A comparison of some metabolic effects of N-methylaspartate stereoisomers, glutamate and depolarization: a multinuclear MRS study.

Exposure of guinea pig brain slices to low concentrations (10 microM) of NMDA caused decreases in PCr and ATP within 30 min, with a slower decrease in NAA and increase in lactate, both detectable after 1 h. Exposure to NMDA for over 1 h or at higher concentrations caused further increases in lactate and decreases in NAA, with no further change in PCr or ATP. The L-isomer, NMLA, and the racemic mixture, NMDLA, caused similar changes in lactate and NAA, but both produced greater decreases in the energy state than NMDA, similar to those caused by prolonged exposure to glutamate. MK-801 prevented the changes in the energy state caused by NMDA, but not those caused by NMLA or by glutamate. The results are compared to previous studies on depolarization and discussed in terms of the role of the NMDA sub-type of glutamate receptor in the excitotoxic hypothesis of neuronal degeneration.

Magnetic resonance spectroscopy studies on changes in cerebral calcium and zinc and the energy state caused by excitotoxic amino acids.

Under control conditions, superfused hippocampal slices exhibited a significantly higher phosphocreatine (PCr)/ATP ratio than cortical slices; the evidence suggests that this is due to lower concentrations of ATP, rather than higher concentrations of PCr. Glutamate caused relatively rapid decreases in PCr and ATP levels to approximately 45%, accompanied or immediately followed by an increased free intracellular calcium concentration ([Ca2+]i) and the release of Zn2+ in the cortex. In the hippocampus PCr and ATP decreased further to approximately 20% of control values, but the changes in [Ca2+]i and Zn2+ content were slower. This is in contrast to the effects of depolarisation, which produced the same rapid changes in the energy state and [Ca2+]i, with no detectable Zn2+, in both tissues. NMDA causes effects similar to those of glutamate in the cortex (decreases in the energy state, increased [Ca2+]i, and release of Zn2+). Pretreatment of the cortex for 1 h with the NMDA blocker MK-801 prevented all of the observed effects of NMDA. In contrast, pretreatment with MK-801 had no detectable effect on the increase in [Ca2+]i or the decreases in PCr and ATP caused by glutamate, although it prevented the release of zinc. The results are discussed in relation to the function of the NMDA subtype of glutamate receptor in excitotoxicity.

Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of uncaged dihydrosphingosine evoked responses which were attenuated by photolysis of the caged Ca2+ chelator Diazo-2. Experiments also suggested that extracellular application of dihydrosphingosine can activate membrane conductances. We conclude that dihydrosphingosine directly or indirectly mobilises Ca2+ from ryanodine-sensitive intracellular stores in cultured sensory neurones.

Amyotrophic lateral sclerosis associated with genetic abnormalities in the gene encoding Cu/Zn superoxide dismutase: molecular pathology of five new cases, and comparison with previous reports and 73 sporadic cases of ALS.

Molecular pathology has identified 2 distinct forms of neuronal inclusion body in Amyotrophic Lateral Sclerosis (ALS). ALS-type inclusions are skeins or small dense filamentous aggregates which can only be demonstrated by ubiquitin immunocytochemistry (ICC). In contrast hyaline conglomerates (HC) are large multifocal accumulations of neurofilaments. Previous reports have failed to clarify the distinction and relationship between these inclusions. Correlation of molecular pathology with sporadic and familial cases of ALS will detect specific associations between molecular lesions and defined genetic abnormalities; and determine the relevance of molecular events in familial cases to the pathogenesis of sporadic disease. We describe the molecular pathology of 5 ALS cases linked to abnormalities of the SOD1 gene, in comparison with a series of 73 sporadic cases in which SOD1-gene abnormalities were excluded. Hyaline conglomerate inclusions were detected only in the 2 cases with the SOD1 I113T mutation and showed a widespread multisystem distribution. In contrast ALS-type inclusions characterized sporadic cases (70/73) and were restricted to lower motor neurons. Hyaline conglomerates were not seen in sproadic cases. Confocal microscopic analysis and ICC shows that HC contain equally abundant phosphorylated and nonphosphorylated neurofilament epitopes, indicating that phosphorylation is not essential for their formation. In contrast neurofilament immunoreactivity is virtually absent from typical ALS-type inclusions. The SOD1-related cases all had marked corticospinal tract and dorsal column myelin loss. In 4 cases the motor cortex was normal or only minimally affected. This further illustrates the extent to which upper motor neuron damage in ALS is usually a distal axonopathy. Previously reported pathological accounts of SOD1-related familial ALS (FALS) are reviewed. Hyaline conglomerates are so far described in cases with mutations A4V, I113T and H48Q. In only 1 of 12 cases (H48Q) reported were both HC and ALS-type inclusions present in the same case. These findings suggest the possibility that the molecular pathology of neuronal inclusions in ALS indicates 2 distinct pathogenetic cascades.

