A monoclonal antibody, F4, has been produced which reacts with an epitope possessing an unusual subcellular distribution. It binds to the external surface of the neuronal plasma membrane only in the region of the synapse. This is evidenced by binding of F4 to presynaptic terminals in unfixed cultures of rat cerebellum and to preparations of unfixed synaptosomes. In addition, much larger amounts of the epitope are present intracellularly. In fixed nervous tissue, the epitope is found in many neurons, and is associated mainly with presynaptic plasma membranes, synaptic vesicles, postsynaptic densities (cerebral cortex and hippocampus, but not cerebellum), rough endoplasmic reticulum, and the Golgi apparatus. The epitope is especially abundant in large neurons (e.g. pyramidal cells). Similar amounts of epitope are present in the chromaffin cells of the adrenal medulla. It is also expressed in ependymal cells in the brain, and in epithelial cells present in ducts of the medulla, but not cortex, of the kidney. However, the epitope is not found in glial cells in the brain, or in either liver, spleen, skeletal muscle, or testes. F4 is not species specific, as it binds to postmortem adult human cerebral cortex and neonatal cerebellum in a manner as described for the rat. It also binds to homogenates of brains of fish, chicken and mouse. The appearance of the epitope during development of the cerebellum in vivo and in vitro occurs in parallel with the differentiation of neurons and formation of synapses, though small amounts are also present in neuronal precursor cells. The F4 antibody can detect nanogram amounts of pp60v-src on immunodots. The strength of this reaction is high enough that F4 can be used to demonstrate pp60v-src-like immunoreactivity in Rous Sarcoma virus-transformed chick embryo fibroblasts. However, present evidence suggests that it may be premature to assign the immunocytochemical reactivity of F4 in the brain exclusively to pp60c-src. This conclusion is based on the fact that F4 reacts with several polypeptides from synaptic plasma membranes on Western blots of renaturing, two-dimensional gels that are dissimilar in size to pp60c-src, and from the fact that it can cross-react, albeit weakly, with several other serine protein kinases in an immunodot assay. Appreciation of this cross-reactivity, and of the evolutionary conservation of the epitope, as well as its sensitivity to denaturation, has led to our working hypothesis that F4 binds to a conformational epitope present on several polypeptides that may be most perfectly represented by some aspect of the catalytic domain of tyrosine protein kinases.

Several cations were examined for their ability to specifically affect one of the 3 L-glutamate (L-Glu) binding sites in rat forebrain synaptic plasma membranes (i.e. Na+-dependent, Cl--dependent and Cl--independent). Na+-dependent binding was potently inhibited by K+ and NH4+ ions. Other monovalent cations tested (Cs+, Li+, triethylammonium) had no effect on this binding site. Polyvalent cations (Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Cr3+) also had little effect on the Na+-dependent L-Glu binding site. Cl--dependent L-Glu binding was potently inhibited by Na+ ions but was not affected by other monovalent ions. All of the divalent cations were potent inhibitors of both Cl--dependent and -independent binding. The results show that these binding sites of L-Glu can be distinguished by their response to cations and suggest possible novel modes of regulation in vivo.

Both K+ and NH4+ are potent inhibitors of Na+-dependent l-glutamate binding to synaptic plasma membranes. Thus, the effects of these ions on Na+-dependent l-glutamate uptake were examined in rat forebrain synaptosome preparations. KCl (2 to 4 mM) stimulated l-glutamate uptake 34% over that in K+-free Krebs bicarbonate buffer; NH4+ was without effect. However, in the presence of 4 mM K+, NH4+ blocked the K+-stimulated component of Na+-dependent l-glutamate uptake. These effects were unrelated to ionic strength of Cl− as added Na+ or tris chloride had no effect on l-glutamate uptake. The results suggest that NH4+ could exert some of its toxic effects by blocking a specific l-glutamate uptake site, thereby elevating l-glutamate in the central nervous system.

