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E K Michaelis

Publications and source records attributed to E K Michaelis.

At least 37 records · Page 2Linked to original sources
PubMed1995

Developmental expression of N-methyl-D-aspartate (NMDA)-induced neurotoxicity, NMDA receptor function, and the NMDAR1 and glutamate-binding protein subunits in cerebellar granule cells in primary cultures.

Cerebellar granule cells maintained in vitro as primary cultures are a relatively homogeneous neuronal population that can be used to evaluate the developmental expression of neurotransmitter receptors and to assess their role in cell survival and degeneration. The toxicity induced by N-methyl-D-aspartate (NMDA) in granule cells maintained under partially depolarizing conditions and in the presence of physiologic extracellular concentrations of Mg2+ was greatest for the neurons maintained for 14 days in vitro (DIV). However, following NMDA receptor activation neurons as young as 5 DIV exhibited increases in the concentration of intracellular free Ca2+ which were as large as those achieved with cells at 8-9 or 13-14 DIV. The less mature neurons exhibited a "down-regulation" of responses to increasing concentrations of NMDA and the more mature cells maintained elevated intracellular Ca2+ levels during the inter-stimulus periods. Immunochemical analyses of the expression of the NMDA receptor-associated proteins NMDAR1 and glutamate-binding protein (GBP) in granule cells indicated a developmental increase in both proteins, albeit the pattern of expression of NMDAR1 was the more complex. No definite correlation has yet been established between toxicity induced by NMDA and the expression of these two proteins. Finally, although the developmental expression of nitric oxide synthase, an enzyme that catalyzes the formation of the potentially neurotoxic radicals nitric oxide and superoxide anion, increased progressively with the maturation of neurons in culture, an inhibitor of this enzyme did not protect neurons from NMDA-induced toxicity. Therefore, the developmental changes in granule cells that lead to increased vulnerability following excessive activation of NMDA receptors are not yet completely defined.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1994

Purification and pharmacological and immunochemical characterization of synaptic membrane proteins with ligand-binding properties of N-methyl-D-aspartate receptors.

A method was developed for the solubilization of approximately 50% of proteins in synaptic membranes that have ligand-binding characteristics of N-methyl-D-aspartate (NMDA) receptors. Affinity chromatographic separation of the solubilized proteins through L-glutamate-derivatized matrices and subsequent elution by NMDA-containing buffers led to the purification of four predominant proteins with estimated sizes of 67-70, 53-62, 41-43, and 28-36 kDa. The co-purification of NMDA-sensitive L-glutamate binding, dizocilpine-sensitive thienylcyclohexyl piperidine (TCP)-binding, and strychnine-insensitive glycine-binding proteins was achieved by this affinity chromatographic procedure. Glutamate, glycine, and the polyamine spermidine increased both the "on" rate and the equilibrium level of [3H]TCP binding to the isolated proteins. The group of proteins eluted by NMDA from the glutamate-derivatized matrices could be further purified through size exclusion chromatography of the NMDA-sensitive glutamate-binding from the dizocilpine-sensitive TCP-binding proteins. Polyclonal and monoclonal antibodies to the cloned NMDA receptor protein NMDAR1 did not react with any proteins in the solubilized membrane proteins or the purified fractions. However, immunoreaction of antibodies raised against a glutamate-binding protein and a phosphonoaminocarboxylic acid-binding protein indicated that these are two of the major proteins in the purified fractions. These studies indicate that these two proteins might be components of a complex that has some of the characteristics of NMDA receptors and that neuronal membranes may contain varieties of NMDA-like receptors composed of protein subunits that differ from the NMDAR1 and NMDAR2 receptor proteins.

Amino Acid Sequence↗
PubMed1994

Direct measurement of glutamate release in the brain using a dual enzyme-based electrochemical sensor.

