AMPA receptor protein expression and function in astrocytes cultured from hippocampus.

Glutamate receptors guide the proliferation, migration, and differentiation of glial cells. Here, we characterize AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA receptor protein expression and function and mRNA expression in hippocampal glial cultures. By immunocytochemistry, GluR2 (the subunit that limits the Ca(2+) permeability of AMPA receptors) exhibited prominent labeling in hippocampal glial cultures. Double-labeling of GluR2 with GFAP and with A2B5 revealed GluR2 subunit expression on type-1 and type-2 astrocyte lineage cells. GluR1 subunit expression was more prominent in type-1 than in type-2 astrocytes. To characterize functional properties of glutamate receptors expressed in cultured hippocampal astrocytes, we performed whole-cell patch clamp recording. Application of L-glutamate, AMPA, and kainate, but not NMDA, to small, rounded cells (morphologically identified as type-2 astrocytes) elicited inward currents which were blocked by the AMPA/kainate antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX). Cyclothiazide potentiated AMPA- and kainate-elicited currents, indicative of AMPA-preferring receptors. Current voltage analysis indicated that type-2 astrocyte AMPA receptors were electrically linear, indicative of GluR2-containing, Ca(2+)-impermeable AMPA receptors. By Northern blot analysis, GluR1 mRNA was highest in astrocyte cultures from cerebellum and hippocampus and moderate in astrocyte cultures from neocortex and striatum. GluR3 mRNA was detectable in astrocyte cultures from cerebellum and neocortex. GluR2 and NR1 mRNA expression were not detected in astrocytes cultured from any brain region examined. In situ hybridization studies showed wide expression of GluR1 mRNA in cultured astrocytes; GluR2 and GluR3 mRNAs were near background levels. Thus, cultured type-2 astrocytes express functional AMPA receptors in a cell-specific and region-specific manner, consistent with their role in neuronal-glial communication.     

Glial processes are glued to synapses via Ca(2+)-permeable glutamate receptors.

Bergmann glia in the cerebellum elaborately ensheathe Purkinje cell dendrites and synapses, and express high levels of Ca(2+)-permeable glutamate receptors (GluRs). Suppression of the Ca(2+) permeability by virus-mediated GluR2 transfer has revealed that GluR-mediated Ca(2+) signaling is essential to maintain both structural and functional connections between the glial cell and the glutamatergic synapses.     

Subcellular localization of calcium-permeable AMPA receptors in spinal motoneurons

Activation of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been linked to potent effects on survival and dendritic outgrowth of spinal motoneurons. Ca(2+) permeability of AMPA receptors is controlled by the GluR2 subunit. Whole-cell electrophysiological studies have suggested that GluR2-containing and GluR2-lacking AMPA receptors may coexist in individual motoneurons. However, there has not been a direct demonstration of heterogeneity in AMPA receptor subunit composition in single motoneurons, nor of distinct subcellular distributions of GluR2-containing and GluR2-lacking receptors. In the present study, we have used confocal microscopy, immunocytochemistry and Ca(2+) imaging to characterize the subcellular localization of AMPA receptors in cultured rat spinal motoneurons. Immunoreactivity for GluR2 and GluR4 was concentrated in clusters, the vast majority of which were found in dendrites at synapses. Double-labelling for GluR2 and GluR4 revealed variability in relative expression of GluR2 and GluR4 between clusters within individual motoneurons; most AMPA receptor clusters were immunoreactive for both GluR2 and GluR4, but a significant minority of clusters were immunoreactive for GluR2 only or for GluR4 only. The majority of GluR2-immunonegative AMPA receptor clusters was present in dendrites, but the relative proportion of GluR2-immunonegative and GluR2-immunopositive clusters was similar in dendrites and soma. Imaging of [Ca(2+)](i) rises triggered by AMPA receptor activation confirmed Ca(2+) influx in motoneuron dendrites. These findings strongly support a model in which GluR2-containing and GluR2-lacking AMPA receptors coexist in motoneurons, clustered at synapses, and mixed in a relative proportion that varies considerably between cell membrane microdomains.     

