSD segments characteristic of TC synapses. At CT synapses the number and density of GluR4 particles were 2.5 times greater than GluR3 particles. At the larger TC synapses, the number of GluR3 particles exceeded that of GluR4 particles but density of GluR4 particles was lower than at CT synapses while density of GluR3 particles was similar. Despite enrichment of GluR4 subunits at CT synapses, larger conductances prevailed at thalamocortical collateral synapses, probably reflecting both larger overall numbers of AMPA receptors and a greater number of release sites represented by the split PSDs. Variability in amplitudes of TC EPSCs may reflect variability in the number of release sites; lower variability in CT EPSC amplitudes may reflect a more constant number of release sites.

Differentiation of corticothalamic and collateral thalamocortical synapses on mouse reticular nucleus neurons by EPSC amplitude and AMPA receptor subunit composition

Differentiation of corticothalamic and collateral thalamocortical synapses on mouse reticular nucleus neurons by EPSC amplitude and AMPA receptor subunit composition

Intraischemic but not postischemic hypothermia prevents non-selective hippocampal downregulation of AMPA and NMDA receptor gene expression after global ischemia

Hypothermia may afford histological neuroprotection induced by ischemia by preventing aberrant Ca2+ influx through NMDA (N-methyl-d-aspartic acid) or Ca2+-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors. Expression of hippocampal GluR1(A), GluR2(B), GluR3(C) and NMDAR1 (NR1) subunits was investigated by in situ hybridization at 1 and 7 days after 10-min transient global ischemia in the presence and absence of intraischemic or postischemic brain hypothermia (30°C). At 1 day, normothermic ischemia markedly suppressed the expression of GluR1(A), GluR2(B), and GluR3(C) receptor mRNAs to a similar degree in the vulnerable CA1. Less vulnerable CA3a–c subregions were also acutely downregulated. NR1 mRNA expression was reduced in CA1 but to a lesser extent than AMPA mRNAs. At 7 days after normothermic ischemia, a time of marked CA1 cell loss, all three AMPA transcripts were nearly absent in CA1 while a percentage (33.9±7.2%) of NR1 mRNA remained. Intraischemic hypothermia fully blocked the damage and non-selective mRNA downregulations at 1 and 7 days. By contrast, postischemic hypothermia postponed neurodegeneration but only partially rescued the expression of AMPA and NR1 mRNAs at 7 days and not at 1 day after the insult. Therefore, hippocampal AMPA receptor mRNAs decline at a relatively similar rate after normothermic global ischemia and cellular neuroprotection by intraischemic hypothermia occurred independently of altered subunit composition of AMPA receptors. Since decreases persist within resistant neurons under the postischemic condition, AMPA receptor-mediated Ca2+ currents probably do not contribute to selective vulnerability.

Subunit composition of AMPA receptors expressed by single hippocampal neurons.

Using a patch-clamp, reverse transcription-polymerase chain reaction (RT-PCR) method, we determined AMPA receptor subunits expressed in neurons in rat hippocampal slices at the single-cell level. In a population of non-pyramidal neurons (type II neurons) expressing AMPA receptors with strong inward rectification and Ca2+ permeability, no GluR2 was detected, and GluR1 and GluR4 were the most frequently detected subunits. Analysis of the splicing variants revealed that only the flop form of GluR4 was expressed, but both flip and flop forms of GluR1 were expressed in these neurons. In contrast, GluR2 was the most frequently detected subunit both in CA1 pyramidal cells and type I non-pyramidal cells possessing AMPA receptors with outwardly rectifying current-voltage relationships and little Ca2+ permeability.

Developmental Changes in Synaptic AMPA and NMDA Receptor Distribution and AMPA Receptor Subunit Composition in Living Hippocampal Neurons

AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have developed antibodies that recognize all AMPA or all NMDA receptor variants on the surface of living neurons. AMPA receptor variants were identified with a polyclonal antibody recognizing the conserved extracellular loop region of all four AMPA receptor subunits (GluR1-4, both flip and flop), whereas NMDA receptors were immunolabeled with a polyclonal antibody that binds to an extracellular N-terminal epitope of the NR1 subunit, common to all splice variants. In non-fixed brain sections these antibodies gave labeling patterns similar to autoradiographic distributions with particularly high levels in the hippocampus. Using these antibodies, in conjunction with GluR2-specific and synaptophysin antibodies, we have directly localized and quantified surface-expressed native AMPA and NMDA receptors on cultured living hippocampal neurons during development. Using a quantitative cell ELISA, a dramatic increase was observed in the surface expression of AMPA receptors, but not NMDA receptors, between 3 and 10 d in culture. Immunocytochemical analysis of hippocampal neurons between 3 and 20 d in vitro shows no change in the proportion of synapses expressing NMDA receptors (approximately 60%) but a dramatic increase (approximately 50%) in the proportion of them that also express AMPA receptors. Furthermore, over this period the proportion of AMPA receptor-positive synapses expressing the GluR2 subunit increased from approximately 67 to approximately 96%. These changes will dramatically alter the functional properties of hippocampal synapses.