The in vivo measurement of the rapid changes in the extracellular concentrations of L-glutamic acid in the mammalian brain during normal neuronal activity or following excessive release due to episodes of anoxia or ischemia has not been possible to this date. Current techniques for the measurement of the release of endogenous glutamate into the extracellular space of the central nervous system are relatively slow and do not measure the actual concentration of free glutamate in the extracellular space. An enzyme-based electrode with rapid response times (about 1 s) and high degree of sensitivity (less than 2 microM) and selectivity for L-glutamic acid is described in this paper. This electrode has both L-glutamate and ascorbate oxidase immobilized on its surface. The latter enzyme removes almost completely any interferences produced by the high levels of extracellular ascorbate present in brain tissue. The response of the electrode to glutamate and other potentially interfering substances was fully characterized in vitro and its selectivity, sensitivity and rapidity in responding to a rise in extracellular glutamate concentrations was also demonstrated in vivo. Placement of the electrode in the dentate gyrus of the hippocampus led to the detection of both KCl-induced release of L-glutamic acid and the release induced by stimulation of the axons in the perforant pathway. The development of this selective, sensitive and rapidly responding glutamate sensor should make it now possible to measure the dynamic events associated with glutamate neurotransmission in the central nervous system.

Animals↗
PubMed1994

Effects of ethanol on NMDA receptors in brain: possibilities for Mg(2+)-ethanol interactions.

The major excitatory neurotransmitter in the CNS is L-glutamate, and one of the subtypes of L-glutamate receptors, the N-methyl-D-aspartate (NMDA) subtype, has been found to be quite sensitive to inhibition by low concentrations of ethanol (5-50 mM). The NMDA receptor-ion channels are unique in that they exhibit a voltage-dependent blockade by physiological concentrations of Mg2+, a blockade that is relieved as the cell membrane is depolarized. Several lines of evidence also suggest that the activity of this receptor-channel complex may be regulated through a high-affinity Mg2+ site, which is distinct from the channel-blocking site and could even be located on the extracellular domain of the protein. This high-affinity Mg2+ site has been shown to increase the binding of N-[1-(2-thienyl) cyclohexyl]piperidine within the ion channel, as well as the binding of competitive antagonist such as 3-(+/-)-carboxypiperazine-4-yl)-[1,2]-propyl-1-phosphonic acid and the receptor coactivator glycine. The relationship between the acute effects of ethanol on receptor activation and the regulatory properties of Mg2+ is not yet known, although the hypomagnesemia that occurs in chronic alcoholism could certainly have implications for receptor function. A significant amount of molecular characterization of the multiple isoforms of the NMDA receptor-ion channel will be required before the role of Mg2+ can be clarified and any relationship between Mg2+ regulation and ethanol inhibition established.

Alcoholism↗
PubMed1993

Immunochemical and immunohistochemical characterization of a synaptic membrane protein that binds the competitive antagonists of NMDA receptors.

An approximately 54-kDa protein that has binding sites for the competitive N-methyl-D-aspartate (NMDA) receptor antagonists 3-((+-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) and (+-)-(E)-2-amino-4-propyl-5-phosphonopentenoic acid (CGP 39653) was purified from rat brain synaptic membranes. Polyclonal antibodies to this protein reacted specifically with an approximately 54-kDa protein in synaptic membranes and immunoextracted approximately 60% of [3H]CGP 39653 binding sites associated with solubilized membrane proteins. The antibodies also labeled antigenic sites in the perikaryon and apical and basilar dendrites of pyramidal neurons of the hippocampus and cerebral cortex.

2-Amino-5-phosphonovalerate↗
PubMed1993

Two different families of NMDA receptors in mammalian brain: physiological function and role in neuronal development and degeneration.

The data from purification, ligand binding, reconstitution and immunochemical studies indicate that there is a group of small molecular size proteins (30 to 70 kDa) that form what appear to be NMDA receptor complexes. Based on cell biological studies of the expression and localization of one of the subunits of this complex, the glutamate-binding subunit, it appears that this putative NMDA receptor plays a key role in neuronal sensitivity to NMDA and in neuronal survival in early development. However, brain neurons quite clearly express another family of proteins which have all functional characteristics of an NMDA receptor plus a great degree of variability that can account for the varieties of NMDA receptors found in brain. This family of NMDA receptors, the NMDAR1 and NMDAR2, are not homologous to the small molecular size proteins of the previously described receptor complex that was isolated from synaptic membranes. If brain neurons are indeed expressing two very diverse families of proteins that function as glutamate/NMDA receptors, this must be an indication that either there is a very selective expression of one of these forms in specific neurons or neuronal compartments, or that one of these forms of the receptor plays an important role in unique functions of the cell, such as synaptic plasticity or neurodegeneration. As more information is gathered about the structure and function of these receptors, a better understanding of the expression and role of these families of receptor proteins in normal neuronal excitability will be achieved. This enhanced level of understanding about the function and activity of these receptors will be needed in order to identify more precisely the NMDA receptors most affected in pathological conditions such as hyperammonemia.