Sindbis viral-mediated expression of Ca2+-permeable AMPA receptors at hippocampal CA1 synapses and induction of NMDA receptor-independent long-term potentiation

Gene manipulation in order to artificially express a particular gene in neurons in the central nervous system is a powerful tool for the analysis of brain function. Sindbis viral vectors have been developed to express high levels of foreign genes in postmitotic brain neurons with little transfection of glial cells. In this study, we expressed the gene encoding the unedited GluR2 (GluR-B) subunit of the AMPA-type glutamate receptor that forms inwardly rectifying and Ca2+-permeable channels, in rat CA1 hippocampal neurons in slice cultures using Sindbis viral vectors. The pyramidal cell layer of the CA1 region was injected with recombinant Sindbis viruses encoding both enhanced green fluorescent protein (GFP) and unedited GluR2. The GFP fluorescence from CA1 neurons could be detected as early as 6 h and reached a maximal level about 48 h postinfection. The inwardly rectifying and Ca2+-permeable AMPA receptors were expressed in most CA1 pyramidal cells expressing GFP. These AMPA receptors expressed by gene transfer were involved in fast excitatory neurotransmission elicited by electrical stimulation of the Schaffer collaterals in the stratum radiatum. Tetanic stimulation of Schaffer collaterals induced NMDA receptor-independent, long-term potentiation due to Ca2+ influx through the newly expressed AMPA receptors in the area densely stained with GFP. Thus, the combined use of Sindbis viral vectors with the GFP reporter allowed physiological examination of the roles of a specific gene product in synaptic function in well-characterized brain neurons.     

Extension of glial processes by activation of Ca2+-permeable AMPA receptor channels

AMPA type-glutamate receptor channels (AMPARs) assembled without the GluR2 (GluR-B) subunit are characterized by high Ca2+ permeability, and are expressed abundantly in cerebellar Bergmann glial cells. Here we show that the morphology of cultured Bergmann glia-like fusiform cells derived from the rat cerebellum was changed by manipulating expression of Ca2+-permeable AMPARs using adenoviral vector-mediated gene transfer. Converting endogenous Ca2+-permeable AMPARs into Ca2+-impermeable channels by viral-mediated transfer of GluR2 gene induced retraction of glial processes. In contrast, overexpression of Ca2+-permeable AMPARs markedly elongated glial processes. The process extension was blocked by 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX), a specific antagonist of AMPAR. These results indicate that glutamate regulates the morphology of glial processes by activating Ca2+-permeable AMPARs.     

Ca2+-permeable AMPA receptors in synaptic plasticity and neuronal death

AMPA receptors mediate fast synaptic transmission at excitatory synapses in the CNS and are crucial during neuronal development, synaptic plasticity and structural remodeling. AMPA receptors lacking GluR2 subunits are permeable to Ca(2+) and Zn(2+). Ca(2+) permeation through AMPA receptors is crucial in several forms of synaptic plasticity and cell death associated with neurological diseases and disorders. The subunit composition and Ca(2+) permeability of AMPA receptors are not static, but they are dynamically remodeled in a cell- and synapse-specific manner during development and in response to neuronal activity, sensory experience and neuronal insults. Exciting new research shows that these changes arise not only because of regulated expression of the AMPA receptor subunit GluR2, but also as a consequence of RNA editing, receptor trafficking and dendritic protein synthesis. This article reviews new insights into the role of Ca(2+)-permeable AMPA receptors in neuronal function and survival.     