Extracellular or intracellular application of argiotoxin-636 has inhibitory actions on membrane excitability and voltage-activated currents in cultured rat sensory neurones.

The whole cell variant of the patch clamp technique was used to investigate the actions of the polyamine amide spider toxin, argiotoxin-636, on the excitability of cultured dorsal root ganglion neurones. Synthesized argiotoxin-636 (0.1-100 microM) reduced neuronal excitability when applied to the extracellular environment by low pressure ejection or to the intracellular environment via the patch pipette solution. The toxin prolonged the duration of evoked action potentials and reduced the peak amplitude of action potentials. Intracellular and extracellular application of argiotoxin-636 also decreased the number of action potentials evoked in response to 800-ms depolarizing current commands. This action of the toxin was mimicked by 100 microM tetraethylammonium. Extracellular application of argiotoxin-636 inhibited voltage-activated K currents in a dose-dependent manner over the complete voltage range. This inhibition occurred without any significant changes in the voltage dependence of activation or inactivation. Intracellular application of argiotoxin-636, during 5-10 min of whole cell recording, also inhibited voltage-activated K+ currents without changing the voltage dependence of activation or steady-state inactivation. Extracellular or intracellular spermidine (250 microM) reversibly attenuated the inhibitory actions of extracellular argiotoxin-636. Argiotoxin-636 also inhibited voltage-activated Na + currents; this effect was dependent on repeated activation of the currents and the period during which the neurones were in culture. We conclude that application of argiotoxin-636 to either the extracellular or intracellular environment reduced excitability of cultured sensory neurones from neonatal rats and that this involved inhibition of both voltage-activated K+ and Na+ currents. The data suggest that the toxin was more effective at attenuating action potentials when neurones were repeatedly excited, and that access to inhibitory sites of action on the voltage-activated ion channels can be achieved from the inside of the neurone.

Selective loss of neurofilament proteins after exposure of differentiated human IMR-32 neuroblastoma cells to oxidative stress.

Millimolar concentrations of ascorbate in the presence of iron can cause neuronal cell death. This study shows that the human neuronal cell line IMR-32 is sensitive to ascorbate and that cytotoxicity can be blocked by the antioxidant enzymes Cu/Zn-superoxide dismutase and catalase. There was a selective loss of neurofilament proteins after exposure to 5 or 10 mM ascorbate, as assessed by immunostaining and by Western blotting. Loss of actin or tubulin was not seen, suggesting that loss of neurofilaments is a sensitive and selective marker for free radical damage in these cells.

Neither moderate hypoxia nor mild hypoglycaemia alone causes any significant increase in cerebral [Ca2+]i: only a combination of the two insults has this effect. A 31P and 19F NMR study.

(1) The energy state and free intracellular calcium concentration ([Ca2+]i) of superfused cortical slices were measured in moderate hypoxia (approximately 65 microM O2), in mild hypoglycaemia (0.5 mM glucose), and in combinations of the two insults using 19F and 31P NMR spectroscopy. (2) Neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca2+]i. Hypoxia caused a 40% fall in phosphocreatine (PCr) content but not in ATP level, and hypoglycaemia produced a slight fall in both (as expected from previous studies). These changes in the energy state recovered on return to control conditions. (3) A combined sequential insult (hypoxia, followed by hypoxia plus hypoglycaemia) produced a 100% increase in [Ca2+]i and a decrease in PCr level to approximately 25% of control. The reverse combined sequential insult (hypoglycaemia, followed by hypoglycaemia plus hypoxia) had the same effect. On return to control conditions there was some decrease in [Ca2+]i and a small increase in PCr content, but neither recovered to control levels. (4) Exposure of the tissue to the combined simultaneous insult (hypoxia plus hypoglycaemia) immediately after the control spectra had been recorded resulted in a fivefold increase in [Ca2+]i and a similar decrease in PCr level to 20-25% of control. There was little if any change of [Ca2+]i or PCr level on return to control conditions. (5) These results are discussed in terms of metabolic adaptation of some but not all of the cortical cells to the single type of insult, which renders the tissues less vulnerable to the combined insult.