Recent studies have shown that Cl− and Ca2+ ions increase [3H]glutamate binding to rat forebrain synaptic plasma membranes by expressing a new class of glutamate receptors. We examined the regional distribution of these two classes of glutamate binding sites and further characterized their ionic requirements. Significant differences in both Cl−/Ca2+-independent (basal) and Cl−/Ca2+-activated receptors, as well as the ratios of these two receptor classes were observed among different areas of the CNS. Cl− and Ca2+ appeared to act synergistically, with Cl− ion an absolute requirement for Ca2+ stimulation, in expressing these additional binding sites. Ca2+ alone did not affect glutamate binding.

Cl−/Ca2+-dependent and Cl−/Ca2+-independent L-[3H] glutamate binding sites in rat brain synaptic membranes showed marked differences in their pharmacological properties. One site resembled L-2-amino-4-phosphonobutyrate (L-APB)-sensitive receptors and the other N-methyl-D-aspartate (NMDA) receptors. Inhibition studied demonstrated that L-aspartate was more potent at Cl−/Ca2+-independent than at Cl−/Ca2+-dependent sites although L-glutamate was of similar potency at both sites; the D-isomers of aspartate, glutamate and α-aminoadipate exhibited the opposite trend. Quisqualate and ibotenate showed high and low affinity inhibition components in the presence of Cl− and Ca2+, and only low affinity inhibition at Cl−/Ca2+-independent sites. For a series of α-amino-ω-phosphono carboxylic acids (propionate-heptanoate), peaks of inhibitory activity in the presence of Cl− and Ca2+ were shifted to 1-carbon shorter homologues than in the absence of these ions. These data indicate that the ionic environment is of critical importance for the activity of different physiological receptor populations in vitro.

The binding of l-[3H]glutamate (l-Glu) to freeze-thawed synaptic membranes (SPMs) exhibited saturation kinetics, with Kd 507 nM and Bmax 6.99 pmol/mg protein. The effects of ions, the susceptibility to Triton X-100 and the pharmacological properties of the binding indicated that those sites detected in freeze-thawed SPMs were only of the C1−/Ca2+-independent type. The C1−/Ca2+-dependent (2-amino-4-phosphonobutyrate-sensitive) l-Glu binding sites which are additionally present in fresh SPMs are abolished by freezing.

The concanavalin A--binding glycoproteins of the rat synaptic junction were isolated by affinity chromatography. These glycoproteins had molecular weights of 160,000, 123,000, 110,000, and 95,000. The tryptic peptide maps of these glycoproteins showed that the three largest glycoproteins contained a high percentage of identical peptides. This indicates that the amino acid sequences of these glycoproteins have a high degree of homology. The 95,000-dalton glycoprotein was unrelated to the other three. These findings suggest that homologous glycoproteins may participate in synapse formation or maintenance, or both.

Chloride ions increased L-glutamate (L-Glu) binding to synaptic membranes. The binding was saturable and resulted in a 2.5-fold enhancement at concentrations of 20--40 mM chloride. Sodium and potassium ions inhibited only chloride stimulated L-Glu binding. Calcium ions also increased L-Glu binding but this was observed only in the presence of chloride. The anion selectivity of the enhancement of L-Glu binding was similar to that reported for the membrane chloride channel, suggesting that some L-Glu binding sites may be associated with this channel.

Removal of the entorhinal cortical projection to the hippocampus in adult rats decreased the density of muscarinic cholinergic receptors in the denervated dentate gyrus outer molecular layer at two days postlesion. Thirty days following the lesion (in adults and neonates) there is a small receptor density increase in the outer molecular layer (may be due to tissue shrinkage), and a larger increase in the lacunosum-moleculare. The receptor density decrease seen two days postlesion suggests the presence of presynaptic muscarinic receptors on the lost entorhinal cortical fibers. The distribution and extent of the receptor changes seen at 30 days postlesion are inconsistent with the cholinergic fiber reorganization which follows an entorhinal cortical lesion, but are consistent with a proposed model of non-cholinergic afferent mediated control of muscarinic receptor density in the rat hippocampus.