Alternative Splicing↗
PubMed1993

Glutamate receptor changes in brain synaptic membranes during chronic alcohol intake.

Ethanol inhibits the receptor-ion channels activated by the glutamate analog N-methyl-D-aspartate (NMDA) in mammalian brain neurons. The high degree of sensitivity of these receptors to the acute effects of ethanol has led to the development of hypotheses that brain neurons may adapt to chronic exposure to ethanol either by increasing the numbers of NMDA receptors, or by expressing receptors with decreased sensitivity to ethanol, or by expressing receptors with increased affinity for the endogenous activator of these entities, L-glutamic acid. Studies performed with experimental animal models of chronic ethanol intake are indicative of an adaptive response that entails the expression in neuronal membranes of greater numbers of L-glutamate and NMDA receptors. When similar studies were performed with synaptic membranes isolated from the brains of human alcoholics, there was an apparent increase in the total number of glutamate receptors, but a decrease in the density of the subpopulation of these receptors that are activated by NMDA. These results are interpreted to suggest that there are more subtle and complex adaptations made in neurons in the brains of human alcoholics as compared with those occurring in animal models of chronic alcoholism.

Adaptation, Physiological↗
PubMed1993

Basic FGF regulates the expression of a functional 71 kDa NMDA receptor protein that mediates calcium influx and neurotoxicity in hippocampal neurons.

Basic fibroblast growth factor (bFGF) was recently found to modulate the outgrowth-regulating effects of glutamate, and protected neurons from several brain regions against excitotoxi/ischemic damage. We provide evidence that the excitoprotective mechanism of bFGF involves suppression of the expression of a 71 kDa NMDA receptor protein (NMDARP-71). NMDARP-71 protein and mRNA levels were reduced in neurons in bFGF-treated hippocampal cell cultures. The levels of the NMDARP-71 were not reduced by NGF or epidermal growth factor, and bFGF did not reduce the level of mRNA for the GluR1 kainate/AMPA receptor, demonstrating the specificity of the effect of bFGF on the NMDARP-71. The reduction in NMDARP-71 expression in bFGF-treated neurons was correlated with reduced vulnerability to NMDA neurotoxicity. A major role for NMDARP-71 in calcium responses to NMDA and excitotoxicity was demonstrated using antisense oligonucleotides directed against NMDARP-71. Northern and Western blot analysis and immunocytochemistry showed that NMDARP-71 antisense oligonucleotides caused a selective suppression of NMDARP-71 mRNA and protein levels during 12-44 hr exposure periods. Elevations in intracellular calcium levels normally caused by glutamate and NMDA were attenuated in neurons exposed to NMDARP-71 antisense oligonucleotide; calcium responses to kainate were relatively unaffected. NMDARP-71 antisense oligonucleotides protected the neurons against excitotoxicity. Thus, NMDARP-71 is a necessary component of an NMDA receptor mediating calcium responses and neurotoxicity in hippocampal neurons. Taken together, these data identify a mechanism whereby bFGF can modify neuronal responses to glutamate, and suggest that regulating the expression of excitatory amino acid receptors may provide a means for growth factors to influence the plasticity and degeneration of neural circuits.

Animals↗
PubMed1992

Molecular aspects of glutamate receptors and sodium-calcium exchange carriers in mammalian brain: implications for neuronal development and degeneration.

N-Methyl-D-aspartate (NMDA) and L-glutamate activate membrane receptors that produce substantial permeation of Na+, K+ and Ca2+ through the neuronal membrane. These ionic fluxes are intimately linked to processes that regulate neuronal survival, growth and differentiation. Intracellular free Ca2+ concentrations are thought to be particularly important determinants of the vulnerability of neurons to excessive excitatory stimulation produced through activation of NMDA receptors. In order to understand the molecular events involved in both NMDA receptor activation and regulation of intracellular Ca2+ levels, we have purified and reconstituted the protein complexes that form the NMDA/glutamate receptors in rat brain synaptic membranes and those that constitute the Na(+)-Ca2+ antiporters in bovine brain synaptic membrane. The molecular properties of these protein complexes are described, and information from the most recent studies of exploration of the molecular structures of these receptors and transport carriers is summarized.