Blockade of ion channels as an approach to studying AMPA receptor subtypes

This article reviews current progress in studies of the relationship between the molecular structure of different subtypes of AMPA receptors and their functional properties. Differences in the subunit composition of AMPA receptors involved in glutamatergic synaptic inputs to efferent (main) neurons and interneurons are discussed with reference to neurons isolated from the hippocampus, striatum, and cerebellum. Data on the possibility of selective pharmacological actions on the ion channels of different AMPA receptor subtypes are presented; this allows these receptors to be identified and their functions to be studied in greater depth in normal and pathological conditions.

Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning.

Gene-targeted mice lacking the L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR-A exhibited normal development, life expectancy, and fine structure of neuronal dendrites and synapses. In hippocampal CA1 pyramidal neurons, GluR-A-/- mice showed a reduction in functional AMPA receptors, with the remaining receptors preferentially targeted to synapses. Thus, the CA1 soma-patch currents were strongly reduced, but glutamatergic synaptic currents were unaltered; and evoked dendritic and spinous Ca2+ transients, Ca2+-dependent gene activation, and hippocampal field potentials were as in the wild type. In adult GluR-A-/- mice, associative long-term potentiation (LTP) was absent in CA3 to CA1 synapses, but spatial learning in the water maze was not impaired. The results suggest that CA1 hippocampal LTP is controlled by the number or subunit composition of AMPA receptors and show a dichotomy between LTP in CA1 and acquisition of spatial memory.

Developmental changes in EPSC quantal size and quantal content at a central glutamatergic synapse in rat.

1. Developmental changes in amplitude and time course of single-fibre-evoked and spontaneous EPSCs mediated by AMPA and NMDA receptors at the endbulb-bushy cell synapse of rats from 4 to 22 days of age were recorded using whole-cell patch-clamp methods in in vitro slices of cochlear nucleus. 2. The mean conductance of the AMPA component of evoked EPSCs increased by 66 %, while that of the NMDA component decreased by 61 %, for 12- to 18-day-old rats cf. 4- to 11-day-old rats. 3. The mean AMPA spontaneous EPSC conductance increased by 54 %, while mean NMDA spontaneous EPSC conductance decreased by 83 %, for 12- to 22-day-old rats cf. 4- to 11-day-old rats. The mean number of quanta contributing to peak evoked AMPA conductance also increased by 78 % in the older age group, after correction for the asynchrony of evoked quantal release. 4. The decay time constant of spontaneous AMPA EPSCs showed a small decrease in older animals, while the decay time constant of spontaneous NMDA EPSCs was markedly decreased in older animals. The decay time constants of evoked NMDA EPSCs showed a quantitatively similar decrease to that of spontaneous NMDA EPSCs. This suggests that AMPA receptor subunit composition is unlikely to undergo developmental change, while NMDA receptor subunit composition may be substantially altered during synaptic maturation. 5. These data are consistent with a developmentally increased efficacy of AMPA receptor-mediated synaptic transmission at the endbulb-bushy cell synapse, due to an increase in underlying AMPA-mediated quantal size and content during the same period as a transient co-localization of NMDA receptors.

Subunit Composition, Kinetic, and Permeation Properties of AMPA Receptors in Single Neocortical Nonpyramidal Cells

Native AMPA receptors (AMPARs) were investigated in neocortical fast-spiking (FS) and regular-spiking nonpyramidal (RSNP) cells. The onset of and recovery from desensitization as well as current rectification and single-channel conductance were studied by using fast glutamate application to outside-out patches. The GluR1-4 subunit, flip/flop splicing, and R/G editing expression patterns of functionally characterized cells were determined by single-cell reverse transcription-PCR to correlate the subunit composition of native AMPARs with their functional properties. Our sample, mostly constituted by RSNP neurons, predominantly expressed GluR3 flip and GluR2 flop. In individual cells, flip/flop splicing of each subunit appeared to be regulated independently, whereas for R/G editing all subunits were either almost fully edited or unedited. We confirmed that the relative GluR2 expression controls the permeation properties of native AMPARs, whereas none of the single molecular parameters considered appeared to be a key determinant of the kinetics. FS neurons displayed AMPARs with relatively homogeneous functional properties characterized by fast desensitization, slow recovery from desensitization, marked inward rectification, and large single-channel conductance. In contrast, these parameters varied over a wide range in RSNP neurons, and their combination resulted in various AMPAR functional patterns. Indeed, in different cells, fast or slow desensitization was found to be associated with either slow or fast recovery from desensitization. Similarly, fast or slow kinetics was associated with either strong or weak rectification. Our results suggest that kinetic and permeation properties of native AMPARs can be regulated independently in cortical neurons and probably do not have the same molecular determinants.

