Elek Molnar

Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus.

It has been suggested that some glutamatergic synapses lack functional AMPA receptors. We used quantitative immunogold localization to determine the number and variability of synaptic AMPA receptors in the rat hippocampus. Three classes of synapses show distinct patterns of AMPA receptor content. Mossy fiber synapses on CA3 pyramidal spines and synapses on GABAergic interneurons are all immunopositive, have less variability, and contain 4 times as many AMPA receptors as synapses made by Schaffer collaterals on CA1 pyramidal spines and by commissural/ associational (C/A) terminals on CA3 pyramidal spines. Up to 17% of synapses in the latter two connections are immunonegative. After calibrating the immunosignal (1 gold = 2.3 functional receptors) at mossy synapses of a 17-day-old rat, we estimate that the AMPA receptor content of C/A synapses on CA3 pyramidal spines ranges from <3 to 140. A similar range is found in adult Schaffer collateral and C/A synapses.

High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus

The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal sequence of the protein (residues 869-881), conjugated to carrier protein and absorbed to colloidal gold for immunization. The purified antibodies immunoprecipitated about 25% of 3[H]AMPA binding activity from the hippocampus, cerebellum or whole brain, but very little from neocortex. These antibodies did not precipitate a significant amount of 3[H]kainate binding activity. The antibodies also recognize the GluRD subunit, but not the other AMPA receptor subunits, when expressed in transfected COS-7 cells and only when permeabilized with detergent, indicating an intracellular epitope. All subunits were enriched in the neuropil of the dendritic layers of the hippocampus and in the molecular layer of the dentate gyrus. The cellular distribution of the GluRD subunit was studied more extensively. The strata radiatum, oriens and the dentate molecular layer were more strongly immunoreactive than the stratum lacunosum moleculare, the stratum lucidum and the hilus. However, in the stratum lucidum of the CA3 area and in the hilus the weakly reacting dendrites were surrounded by immunopositive rosettes, shown in subsequent electron microscopic studies to correspond to complex dendritic spines. In the stratum radiatum, the weakly reacting apical dendrites contrasted with the surrounding intensely stained neuropil. The cell bodies of pyramidal and granule cells were moderately reactive. Some non-principal cells and their dendrites in the pyramidal cell layer and in the alveus also reacted very strongly for the GluRD subunit. At the subcellular level, silver intensified immunogold particles for the GluRA, GluRB/C and GluRD subunits were present at type 1 synaptic membrane specializations on dendritic spines of pyramidal cells throughout all layers of the CA1 and CA3 areas. The most densely labelled synapses tended to be on the largest spines and many smaller spines remained unlabelled. Immunoparticle density at type 1 synapses on dendritic shafts of some non-principal cells was consistently higher than at labelled synapses of dendritic spines of pyramidal cells. Synapses established between dendritic spines and mossy fibre terminals, were immunoreactive for all studied subunits in stratum lucidum of the CA3 area. The postembedding immunogold method revealed that the AMPA type receptors are concentrated within the main body of the anatomically defined type 1 (asymmetrical) synaptic junction. Often only a part of the membrane specialization showed clustered immunoparticles. There was a sharp decrease in immunoreactive receptor density at the edge of the synaptic specialization. Immunolabelling was consistently demonstrated at extrasynaptic sites on dendrites, dendritic spines and somata. The results demonstrate that the GluRA, B/C and D subunits of the AMPA type glutamate receptor are present in many of the glutamatergic synapses formed by the entorhinal, CA3 pyramidal and mossy fibre terminals. Some interneurons have a higher density of AMPA type receptors in their asymmetrical afferent synapses than pyramidal cells. This may contribute to a lower activation threshold of interneurons as compared to principal cells by the same afferents in the hippocampal formation.

