Studies of NMDA- and non-NMDA-mediated neurotoxicity in cultured neurons.

The neurotoxic effects of various glutamate agonists were studied using whole fetal rat brain cultures. The results showed that L-glutamate (L-glu) and N-methyl-D-aspartate (NMDA) were the most potent agonists for inducing neurotoxicity, producing significant toxicity at 0.10 and 0.01 mM concentrations, respectively. Kainic acid (KA) and quisqualic acid (QA) also produced neurotoxicity, but only at a relatively high concentration (1.0 mM). No other glutamate agonist tested produced neurotoxicity in the cultures following brief incubations. The effects of each agonist were found to be Ca2+ dependent, and the selective NMDA Ca2+ channel agonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine hydrogen maleate (MK-801), blocked the toxicity produced by all the glutamate agonists. Thus, the results of this study found little or no evidence for a direct non-NMDA receptor mediated neurotoxicity. These results suggest that the neurotoxicity produced by the non-NMDA agonists may be due to one of the following mechanisms: (i) non-specific binding of non-NMDA agonists to NMDA receptor; (ii) release of L-glu via non-NMDA agonists induced depolarization of cell membrane and subsequent activation of NMDA receptor by released L-glu; (iii) inhibition of L-glu uptake by non-NMDA agonists resulting in activation of L-glu receptors including NMDA receptors.

Purification and characterization of a low molecular weight endogenous glutamate binding inhibitor (LGBI) in porcine brain.

One of the endogenous substances which modulate glutamate receptor binding was isolated and highly purified from porcine brain. The purification involved extraction of brain tissue with doubled distilled water, followed by gel filtration, anion exchange, cation exchange, and several steps of C18 reverse-phase high performance liquid chromatography (HPLC). A low molecular weight glutamate binding inhibitor (LGBI) was purified to apparent homogeneity as judged from the elution profile of an HPLC column, in which a symmetrical peak was obtained when the eluate was monitored at 220 nm. The LGBI appears to be a small molecule (< 2 kD) that is heat- and acid/base-stable. The highly purified LGBI has no effect on GABAA and benzodiazepine receptor binding. The LGBI is not L-glutamate, L-aspartate or other negatively charged endogenous substances, since they are clearly separated from the LGBI in anion exchange chromatography. The inhibitory effect of the LGBI on [3H]L-glutamate binding is reversible, and it only changes the Bmax while the Kd remains the same. Since the membrane preparations used for [3H]L-glutamate binding assays for the detection of LGBI activity were enriched with quisqualate (QA)-sensitive subtypes, it was suggested that the LGBI could be a modulator of the QA receptor. Some amino acids which produce significant inhibition of glutamate binding activity were also compared with the LGBI, and they all showed no resemblance to the LGBI. The chemical structure of the LGBI remains to be determined.

Purification and Characterization of a High-Molecular-Weight Endogenous Glutamate-Binding Inhibitor in Porcine Brain.

A high-molecular-weight glutamate-binding inhibitor (HGBI) from porcine brain extract was purified to homogeneity. The results of this purification process show that glutamate receptor activity can be regulated by a high-molecular-weight protein, which inhibits [(3)H]L-glutamate binding to excitatory amino acid (EAA) receptors. The purified HGBI appears to be a protein with a molecular weight of approximately 70 kD. The purified HGBI is negatively charged, suggesting that it may contain acidic amino acids, and most likely, L-glutamate- and L-aspartate-enriched regions, responsible for its surface charge as well as for its binding to glutamate receptors. Inhibition of [(3)H]L-glutamate binding by the purified HGBI is reversible, and appears to change the binding kinetics. This endogenous ligand for glutamate receptors has unique characteristics separating it from all the other ligands found so far in the EAA receptor system. This HGBI represents a new class of modulator for the EAA receptor, thus further investigation of the function and structure of the HGBI should provide new understanding of the mechanisms of EAA-mediated neutrotransmission. Copyright 1994 S. Karger AG, Basel

Role of Ca2+ in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-mediated polyphosphoinositide turnover in primary neuronal cultures.