Postsynaptic expression of Ca2+-permeable AMPA-type glutamate receptor channels by viral-mediated gene transfer

The ability to artificially express a particular receptor protein in the postsynaptic sites of neurons in the central nervous system (CNS) would be useful for the study of synaptic function of cloned receptor genes as well as for gene therapy of neurological disorders caused by dysfunction of postsynaptic receptors. In this study, we aimed to express the cDNA of unedited GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor that forms inwardly rectifying and Ca2+-permeable channel in CNS neurons by using adenoviral-mediated gene transfer. For this purpose, we have constructed a recombinant adenovirus bearing an expression-switching unit, where the unedited GluR2 cDNA can be activated by the Cre recombinase-mediated excisional deletion of a stuffer DNA interposed between the promotor and the coding region. When PC12 cells were infected with this recombinant adenovirus together with an adenovirus expressing Cre recombinase, the inwardly rectifying and Ca2+-permeable AMPA receptor channels were expressed in nearly 100% of infected cells. Two days after co-infection of cultured rat hippocampal neurons with these adenoviruses, fast excitatory neurotransmission in the glutamatergic synapse was mediated predominantly by the inwardly rectifying and Ca2+-permeable AMPA receptor channels. This indicates that the native AMPA receptors in the postsynaptic sites of the glutamatergic synapse are replaced rapidly with recombinant receptors newly produced by the viral-mediated gene transfer.     

A high GluR1 : GluR2 expression ratio is correlated with expression of Ca2+-binding proteins in rat forebrain neurons

alpha-Amino-3-hydroxy-5-methyl-4-isoxazle propionic acid (AMPA) receptors are ubiquitously expressed; however, their subtypes and abundance vary from region to region. We classified the neurons in various forebrain regions (hippocampus, striatum, amygdala, piriform cortex and somatosensory cortex) into six types: [R1+/R2+], [R1-/R2+], [R1+/R2-], [R1-/R2-], [R1++/R2+] and [R1++/R2-], and analysed the expression of Ca2+-binding proteins, such as parvalbumin and calbindin-D28k, using a triple-staining method. The neurons showing a high GluR1 : GluR2 expression ratio, [R1+/R2-], [R1++/R2+] and [R1++/R2-] neurons, comprised 13-30% of the total neuronal population. In addition, the expression of Ca2+-binding proteins was mainly observed in these three types of neurons. The results suggest that Ca2+-binding protein-positive neurons express Ca2+-permeable AMPA receptors, because the Ca2+-permeability of AMPA receptors is enhanced by the relative scarcity of the GluR2 subunit. To directly test the possibility that Ca2+-binding protein-positive neurons express Ca2+-permeable AMPA receptors, we performed Ca2+-imaging experiments in cultured cortical neurons. Ca2+ influx through AMPA receptors was measured selectively by addition of AMPA together with cyclothiazide in the presence of blockers of other Ca2+ influx routes. More than half of the calbindin-D28k-positive neurons showed a large increase in the intracellular Ca2+ concentration ([Ca2+]i), whilst most of the calbindin-D28k-undetectable neurons exhibited only a slight rise in [Ca2+]i after AMPA addition. These results suggest that the expression of calbindin-D28k is related to the expression of Ca2+-permeable AMPA receptors.     

Functional α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in differentiating embryonic neural progenitor cells

Glutamate-responsive α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are considered to play a significant role in neurogenesis. We have studied the functional expression of these receptors in migrating embryonic neural progenitor cells (NPCs). The majority of neurosphere-derived NPCs express AMPA receptors already during the first day of differentiation, based on mRNA quantification, immunocytochemistry, and Ca2+ imaging. The expression of GluR1 mRNA was significantly increased at 5 days of differentiation. The AMPA receptor subunits coexpressed with neuronal markers and were present in all cells at the outer periphery of the migration zone. In migrating NPCs, most of the AMPA receptors were philantotoxin sensitive and Ca2+-permeable, suggesting that in addition to their role in plasticity, the receptors are of importance in NPC differentiation.     