Assembly intracellular targeting and cell surface expression of the human N-methyl-d-aspartate receptor subunits NR1a and NR2A in transfected cells

The intracellular trafficking, assembly, and cell surface targeting of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A has been studied using both transiently and permanently transfected mammalian cell lines. The expression of either NR1a or NR2A alone does not result in significant cell surface expression of either subunit as determined by cell surface biotinylation and immunofluorescence staining. When NR1a is expressed alone large intracellular accumulations of the subunit are formed which do not co-localize with the golgi apparatus markers protein p58 and wheat germ agglutinin, but do co-localize with the endoplasmic reticulum marker calreticulin. Co-expression of NR1a and NR2A results in a reduction of these intracellular accumulations and the appearance of both subunits on the cell surface. Immunoprecipitation of NR1a from in vitro translated subunit proteins showed that NR2A could only be immunoprecipitated with NR1a when both subunits were co-synthesized in the presence of microsomes. When cells expressing NR1a and NR2A were incubated with [35S]methionine in the presence of Brefeldin-A, a drug which prevents protein transport from the endoplasmic reticulum, NR2A could be immunoprecipitated by an antiserum specific for NR1a. Together these results suggest that the NMDA receptor subunits are assembled in the endoplasmic reticulum and that co-synthesis of the subunits is necessary for their association and their successful cell surface targeting.

Metabotropic Glutamate Receptor-Mediated Long-Term Depression: Molecular Mechanisms

The ability to modify synaptic transmission between neurons is a fundamental process of the nervous system that is involved in development, learning, and disease. Thus, synaptic plasticity is the ability to bidirectionally modify transmission, where long-term potentiation and long-term depression (LTD) represent the best characterized forms of plasticity. In the hippocampus, two main forms of LTD coexist that are mediated by activation of either N-methyl-d-aspartic acid receptors (NMDARs) or metabotropic glutamate receptors (mGluRs). Compared with NMDAR-LTD, mGluR-LTD is less well understood, but recent advances have started to delineate the underlying mechanisms. mGluR-LTD at CA3:CA1 synapses in the hippocampus can be induced either by synaptic stimulation or by bath application of the group I selective agonist (R,S)-3,5-dihydroxyphenylglycine. Multiple signaling mechanisms have been implicated in mGluR-LTD, illustrating the complexity of this form of plasticity. This review provides an overview of recent studies investigating the molecular mechanisms underlying hippocampal mGluR-LTD. It highlights the role of key molecular components and signaling pathways that are involved in the induction and expression of mGluR-LTD and considers how the different signaling pathways may work together to elicit a persistent reduction in synaptic transmission.

Concomitant deficits in working memory and fear extinction are functionally dissociated from reduced anxiety in metabotropic glutamate receptor 7-deficient mice.

Metabotropic glutamate receptor 7 (mGluR7), a receptor with a distinct brain distribution and a putative role in anxiety, emotional responding, and spatial working memory, could be an interesting therapeutic target for fear and anxiety disorders. mGluR7-deficient (mGluR7−/−) mice showed essentially normal performance in tests for neuromotor and exploratory activity and passive avoidance learning but prominent anxiolytic behavior in two anxiety tests. They showed a delayed learning curve during the acquisition of the hidden-platform water maze, and three interspersed probe trials indicated that mGluR7−/− mice were slower to acquire spatial information. Working memory in the water maze task and the radial arm maze was impaired in mGluR7−/− mice compared with mGluR7+/+. mGluR7−/− mice also displayed a higher resistance to extinction of fear-elicited response suppression in a conditioned emotional response protocol. In a non-fear-based water maze protocol, mGluR7−/− mice displayed similar delayed extinction. These observed behavioral changes are probably not attributable to changes in AMPA or NMDA receptor function because expression levels of AMPA and NMDA receptors were unaltered. Extinction of conditioned fear is an active and context-dependent form of inhibitory learning and an experimental model for therapeutic fear reduction. It appears to depend on glutamatergic and higher-level brain functions similar to those involved in spatial working memory but functionally dissociated from those that mediate constitutional responses in anxiety tests.

Identification of functional ionotropic glutamate receptor proteins in pancreatic beta-cells and in islets of Langerhans.

The presence of ionotropic glutamate receptor proteins in islets of Langerhans and pancreatic β‐cell lines (MIN6, HIT T15, RINm5F) was investigated. For this purpose immunoblot analysis of β‐cell membranes was performed with subunit‐specific antibodies. We identified NMDAR1 subunits of the NMDA and KA‐2 subunits of the kainate receptors, but did not detect GluR1 subunits of the AMPA receptor. The receptor subunits present were shown to be glycosylated. β‐cell membranes contained specific binding sites for glutamate receptor ligands, and NMDA increased insulin secretion. These results demonstrate that ionotropic glutamate receptor proteins, similar to those in the central nervous system, are expressed in rat pancreatic β‐cells.