AMPA receptor subunits expressed by single astrocytes in the juvenile mouse hippocampus

The subunit composition of native AMPA receptor (AMPA-R) channels was recently described in several neuronal cell types but less information is available on glial cells. Evidence from recombinant receptor studies suggests that the expression of distinct subunits determines the specific functional properties of the receptor channel. In the present study, we combined the patch clamp technique with the reverse transcription-polymerase chain reaction (RT-PCR) to correlate the expression of gene transcripts with functional properties of AMPA-R in single identified glial cells of the hippocampus. The cells were freshly isolated from the stratum radiatum of the CA1 subregion. We focused on cells expressing AMPA-R with an intermediate Ca 2+ permeability which were identified as immature astrocytes due to their morphological, immunocytochemical and electrophysiological characteristics. After recording, the cells were harvested and RT-PCR was performed with the same individual cell to investigate the composition of their AMPA-R transcripts. Our results suggest the expression of a heteromeric subunit architecture. In all cells, the GluR2 subunit was present, which is known to confer a low Ca 2+ permeability to the receptor complex. Most frequently, we met co-expression of GluR2 and GluR4. This study demonstrates that astrocytes in the hippocampus express a distinct AMPA-R subunit composition which differs from neurons. The glial receptors might be involved in the modulation of gene expression as well as the regulation of proliferation and differentiation.

Predicting survival in cystic fibrosis.

Ionotropic glutamate receptors play important roles in spinal processing of nociceptive sensory signals and induction of central sensitization in chronic pain. Here we applied highly sensitive freeze-fracture replica labeling to...

AN IMMUNOCYTOCHEMICAL STUDY OF THE DISTRIBUTION OF AMPA SELECTIVE GLUTAMATE RECEPTOR SUBUNITS IN THE NORMAL HUMAN MOTOR SYSTEM

Glutamate is the major mediator of fast excitatory neurotransmission in the mammalian central nervous system. Disturbances of this neurotransmitter system have been implicated in chronic degenerative neurological disease. Recently, major advances in our knowledge and understanding of the molecular biology of the glutamatergic receptor system have been made. It is now known that functional glutamate receptors consist of various combinations of some 20 identified subunits. A growing body of circumstantial evidence suggests that the non- N-methyl- d-aspartate subtype of glutamate receptors may mediate, at least in part, the selective motor neuron death seen in the human neurodegenerative disease amyotrophic lateral sclerosis. We have used subunit specific immunocytochemistry to study the distribution and potential subunit composition of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) selective glutamate receptors, (a subgroup of non- N-methyl- d-aspartate selective glutamate receptors formed by combinations of GluR1-4 subunits), in the human motor system. Motor neurons in the spinal cord, brainstem, and motor cortex were relatively strongly immunoreactive with the GluR 23 subunit antibody, moderately so with the GluR4 subunit antibody, and showed relatively low levels of immunoreactivity with the GluR1 subunit antibody.This is the first detailed study of AMPA receptor subunit expression in the human motor system. Motor neurons express a distinct subunit profile when compared with other groups of neurons in the human nervous system. There were no significant differences in the pattern of relative AMPA subunit expression (GluR 23 ⩾ GluR4 > GluR1) between groups of motor neurons typically affected (in the spinal cord and hypoglossal nucleus), or spared (oculomotor and Onuf's nucleus) by the amyotrophic lateral sclerosis disease process. However, oculomotor motor neurons had higher levels of expression of all AMPA subunit proteins which may indicate greater AMPA mediated glutamatergic input in the normal function of this neuronal population.This study does not support a role for differential subunit composition of AMPA receptors in determining the selective vulnerability of motor neurons in amyotrophic lateral sclerosis. However, the overall density of receptors may be of importance. Copyright © 1996 IBRO. Published by Elsevier Science Ltd.

Argiotoxin detects molecular differences in AMPA receptor channels

Argiotoxin, a component of the spider venom from Argiope lobata, blocks AMPA receptor channels expressed in homomeric and heteromeric configuration in Xenopus oocytes. Argiotoxin acts as an open channel blocker in a voltage-dependent manner and discriminates between the functionally diverse AMPA receptors. Importantly, a transmembrane region 2 determinant for divalent cation permeability also determines argiotoxin sensitivity. Subunit-specific differences in the time courses of block and recovery demonstrate that heteromeric AM PA receptors can assemble in variable ratios. Thus, argiotoxin can be used as a tool in analyzing the subunit composition of AMPA receptors in native membranes.

317 Subunit composition of AMPA receptors in the type II hippocampal neuron explains its functional properties

317 Subunit composition of AMPA receptors in the type II hippocampal neuron explains its functional properties