Characterization of AMPA receptor populations in rat brain cells by the use of subunit-specific open channel blocking drug, IEM-1460

Dicationic adamantane derivative, IEM-1460, which selectively blocks GluR2-lacking, Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, was used to characterize the distribution of AMPA receptors among populations of rat brain cells. IEM-1460 inhibited kainate-induced inward currents (at -80 mV) in a dose-dependent manner. IEM-1460 concentrations producing 50% inhibition of kainate-induced current amplitude (IC50) varied greatly depending on the cell type studied. Striatal giant cholinergic interneurons and putative Bergmann glial cells isolated from the cerebellum were found to be highly sensitive to IEM-1460 block (IC50=2.6 microM), indicating the expression of GluR2-lacking AMPA receptor subtype. Among hippocampal and cortical non-pyramidal neurons, there were cell-to-cell differences in the pattern of AMPA receptor subtype expression. Some cells which are known to express AMPA receptors lacking GluR2 subunit exhibited high sensitivity of IEM-1460 block (IC50 about 1 microM) but in the others, the part of AMPA receptor population seemed to be represented by GluR2-having receptor subtype. The latter subtype was mainly expressed by pyramidal neurons isolated from hippocampus (IC50=1102 microM) and sensorimotor cortex (IC50=357 microM) which showed low affinity for IEM-1460 block. In conclusion, IEM-1460 can be utilized as an indicator of the distribution of AMPA receptor subtypes among populations of rat brain cells, and pharmacological detection of the absence of GluR2 subunit in AMPA receptor assembly can provide useful information for the interpretation of physiological events.     

Cultured motor neurons possess calcium-permeable AMPA/kainate receptors

We examined the biology of AMPA/kainate-induced motor neuron degeneration using dissociated spinal cord cultures and motor neuron-specific antibodies which enable characterization of individual motor neurons in culture. Cobalt, which is thought to pass through Ca2+-permeable AMPA/kainate receptors following kainate exposure, labeled motor neurons in spinal cord cultures. The analysis of AMPA subunit distribution in dissociated motor neurons revealed a unique pattern of glutamate receptor (GluR) subunits in those cells; the GluR1 subunit was found in all spinal cord neurons, but the GluR2 subunit was not found in identified dissociated motor neurons. These data suggest that selective sensitivity of motor neurons to non-NMDA receptor activation is due, at least in part, to the presence of Ca2+-permeable AMPA/kainate receptors.     

Long-term potentiation in the hippocampal CA1 area and dentate gyrus plays different roles in spatial learning

NMDA receptor-dependent long-term potentiation (LTP) at hippocampal synapses has been considered a crucial component of the cellular basis for learning and memory. This form of LTP occurs in excitatory synapses in both the CA1 area and the dentate gyrus in the hippocampus. However, differential roles of LTP in these areas have not yet been identified. To address this issue, we enhanced the degree of LTP by expressing Ca2+-permeable AMPA receptors at either hippocampal CA1 or dentate gyrus synapses using Sindbis viral vectors (SINs) encoding both green fluorescent proteins and unedited GluR2 (GluR2Q) subunits, and examined their effects on rat spatial learning. The viral vectors were locally injected into the 8-week-old-rat brain in vivo bilaterally. The postsynaptic expression of Ca2+-permeable AMPA receptors enhanced the degree of LTP, and induced NMDA receptor-independent LTP in the presence of the NMDA receptor antagonist in SIN-infected regions in both CA1 and dentate gyrus in hippocampal slice preparations. However, the regional expression of Ca2+-permeable AMPA receptors caused opposite behavioural consequences on the Morris water maze task: rats with SIN-infected CA1 pyramidal cells showed shorter escape latency and better probe test performance, whereas those with SIN-infected dentate gyrus granule cells showed impaired performance. Thus, it was demonstrated that CA1 and dentate gyrus synapses play different functional roles in spatial learning despite their similar mechanism for LTP induction.     

Quantitative assessment of AMPA receptor mRNA in human spinal motor neurons isolated by laser capture microdissection

Disturbance of glutamate neurotransmission may contribute to the motor neuron injury seen in amyotrophic lateral sclerosis. Previous studies have suggested that human spinal motor neurons express a specific profile of the AMPA subtype of glutamate receptor with low mRNA expression for the GluR2 AMPA receptor subunit but other studies have contested this finding. The present study uses laser capture microdissection to isolate specifically identified neurons coupled with quantitative RT-PCR to demonstrate that the level of expression of the GluR2 subunit is lower in spinal motor neurons than in dorsal horn neurons from the same spinal cord region. Thus, it is likely that human spinal motor neurons express a proportion of Ca2+-permeable AMPA receptors which may contribute to the selective vulnerability of these cells in amyotrophic lateral sclerosis.     