Excitatory amino acid (EAA) receptors and EAA-mediated stimulation of polyphosphoinositide (poly-PI) turnover were studied in cultured neurons at different days in vitro (DIV). Six main observations have emerged from these studies: (a) Neurons increased their sensitivity to EAAs as a function of time in culture, indicated by increasing EAA-mediated poly-PI turnover. (b) Extracellular Ca2+ concentration played an important role in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-stimulated poly-PI turnover in cells at 4 DIV, whereas poly-PI turnover mediated by L-glutamate and trans-1-amino-cyclopentane-1,3-dicarboxylic acid was not Ca(2+)-dependent. (c) A marked stimulation of poly-PI turnover by AMPA was seen in the cultured neurons at 4 DIV, but not at 17 DIV, suggesting that a distinct EAA receptor sensitive to AMPA is transiently expressed. (d) The Ca2+ ionophore A23187 increased poly-PI turnover in cultured neurons, suggesting that Ca2+ entry is involved in stimulating poly-PI turnover. (e) Stimulation of poly-PI turnover by carbachol was greater in neurons at 17 DIV as compared with 4 DIV, and appeared to be Ca(2+)-dependent across DIV. (f) 6-Cyano-7-nitroquinoxaline-2,3-dione, an antagonist for non-N-methyl-D-aspartate ionotropic EAA receptors, inhibited 100% and 35% of AMPA- and quisqualate-induced poly-PI turnover, respectively, suggesting an involvement of ionotropic AMPA/quisqualate receptors in stimulating poly-PI turnover.

An integral membrane protein form of brain L-glutamate decarboxylase: purification, characterization and its relationship to insulin-dependent diabetes mellitus.

A new and novel form of L-glutamate decarboxylase (GAD; EC 4.1.1.15) was purified from whole porcine brain to apparent homogeneity by a combination of column chromatographies on DE-52, ultragel AcA 34, hydroxylapatite and Sephadex G-200, and native gel electrophoresis. The purified GAD was established as an integral membrane protein based on hydrophobic interaction chromatography and membrane extraction studies. This membrane GAD (MGAD) has a native molecular weight of 120 +/- 5 kDa and is a homodimer of 60 +/- 2 kDa. Immunoprecipitation and immunoblotting tests using the sera from insulin-dependent diabetes mellitus (IDDM) patients revealed the presence of antibodies against this newly identified MGAD in IDDM. The role of MGAD in the pathogenesis of IDDM and related autoimmune disorders is also discussed.

Influence of fetal hippocampal grafts on lesions induced by intraventricular infusion of N-methyl-D-aspartate (NMDA) into rats.

Effects of fetal hippocampal transplants were evaluated following a prolonged intraventricular excitotoxic lesion (1.0 mg of N-methyl-D-aspartate over two weeks infusion) in F344 rats. The septum and ipsilateral hippocampus (CA1 and dentate regions) showed extensive cell loss, decreased acquisition of spatial memory was observed and a decrease in AChE positive fiber innervation to the hippocampus was noted following the lesion. Fetal hippocampal transplants into the posterior lateral ventricle resulted in moderate graft survival and physiological analysis of graft-host interconnection in vitro demonstrated evoked field potentials. However, the transplants did not lead to significant improvement in behavior, possibly due to poor synaptic integration of the intraventricular transplants into the host hippocampus. The prolonged intraventricular NMDA lesion may be helpful to understand a mixed lesion model of both septal areas and hippocampus and also as a background lesion in which to assess the connectivity and development of various types of neural grafts.

Age-related alterations in potentiation in the CA1 region in F344 rats.

F344 rats of various ages (2-3 months, 15-16 months, and 24-25 months) were tested on a spatial memory task. The 15- and 24-month-old rat groups showed impaired acquisition and retention of the memory task, compared to the young animals. Extracellular field potential recordings in the CA1 region were subsequently performed in vitro, using hippocampal slices from both these tested rats and similar but untested F344 young and aged rats. Findings included: a) a positive correlation between baseline dendritic EPSP slope values and retention scores across age groups; b) a more rapid decay of both somatic and dendritic short-term potentiation in aged slices; c) decreased somatic but not dendritic long-term potentiation overall in aged slices, regardless of bath Mg2+ level; and d) decreased paired-pulse facilitation in slices from aged rats bathed in 4.0 mM Mg2+ media compared to young controls. These findings suggest an age-related alteration in both presynaptic and postsynaptic potentiation mechanisms, which may relate to the poor spatial memory acquisition and retention in the aged rats. These age-related differences point to substantial changes in neuronal signal processing capabilities and local circuit function in the hippocampus as a function of aging.

Spatial performance correlates with in vitro potentiation in young and aged Fischer 344 rats.

