Graham E. Fagg

Chloride ions enhance L-glutamate binding to rat brain synaptic membranes

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.

Chloride and calcium ions separte L-glutamate receptor populations in synaptic membranes

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 studies 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 alpha-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 alpha-amino-omega-phosphono carboxylic acids (propionate-heptanoate), peaks of inhibitory activity in the presence of Cl- and Ca2+ were shifted to l-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.

Axonal sprouting at the neuromuscular junction of adult and aged rats.

Abstract We compared the extent of the repair response (terminal and nodal sprouting) that followed motor nerve damage in young (3 months old) and aged rats (27 months old). Two weeks after unilateral partial denervation of the soleus muscle (transection of the L4 spinal root), muscles were removed and stained using the zinc iodide-osmium tetroxide procedure. Sprouting was assessed by determining (a) the percentage of end-plates with terminal sprouts, (b) the percentage of preterminal axons with nodal sprouts, and the lengths of (c) the end-plate and (d) terminal sprouts. Relative to control (unoperated) animals, young adults showed significant increases in all four parameters after partial denervation. Aged animals exhibited evidence of considerable sprouting prior to any surgery: end-plates were long and complex, greater than 50% of preterminal axons had nodal sprouts, there was a high degree of terminal sprouting, and terminal sprouts were longer than in young animals. Partial denervation in the aged animals significantly increased only end-plate length and the degree of terminal sprouting. In both age groups, there were no differences between soleus muscles contralateral to the transection and those from control animals. The decreased response to partial denervation in aged animals may possibly be attributed to a longer latency or higher threshold for repair than in adults, or to the existence of an upper limit for axonal growth which has already been attained in the older age group.

Cl-/Ca2+-dependent L-glutamate binding sites do not correspond to 2-amino-4-phosphonobutanoate-sensitive excitatory amino acid receptors.

1 A series of phosphono and phosphino analogues of glutamate were used to compare the pharmacological properties of (a) Cl−/Ca2+‐dependent, 2‐amino‐4‐phosphonobutanoate (AP4)‐sensitive L‐[3H]‐glutamate binding sites in rat brain synaptic plasma membranes (SPMs) and (b) AP4‐sensitive excitatory synaptic responses by use of electrophysiological techniques. 2 In the presence of Cl− and Ca2+, L‐[3H]‐glutamate bound to SPMs with Kd804 nM and Bmax 53 pmol mg−1 protein. The AP4‐sensitive (Ki 7.3 μM) population of binding sites represented 61 % of L‐glutamate specifically bound. co‐Substituted analogues of AP4 were potent inhibitors of L‐[3H]‐glutamate binding (Ki values 2.4–38 μM), whereas N‐substituted compounds or propionic acid derivatives were inactive. Experiments with AP4 alone and in combination with other analogues demonstrated that the primary target of all substances was the AP4‐sensitive population of L‐glutamate binding sites. 3 In the hippocampal slice in vitro, AP4 antagonized lateral perforant path‐evoked field potentials with an IC50 of 2.7 μM. In contrast to their actions at AP4‐sensitive L‐glutamate binding sites, all other compounds (except for the ω‐carboxymethylphosphino analogue, IC50 19 μM) were weak or inactive as antagonists of this synaptic response (IC50 values > 100 μM). Inactive compounds which exhibited activity in the binding assay did not reverse the synaptic depressant effects of AP4, indicating that they were neither agonists nor antagonists at AP4‐sensitive synapses. 4 The lack of correspondence between (a) the Cl−/Ca2+‐dependent, AP4‐sensitive population of L‐[3H]‐glutamate binding sites and (b) AP4‐sensitive synaptic responses indicates that these binding sites are not the receptors through which AP4 exerts its neuropharmacological effects. The possibility that Cl−/Ca2+‐dependent ‘binding sites’ represent transport into resealed SPM vesicles is discussed. 5 Electrophysiological data demonstrate that AP4‐sensitive synaptic receptors display a high degree of ligand selectivity. High antagonist potency is shown only by glutamate analogues with unmodified α‐amino and α‐carboxyl groups, and with a bifunctional (dianionic) ω‐terminal.

Freezing eliminates a specific population of l-glutamate receptors in synaptic membranes☆

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 Cl-/Ca2+-independent type. The Cl-/Ca2+-dependent (2-amino-4-phosphonobutyrate-sensitive) L-Glu binding sites which are additionally present in fresh SPMs are abolished by freezing.

Regeneration of rat hippocampal fimbria fibers after fimbria transection and peripheral nerve or fetal hippocampal implantation

After a unilateral hippocampal fornix-fimbria transection in adult rats, either autologous peripheral nerve or fetal hippocampus was implanted into the transection site. After 2 to 4 weeks, 2 to 3 months, and 6 to 8 months fimbria fiber regeneration was analyzed by acetylcholinesterase (AChE) histochemistry and retrograde transport of horseradish peroxidase after injection into the denervated host hippocampus. Prominent innervation of both types of implant by central AChE-staining axons occurred by 2 to 3 weeks postimplantation and was sustained to at least 8 months. Reinnervation of the adjacent host hippocampal terminal zone was also apparent, but was sparse compared with innervation of implants.

Regulation of glutamate receptors: possible role of phosphatidylserine

The polar head group of the phospholipid phosphatidylserine is similar in structure to the glutamate analogue 2-amino-4-phosphonobutyric acid (APB), an antagonist of excitatory transmission in the brain. When tested in ligand binding assays phosphatidylserine and its polar head group components O-phosphoserine and L-alpha-glycerophosphoserine were good displacers of APB-sensitive L-glutamate binding. The polar head group components were also antagonists of synaptic field potentials in the rat dentate gyrus evoked by stimulation of a presumed glutamate-using pathway. These results suggest that phosphatidylserine may regulate the activity of synaptic L-glutamate receptors.

Identification of Synapse Specific Components: Synaptic Glycoproteins, Proteins, and Transmitter Binding Sites

Abstract: 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.

L-glutamate and l-aspartate bind to separate sites in rat brain synaptic membranes

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.

Structure-activity relationships of l-glutamate receptor ligands: Role of the ω-acidic terminal☆

Acidic amino acid analogues varying in their ω-terminal were evaluated: (1) as neuronal excitants or antagonists of excitatory synaptic transmission, and (2) as inhibitors of l-[3H]glutamate binding to synaptic membranes. ω-Phosphonates were antagonists and inhibited l-glutamate binding to the 2-amino-4-phosphonobutyrate (APB)-sensitive population of binding sites; ω-sulfonates and ω-carboxylates were excitants and inhibited l-glutamate binding to APB-sensitive and -insensitive sites. The data indicate that properties of the ω-acidic group are important for establishing the relative potencies of antagonist substances and the overall excitatory/antagonist activity of these analogues.