Novel toxicity of the unedited GluR2 AMPA receptor subunit dependent on surface trafficking and increased Ca2+-permeability

RNA editing modifies the GluR2 AMPA receptor subunit pore loop at the Q/R site and limits receptor Ca(2+) permeability. Editing failure is implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis. We show that channels with unedited GluR2 are highly toxic in cultured hippocampal neurons. Toxicity exceeds that of other Ca(2+)-permeable AMPA receptor types and is influenced by agonist binding site mutations, ability to desensitize, and extracellular Ca(2+) levels. Significantly, toxicity also depends on GluR2's constitutive surface trafficking, a function dependent on GluR2 C-terminal domain interaction with NSF, a specialized chaperone. We have exploited the interaction between unedited GluR2 and NSF to reduce GluR2 surface levels. We show that a peptide that blocks the GluR2-NSF interaction reduces unedited GluR2 toxicity by reducing receptor surface expression. Peptide block of trafficking provides a model for design of drugs to reduce unedited GluR2 excitotoxicity in neurodegenerative diseases that result from editing failure.     

The lethal expression of the GluR2flip/GluR4flip AMPA receptor in HEK293 cells

alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) -type glutamate receptors play a critical role in excitotoxicity associated with cerebral hypoxia, ischaemia and other acute brain insults. AMPA receptors are composed of GluR1-GluR4 subunits in homomeric and heteromeric assemblies, forming nonselective cation channels. In addition, each subunit has alternative splice variants, flip and flop forms. Heterologous expression studies showed that the AMPA receptor channels exhibit diverse properties depending on subunit/variant composition. For example, the absence of the GluR2 subunit makes AMPA receptor assemblies Ca2+-permeable. Excitotoxicity induced by activating AMPA receptor channels has been linked to excessive Ca2+ influx through the GluR2-lacking channels. Here we demonstrate that coexpression of the AMPA receptor GluR2flip and GluR4flip subunits exerts a lethal effect on HEK293 cells, whereas no lethal activity is observed in other homomeric or heteromeric combinations of AMPA receptor subunits. Patch clamp recordings and Ca2+ imaging analyses have revealed that this GluR2flip/GluR4flip receptor exhibits a low Ca2+ permeability. This subunit combination, however, showed prolonged Na+ influx following AMPA stimulation, even in the absence of cyclothiazide, which attenuates AMPA receptor desensitization. Furthermore, the GluR2flip/GluR4flip-mediated lethality was potentiated by the interruption of cellular Na+ extrusion mechanisms using ouabain or benzamil. These observations suggest that the GluR2flip/GluR4flip receptor-mediated excitotoxicity is attributed to Na+ overload, but not Ca2+ influx.     

Selective and AMPA receptor-dependent astrocyte death following prolonged blockade of glutamate reuptake in rat cerebellar cultures