Animals↗
PubMed1992

Isolation of glutamate-binding proteins from rat and bovine brain synaptic membranes and immunochemical and immunocytochemical characterization.

We previously isolated two glutamate-binding proteins from rat brain synaptic membranes of 71,000 and 63,000 mol. wt [Chen et al. (1988) J. biol. Chem. 263, 417-426]. In the present study, the 71,000 and 63,000 mol. wt glutamate-binding proteins were purified from rat and bovine brain synaptic membranes by affinity chromatographic separation on an L-glutamate-derived Trisacryl matrix. The major protein component in the purified fractions was a 71,000 mol. wt protein as judged by sodium dodecyl sulfate gel electrophoresis. This fraction represented enrichment of bovine brain glutamate-binding proteins by a factor of 7000-8000 when compared with brain homogenates. The ligand binding characteristics of the proteins purified by this new procedure are very similar to those of the proteins purified by the procedures we described in previous studies. Polyclonal antibodies were raised in mice against the purified, native rat and bovine brain glutamate-binding proteins. These two sets of antibodies interacted specifically with the glutamate-binding proteins but not with any glutamate-metabolizing enzymes. Both sets of antibodies labeled a 71,000 mol. wt protein in Western blots of synaptic membranes obtained from either rat or bovine brain, an indication of homology between these proteins. Both sets of antibodies produced immunoprecipitation of approximately 70-75% of all glutamate-binding proteins from solubilized synaptic membrane proteins. These observations are an indication that the glutamate-binding proteins described above are the most common glutamate-binding entities in both bovine and rat brain synaptic membranes and that they can be easily purified in a one-step chromatographic procedure.

Animals↗
PubMed1992

Characterization of organophosphate interactions at N-methyl-D-aspartate receptors in brain synaptic membranes.

Several competitive antagonists of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors are phosphonate analogs of L-glutamic acid. The position of the phosphonate has been shown to be important in the structure-activity relationships of these analogs. To investigate whether other phosphorous-containing compounds had activity at the NMDA receptor, several organophosphates were tested for the ability to inhibit the specific binding to brain synaptic membranes of 3-((+-)-2-carboxypiperazin-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP), a selective antagonist of NMDA receptors. Diisopropylfluorophosphate (DFP), dichlorvos, cyanophos, mipafox, and o-ethyl o-4-nitrophenyl phenylphosphonothioate are relatively potent inhibitors of [3H]CPP binding to synaptic membranes. The inhibition produced by DFP is selective for the NMDA subtype of excitatory amino acid receptors, is irreversible, and can be prevented by preincubation with excess CPP, 2-amino-7-phosphonoheptanoic acid, or L-glutamate. Rat brain synaptic membranes have a population of phosphonate-sensitive [3H]DFP binding sites that are covalently labeled by [3H]DFP. Two protein bands of synaptic membrane proteins subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis are labeled by [3H]DFP in a 2-amino-5-phosphonopentanoic acid-sensitive manner. These proteins have an average molecular size of 47-50 and 32 kDa. Proteins of nearly identical molecular sizes have been shown in other studies to be components of an NMDA receptor complex. These observations are indicative of an interaction between the organophosphates and the NMDA receptor protein complex and suggest that DFP may be another important pharmacological tool that can be used in the elucidation of the molecular structure of the NMDA receptor complex.

Animals↗
PubMed1991

Ion channel sensors for glutamic acid.