Young adult (2-4 months old) and aged (24-26 months old) Fischer 344 (F344) rats were trained for spatial behavior (locating a hidden escape platform) in a circular water maze. The aged rats showed deficits in both the acquisition and retention of the learned response. Following the behavioral training, hippocampal slices from the rats were prepared. Potentiation of CA1 extracellular, somatic field potentials was studied in vitro following either a short stimulus train (4 pulses) or a longer train (50 pulses). Slices from the aged rats showed less short-term potentiation (124.8 +/- 4.9% baseline, mean +/- S.E.M.) at 1 min following the short train in comparison to slices from the young rats (151.8 +/- 7.5%, P less than 0.05). However, following the longer train, no differences were found between the groups in the degree of either short-term (measured at 1 min after stimulation) or long-term potentiation (measured at 60 min). The amount of potentiation seen at various time points after either train correlated with the behavioral measure of retention. These results indicate that F344 rats exhibit age-related behavioral deficits, and age-related synaptic potentiation deficits in response to short stimulation trains. The correlation between the degree of potentiation (both short-term and long-term) and retention of a behavioral task adds strength to the hypothesis that potentiation mechanisms may underlie memory processes.

Functional elongation of CA1 hippocampal neurons with aging in Fischer 344 rats.

Dendritic function of CA1 pyramidal cells was measured during intracellular recording in vitro and correlated with in vivo behavior in Fischer 344 rats. The aged rats (greater than 26 months) were significantly impaired on a water maze test of hippocampal behavioral function. CA1 neurons from these aged rats demonstrated an elevated action potential threshold compared to the young rats. Electrotonic length (L, in lambda), calculated independently from physiological transients and electrotonic cell reconstructions, was significantly longer in neurons from aged rats (L = 0.73 +/- 0.02 lambda; mean +/- SEM) than in neurons from young rats (L = 0.66 +/- 0.02 lambda). Analysis of proximal and distal synaptic potentials pointed to a more distal electrotonic siting of all dendritic synapses in the aged neurons. Thus, electrical lengthening of dendrites, alterations in synaptic location and decreased excitability in neurons from aged rats with behavioral impairment may represent an endpoint of neuronal reactive mechanisms in response to the aging process.

Far-field potentials generated by action potentials of isolated frog sciatic nerves in a spherical volume.

Previous results in cylindrical volumes have shown that action potentials generate far-field potentials when experimental conditions are such that quadrupolar components of the action potential are reduced to an equivalent dipole. We now show that the same conclusions are also reached within a spherical volume, again recording far-field potentials from isolated bullfrog nerves. A mathematical proof is given that shows that in a sphere, antipodal electrodes primarily detect far-field potentials from dipole generators and not quadrupole generators. A revised conception of the 'far-field' in evoked responses is discussed which equates far-field recordings with dipole detection.

Far-field potentials recorded from action potentials and from a tripole in a hemicylindrical volume.

There is growing evidence in support of the hypothesis that far-field potentials are recorded when action potentials encounter discontinuities in the surrounding volume. The present study found further support for this hypothesis using two methods of experimentation. The first method recorded potentials when the action potential from an isolated bullfrog sciatic nerve in a hemicylindrical volume (i) encountered a change in the shape of the surrounding volume, (ii) crossed a boundary between 2 volumes of differing resistivities, (iii) reached a bend in the nerve, or (iv) reached the functional end of the nerve. In the second method, potentials were recorded when an electrical tripole, constructed in a way to produce the electrical equivalent of an action potential, encountered the same discontinuities as well as when it was configured to simulate a curved nerve. These results are consistent with the hypothesis that dipole components of an action potential predominant in far-field recordings.

Far-field potentials due to action potentials traversing curved nerves, reaching cut nerve ends, and crossing boundaries between cylindrical volumes.

A previously published computer simulation was tested in a biological preparation by recording action potentials from frog sciatic nerves within a volume conductor filled with Ringer's solution. Traveling in a straight line, nerve action potentials traversed a constricted cylinder before crossing into a larger, hemicylindrical volume. Recordings from widely spaced electrodes in the larger volume demonstrated a potential associated with the action potential crossing the boundary between the two volumes. Another potential was associated with the action potential reaching the nerve's cut end. These potentials did not diminish in amplitude with increasing distance from the source. In other recordings, a potential associated with a bend in the nerve was found which was dependent upon the angle of the bend. These results indicate that the simple model of a dipole in a bounded sphere in which potentials decrease as a function of distance from the generator does not explain all potentials that can be observed under conditions that approximate human and animal recordings.