In this study we examined the effects of prolonged l-trans-pyrrolidine-2,4-dicarboxylate (PDC)-induced glutamate reuptake blockade on the viability of glial cells in cerebellar granule cell cultures. Immunofluorescence staining for the glial-specific intermediate filament protein, GFAP, revealed that the PDC- induced increase of extracellular glutamate concentration was accompanied by increased astrocyte death, while neurons and oligodendrocytes remained intact and viable. Astrocytic cell death was manifested as fragmentation of processes and cell bodies. The selective astrocyte death was completely prevented by the competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, NBQX (10 microM), whereas MK-801 (10 microM), a noncompetitive blocker of N-methyl-D-aspartate receptors, gave only partial protection. Double staining for GFAP and the AMPA receptor subunits GluR2/3 showed that astrocytes had much higher immunoreactivity for GluR2/3 than neurons or oligodendrocytes, suggesting that the number of AMPA receptors is likely to be higher on astrocytes. Furthermore, we employed real-time RT-PCR to measure GluR1-4 subunit mRNA expression in control and PDC-exposed cultures. Following treatment with PDC, GluR1 and GluR4 mRNAs were reduced by 40% and GluR3 was reduced by 70% relative to control levels. In contrast, GluR2 expression was not affected by the PDC treatment, indicating that GluR3 was the dominant type of AMPA receptor subunit expressed on astrocytes. Our results show that astrocytes appear to be more vulnerable than neurons or oligodendrocytes to a gradual increase in the extracellular glutamate concentration, suggesting that astrocytes may be critically involved in the pathophysiology of slowly developing chronic neurodegenerative disorders.     

Subpopulations of GABAergic and non-GABAergic rat dorsal horn neurons express Ca2+-permeable AMPA receptors.

Subpopulations of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors that are either permeable or impermeable to Ca2+ are expressed on dorsal horn neurons in culture. While both mediate synaptic transmission, the Ca2+ -permeable AMPA receptors provide a Ca2+ signal that may result in a transient change in synaptic strength [Gu, J.G., Albuquerque, C., Lee, C.J. & MacDermott, A.B. (1996) Nature, 381, 793]. To appreciate the relevance of these receptors to dorsal horn physiology, we have investigated whether they show selective expression in identified subpopulations of dorsal horn neurons. Expression of Ca2+-permeable AMPA receptors was assayed using the kainate-induced cobalt loading technique first developed by Pruss et al. [Pruss, R.M., Akeson, R.L., Racke, M.M. & Wilburn, J.L. (1991) Neuron, 7, 509]. Subpopulations of dorsal horn neurons were identified using immunocytochemistry for gamma-aminobutyric acid (GABA), glycine, substance P receptor (NK1 receptor) and the Ca2+-binding proteins, calretinin and calbindin D28K. We demonstrate that, in dorsal horn neurons in culture, kainate-induced cobalt uptake is selectively mediated by Ca2+-permeable AMPA receptors, and that a majority of GABA and NK1 receptor-expressing neurons express Ca2+-permeable AMPA receptors. GABAergic dorsal horn neurons are important in local inhibition as well as in the regulation of transmitter release from primary afferent terminals. NK1 receptor-expressing dorsal horn neurons include many of the projection neurons in the nociceptive spino-thalamic pathway. Thus, we have identified two populations of dorsal horn neurons representing important components of dorsal horn function that express Ca2+-permeable AMPA receptors. Furthermore, we show that several subpopulations of putative excitatory interneurons defined by calretinin and calbindin expression do not express Ca2+-permeable AMPA receptors.     

GluR2 deficiency accelerates motor neuron degeneration in a mouse model of amyotrophic lateral sclerosis

AMPA receptor-mediated excitotoxicity has been implicated in the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Motor neurons in vitro are particularly vulnerable to excessive AMPA receptor stimulation and one of the factors underlying this selective vulnerability is the presence of a large proportion of Ca2+ -permeable (i.e. GluR2-lacking) AMPA receptors. However, the precise role of GluR2-lacking AMPA receptors in motor neuron degeneration remains to be defined. We therefore studied the impact of GluR2 deficiency on motor neuron death in vitro and in vivo. Cultured motor neurons from GluR2-deficient embryos displayed an increased Ca2+ influx through AMPA receptors and an increased vulnerability to AMPA receptor-mediated excitotoxicity. We deleted the GluR2 gene in mutant SOD1G93A mice by crossbreeding them with GluR2 knockout mice. GluR2 deficiency clearly accelerated the motor neuron degeneration and shortened the life span of mutant SOD1G93A mice. These findings indicate that GluR2 plays a pivotal role in the vulnerability of motor neurons in vitro and in vivo, and that therapies that limit Ca2+ entry through AMPA receptors might be beneficial in ALS patients.