Coulometric biosensors using glutamate receptor (GluR) ion channel protein as a signal-amplifying sensory element that exploit the glutamate-triggered Na+ ion current through bilayer lipid membranes have been fabricated. The formation of stable planar bilayer lipid membranes was achieved by applying the folding method across a small circular aperture bored through a thin polyimide film. The multichannel type sensing membranes, formed across an aperture of ca. 120 microns diameter, contained more than 10 GluR proteins and showed L-glutamate-triggered response as a composite of individual single-channel currents. The single-channel type sensing membranes, formed across an aperture of ca. 20 microns diameter, contained a sufficiently small number of GluR proteins so that the response was observed as a series of single-channel pulse currents. Dependence of the integrated channel current on the glutamate concentration was examined. A sharp concentration dependence of up to ca. 1.5 x 10(-7) M and 3 x 10(-6) M for the multichannel and single-channel type sensors, respectively, was observed. A high selectivity for L-glutamate compared with D-glutamate for inducing the channel current was observed. A detection limit as low as ca. 3 x 10(-8) M was attained for the multichannel type sensor. This remarkable sensitivity is discussed in terms of the potential use of GluR ion channel protein for a new type of sensing system.

Biosensing Techniques↗
PubMed1991

Developmental expression, compartmentalization, and possible role in excitotoxicity of a putative NMDA receptor protein in cultured hippocampal neurons.

The mechanisms regulating the expression and localization of excitatory amino acid (EAA) neurotransmitter receptors in neurons of the developing mammalian brain, and roles for these receptors in the plasticity and degeneration of neural circuits are not well understood. We previously isolated and characterized a 71 kDa glutamate binding protein (GBP) from rat brain, and have recently obtained evidence that this GBP is a component of a functional N-methyl-D-aspartate (NMDA) receptor-ion channel complex. We have now used antibodies to this putative NMDA receptor protein to examine its expression and localization, and consequences of its activation in cultured embryonic (18 day) rat hippocampal neurons. Immunocytochemistry and Western blots using monoclonal antibodies to the GBP demonstrated an increase in GBP-positive neurons and their staining intensity with time in culture. GBP was localized to the somata and dendrites of pyramidal-like neurons and was sparse or absent in the axons. The expression and compartmentalization of GBP occurred in isolated neurons indicating that direct cell interactions were not required for these processes. Cell surface staining for GBP occurred in patches on the soma and dendrites. The developmental expression of GBP immunoreactivity closely paralleled the expression of sensitivity to NMDA neurotoxicity. There was a direct relationship between GBP immunoreactivity and neuronal vulnerability to glutamate-induced degeneration; vulnerable neurons stained heavily whereas resistant neurons showed either low levels of staining or no staining. Finally, a GBP antiserum greatly reduced NMDA neurotoxicity (but not kainate neurotoxicity). Taken together, these findings demonstrate the expression of presumptive NMDA receptors within a subpopulation of embryonic hippocampal neurons, and their segregation to the soma and dentrites of pyramidal neurons. This spatial distribution of glutamate receptors among and within neurons is likely to play important roles in regulating the structure of neural circuitry during development, and may also be an important determinant of selective neuronal vulnerability in pathological conditions.

Animals↗
PubMed1991

Cloning of cDNA for the glutamate-binding subunit of an NMDA receptor complex.

The amino acids L-glutamic and L-aspartic acids form the most widespread excitatory transmitter network in mammalian brain. The excitation produced by L-glutamic acid is important in the early development of the nervous system, synaptic plasticity and memory formation, seizures and neuronal degeneration. The receptors activated by L-glutamic acid are a target for therapeutic intervention in neurodegenerative diseases, brain ischaemia and epilepsy. There are two types of receptors for the excitatory amino acids, those that lead to the opening of cation-selective channels and those that activate phospholipase C (ref. 11). The receptors activating ion channels are NMDA (N-methyl-D-aspartate) and kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive receptors. The complementary DNAs for the kainate/AMPA receptor and for the metabotropic receptor have been cloned. We report here on the isolation and characterization of a protein complex of four major proteins that represents an intact complex of the NMDA receptor ion channel and on the cloning of the cDNA for one of the subunits of this receptor complex, the glutamate-binding protein.

Amino Acid Sequence↗
PubMed1991

Hydrodynamic properties of the purified glutamate-binding protein subunit of the N-methyl-D-aspartate receptor.