Myelin was found to contain five major glycoproteins with molecular weights of 120000, 95000, 88000, 43000 and 38000. Light myelin contained only 5-7% of the amount of these glycoproteins in whole myelin, whereas heavy myelin and the membrane fraction contained amounts nearly identical with whole myelin. Since all the major and minor glycoproteins, with the exception of 120000-mol-wt. glycoprotein, were detected only after treating the myelin membrane with neuraminidase, N-acetylneuraminic acid is a terminal sugar residue in these glycoproteins.

The pattern of binding of horseradish peroxidase conjugated lectins (Concanavalin A, fucose binding protein, Ricinus communis agglutinin) was examined in the rat hippocampal formation both prior to and following a lesion of the entorhinal cortex. In normal animals, Concanavalin A binding sites were concentrated around the granular and pyramidal cell bodies. Receptors were less concentrated in the stratum radiatum, stratum oriens and the dentate molecular layer. Receptors of fucose binding protein were concentrated in the granular and pyramidal cells, the boundary between the first and second quarters of the molecular layer and at the hippocampal fissure. Ricinus communis agglutinin binding sites were highest in the first 1/4 of the molecular layer and lowest in the stratum lacunosum-moleculare. Three days after an entorhinal lesion, the binding of Concanavalin A and fucose binding protein in the molecular layer was increased and while Ricinus communis agglutinin binding was unchanged. At thirty days post-lesion there was an increase in the binding of Concanavalin A and fucose binding protein in the molecular layer and stratum lacunosum-moleculare, whereas Ricinus communis agglutinin binding sites increased only in the molecular layer.

The specific binding of L-glutamate (L-Glu) and L-aspartate (L-Asp) was measured in rat brain synaptic plasma membranes (SPMs). A distinction between the binding sites for these amino acids was made on the basis of the kinetics, ion effects, pharmacology and chemical susceptibility of the binding. The existence of distinct binding sites for L-Glu and L-Asp is consistent with electrophysiological data that mammalian neurons possess separate receptors for these amino acids.

Synaptic junctions (SJ) were prepared from synaptic plasma membranes (SPM) by extraction with Triton X-100 and density gradient centrifugation. These SJs were enriched in certain Concanavalin A (Con A) binding glycoproteins, the 52,000 Mr postsynaptic density (PSD) protein, and receptor sites for L-glutamate, L-aspartate, kainic acid (KA) but not quinuclidinyl benzilate (QNB). Various other membrane fractions were extracted by means of the same procedure. Those fractions prepared from light SPMs and crude myelin contained identifiable synaptic junctions and were also highly enriched in the synaptic components. The SJ-like fraction from mitochondria did not contain any of the characteristic synaptic macromolecules. However, this fraction from microsomes contained levels of the 52,000 Mr PSD protein and binding sites for L-glutamate (L-Glu) and L-aspartate (L-Asp) similar to true synaptic junctions, although the Con A binding glycoproteins and KA binding sites were nearly absent. On the basis of electron microscopy, the SJ-like fraction from microsomes did not contain structures recognizable as SJs. Thus, the Con A binding glycoproteins and KA binding sites appear to be excellent markers for the SJ.

The heterocyclic compound kainic acid (KA) is a potent excitant when applied to mammalian neurones. Lesions caused by injections of KA into the rat striatum and hippocampus cause similar patterns of damage to those seen in Huntington's chorea and status epilepticus, respectively. Although it was originally thought to be a glutamate agonist, it is now clear that KA does not act on the majority of the receptors for glutamate, and in fact seems to act on a class of receptors which are distinct from those which mediate responses to other excitatory amino acids. The potent and selective neurotoxic effects of this compound may be mediated by these same receptors. At present, the relative distribution of junctional and extrajunctional (non-synaptic) receptors is unknown and resolution of this issue would provide important insights into the action of KA on the central nervous system (CNS). We show here that KA binding sites are greatly enriched in isolated synaptic junctions from rat brain and, using an in vitro autoradiographic technique, we have found that these binding sites are concentrated specifically in terminal fields where KA acts as a potent neurotoxin.