The C-terminal domain of glutamate receptor subunit 1 is a target for calpain-mediated proteolysis

The AMPA receptors are glutamate-gated ion channels mediating synaptic transmission at the majority of excitatory synapses in the mammalian CNS. They are composed of four subunits (GluR1-4) which exist in two alternatively spliced variants (flip and flop) and are generally considered to form pentameric receptors. The transmembrane structure of the receptors remains a matter of controversy as some data suggest a transmembrane topology consisting of five, four, or three membrane spanning regions. Some receptor properties have been shown to be regulated by phosphorylation processes as well as by the phospholipid environment. More recently, we have shown that calcium treatment of thin (10 microns) frozen-thawed brain sections resulted in profound modifications of the immunochemical properties of the AMPA receptors. More specifically, immunolabelling of the AMPA receptors with antibodies directed against the C-terminal domain of GluR1 and GluR2/3 was markedly decreased in dendritic fields following such treatment at 35 degrees C. This effect was temperature-dependent and completely blocked by inhibitors of the calcium-dependent proteases calpains, and we suggested that calpains are involved in the regulation of AMPA receptor properties. The results of the present study demonstrate that calpain activation produces a partial proteolysis in the C-terminal domain of the receptors and generates a new receptor species with an apparent molecular weight of 103,000 mol. wt. Sequence analysis of the GluR1 C-terminal domain suggests a couple of cleavage sites for calpains. These results are of particular interest considering the body of evidence implicating calpains and changes in excitatory amino acid receptors in mechanisms of synaptic plasticity as well as in neurodegenerative processes.

Identification of Lectin-Purified Neural Glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA Receptor Subunits: Conservation of Glycosylation at the Synapse

Abstract: The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin‐affinity chromatography, showed immunoreactivity in the present study with subunit‐specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (pan‐AMPA) receptors. Sensitivities to the glycosidases peptide N‐glycosidase F and endo‐β‐N‐acetylglucosaminidase H on both western blots and silver‐stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for ∼50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N‐glycosidase F and endo‐β‐N‐acetylglucosaminidase H were observed for cell line‐expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.

Developing oligodendrocytes express functional GABAB receptors that stimulate cell proliferation and migration

GABAB receptors (GABABRs) are involved in early events during neuronal development. The presence of GABABRs in developing oligodendrocytes has not been established. Using immunofluorescent co‐localization, we have identified GABABR proteins in O4 marker‐positive oligodendrocyte precursor cells (OPCs) in 4‐day‐old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABAB1abcdf and GABAB2 subunits of the GABABR. Using semiquantitative PCR analysis with GABABR isoform‐selective primers we found that the expression level of GABAB1abd was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABAB2 isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABAB1 and GABAB2 subunits are under independent control during oligodendroglial development. Activation of GABABRs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABABR activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber‐based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABABRs in oligodendrocyte development. The presence of GABABRs in developing oligodendrocytes provides a new mechanism for neuronal–glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.

Membrane Topology of the GluR1 Glutamate Receptor Subunit: Epitope Mapping by Site‐Directed Antipeptide Antibodies

Abstract: In order to define the membrane topology of the GluR1 glutamate receptor subunit, we have examined the location of epitopes. Antibodies were produced against peptides corresponding to putative extracellular and intracellular segments of the rat brain GluR1 glutamate receptor subunit. Immunocytochemistry at the electron microscopic level in the dentate gyrus of the hippocampal formation showed that epitopes for the antiserum to the N‐terminal part of the subunit are located at the extracellular face of the plasma membrane, whereas the antigenic determinants for the antiserum to the C‐terminal part are found at the intracellular face of the postsynaptic membrane. Furthermore, antibodies to the N‐terminal residues 253–267 reacted similarly with both intact and permeabilized synaptosomes, whereas the binding of antibodies to the C‐terminal residues 877–889 increased about 1.6‐fold following permeabilization. Our data suggest that the N‐ and C‐terminal regions are located on the opposite side of the membrane and, therefore, the GluR1 subunit probably has an odd number of membrane spanning segments. The antibody cross‐reactivities in different species and their effect on ligand binding activity were also established.