The hydrodynamic properties of the previously purified glutamate-binding protein from rat synaptic membranes were determined in order to estimate the molecular size of the protein in its native state. This protein is apparently a subunit of a multisubunit complex that forms the N-methyl-D-aspartate subtype of glutamate receptor and has a molecular size of approximately 70 kDa based on electrophoretic migration under denaturing conditions. On the basis of results obtained from H2O/D2O sucrose density gradient sedimentation and gel filtration chromatography of the purified glutamate-binding protein we calculated the partial specific volume of the protein-detergent complex to be 0.766 cc3/g, the Stokes radius of the complex as 4.9 nm, the Mc of the complex as 203,000 +/- 22,000 and the Mr of the protein as 182,000 +/- 19,000. These results are indicative of stable self-association of the glutamate-binding protein and are in agreement with recent studies indicating that more than one molecule of glutamate may be required to activate the N-methyl-D-aspartate receptor-associated ion channel.

Animals↗
PubMed1991

Solubilization, partial purification, and reconstitution of glutamate- and N-methyl-D-aspartate-activated cation channels from brain synaptic membranes.

L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. The affinity batch chromatography procedure described previously [Chen et al. (1988) J. Biol. Chem. 263, 417-427] was used to obtain a fraction enriched in glutamate-binding proteins. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. [14C]Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L-Glutamate caused an increase in the rate of [14C]methylamine influx into liposomes reconstituted with either solubilized membrane proteins or partially purified glutamate-binding proteins. The increase in methylamine influx was dependent on the concentration of L-glutamic acid with an estimated Kact for L-glutamate equal to 0.2 microM for synaptic membrane proteins and 0.32 microM for purified proteins. Of the major glutamate receptor agonists, only N-methyl-D-aspartate activated cation fluxes in liposomes reconstituted with glutamate-binding proteins. Glutamate-activated methylamine flux was completely inhibited by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D-aspartate- or glutamate-induced influx of Na+ led to a transient increase in the influx of the lipid-permeable anion probe S14CN-. Electrophoretic analysis of partially purified proteins reconstituted in liposomes indicated enrichment of several bands, the most prominent being those of molecular size equal to approximately 69, 60, 35, and 25 kDa. Antibodies raised against the purified 71- and 63-kDa glutamate-binding proteins reacted strongly with the approximately 69-kDa band of reconstituted proteins and markedly decreased the initial rate of glutamate-activated cation flux. These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the approximately 69-kDa protein in the function of these ion channels.

Animals↗
PubMed1990

Immunochemical characterization of brain synaptic membrane glutamate-binding proteins.

Two glutamate-binding proteins (71 and 63 kDa) were previously purified from synaptic plasma membranes (Chen, J.-W., Cunningham, M.D., Galton, V., and Michaelis, E. K. (1988) J. Biol. Chem. 263, 417-426). These proteins may play a role in glutamate neurotransmission in brain. Polyclonal antibodies were raised against the denatured glutamate-binding proteins in rabbits, including sets of antibodies against each of the binding proteins. The antibodies reacted specifically against both 71- and 63-kDa proteins. The antibodies recognized the denatured form of the proteins in Western blots and the native state of the proteins in enzyme-linked immunosorbent assays and in immunoaffinity chromatography and extraction procedures. All antibodies labeled most strongly the 71-kDa protein in Western blots, but extracted both proteins from solubilized synaptic membrane preparations. These findings indicate that the two proteins are closely related immunologically but the reactivity on Western blots differs between these two proteins. Immunoextraction of the 71- and 63-kDa proteins led to a approximately 60% decrease in L-[3H]glutamate-binding activity associated with synaptic membrane proteins. Of the brain subcellular fractions examined, the isolated synaptic plasma membranes had the strongest reaction in enzyme-linked immunosorbent assays toward the antiglutamate-binding protein antisera. Electron microscopy combined with gold particle immunohistochemistry revealed the sites labeled by the antibodies as entities present either on the surface or within the postsynaptic membranes and the associated densities of brain nerve ending particles (synaptosomes). Immunohistochemical procedures of gold labeling with silver enhancement of labeled sites revealed selective neuronal labeling in brain regions enriched in glutamate neurotransmitter pathways such as the hippocampus. Labeling was along dendrites and around cell bodies of pyramidal neurons. Based on the pattern of histochemical labeling, the distribution of immune reactivity in synaptic membranes, and the extractions of a major component of membrane glutamate-recognizing proteins by the antibodies, the glutamate-binding proteins must play a role in glutamate neurotransmission.

Animals↗