David G. Nicholls

The release and uptake of excitatory amino acids

In this article, David Nicholls and David Attwell describe recent advances in our understanding of the mechanisms by which excitatory amino acids are released from cells, and of the way in which a low extracellular glutamate concentration is maintained. Glutamate can be released from cells by two mechanism: either by Ca2(+)-dependent vesicular release or, in pathological conditions, by reversal of the plasma membrane uptake carrier. The contrasting pharmacology and ionic dependence of the glutamate uptake carriers in the vesicle membrane and in the plasma membrane explain how glutamate (but probably not aspartate) can function as a neurotransmitter, and why the extracellular glutamate concentration rises to neurotoxic levels in brain anoxia.

Assessing mitochondrial dysfunction in cells

Assessing mitochondrial dysfunction requires definition of the dysfunction to be investigated. Usually, it is the ability of the mitochondria to make ATP appropriately in response to energy demands. Where other functions are of interest, tailored solutions are required. Dysfunction can be assessed in isolated mitochondria, in cells or in vivo, with different balances between precise experimental control and physiological relevance. There are many methods to measure mitochondrial function and dysfunction in these systems. Generally, measurements of fluxes give more information about the ability to make ATP than do measurements of intermediates and potentials. For isolated mitochondria, the best assay is mitochondrial respiratory control: the increase in respiration rate in response to ADP. For intact cells, the best assay is the equivalent measurement of cell respiratory control, which reports the rate of ATP production, the proton leak rate, the coupling efficiency, the maximum respiratory rate, the respiratory control ratio and the spare respiratory capacity. Measurements of membrane potential provide useful additional information. Measurement of both respiration and potential during appropriate titrations enables the identification of the primary sites of effectors and the distribution of control, allowing deeper quantitative analyses. Many other measurements in current use can be more problematic, as discussed in the present review.

Mitochondrial membrane potential and neuronal glutamate excitotoxicity: mortality and millivolts

In the past few years it has become apparent that mitochondria have an essential role in the life and death of neuronal and non-neuronal cells. The central mitochondrial bioenergetic parameter is the protonmotive force, Deltap. Much research has focused on the monitoring of the major component of Deltap, the mitochondrial membrane potential Deltapsim, in intact neurones exposed to excitotoxic stimuli, in the hope of establishing the causal relationships between cell death and mitochondrial dysfunction. Several fluorescent techniques have been used, and this article discusses their merits and pitfalls.

Mitochondria, calcium regulation, and acute glutamate excitotoxicity in cultured cerebellar granule cells

Abstract: Exposure of cultured cerebellar granule cells to 100 µM glutamate plus glycine in the absence of Mg2+ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca2+ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca2+ concentration), and extensive cell death. Glutamate‐evoked Ca2+ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca2+ transport, allows glutamate‐exposed cells to maintain a high ATP/ADP ratio while accumulating little 45Ca2+ and maintaining a low bulk cytoplasmic free Ca2+ concentration determined by fura‐2. It is concluded that mitochondrial Ca2+ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca2+ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca2+ accumulation in a microdomain accessible to the mitochondria.

Calcium-Dependent and-Independent Release of Glutamate from Synaptosomes Monitored by Continuous Fluorometry

Abstract: An enzyme‐linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea‐pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35–0.39 nmol min−1 mg of protein−1 and is not significantly affected by external Ca2+. KC1 depolarization (30 mM KCI) in the absence of Ca2+ doubles this rate, whereas in the presence of Ca2+, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein−1. The final extent of Ca2+‐dependent release amounts to 1.9 nmol mg of protein−1, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30°C for 2 h before depolarization leads to a decrease in Ca2+‐independent release and an increase in Ca2+‐dependent release, consistent with an intrasynaptosomal relocation of the amino acid.

Release of Glutamate, Aspartate, and γ‐Aminobutyric Acid from Isolated Nerve Terminals

Abstract: With the advent of cloning, sequencing, and patchclamping techniques, knowledge of the postsynaptic actions of amino acid neurotransmitters has undergone a dramatic advance. The primary sequences of the inhibitory receptors for γ‐aminobutyric acid (GABA) (Schofield et al., 1987) and glycine (Grenningloh et al., 1987) are now established, and patch‐clamp analysis has elucidated many of the factors that regulate the opening of their ion channels. The excitatory glutamate receptors are being extensively characterized at both the pharmacological (reviewed by Foster and Fagg, 1984) and the electrophysiological (reviewed by Cull‐Candy and Usowicz, 1987) level. In this climate, it is perhaps surprising that the fundamental presynaptic release mechanism for the amino acid neurotransmitters remains controversial.

Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.

A reevaluation of the role of mitochondria in neuronal Ca2+ homeostasis.

Abstract: The ability of mitochondrial Ca2+ transport to limit the elevation in free cytoplasmic Ca2+ concentration in neurones following an imposed Ca2+ load is reexamined. Cultured cerebellar granule cells were monitored by digital fura‐2 imaging. Following KCI depolarization, addition of the protonophore carbonylcyanide m‐chlorophenylhydrazone (CCCP) to depolarize mitochondria released a pool of Ca2+ into the cytoplasm in both somata and neurites. No CCCP‐releasable pool was found in nondepolarized cells. Although the KCI‐evoked somatic and neurite Ca2+ concentration elevations were enhanced when CCCP was present during KCI depolarization, this was associated with a collapsed ATP/ADP ratio. In the presence of the ATP synthase inhibitor oligomycin, glycolysis maintained high ATP/ADP ratios for at least 10 min. The further addition of the mitochondrial complex I inhibitor rotenone led to a collapse of the mitochondrial membrane potential, monitored by rhodamine‐123, but had no effect on ATP/ADP ratios. In the presence of rotenone/oligomycin, no CCCP‐releasable pool was found subsequent to KCI depolarization, consistent with the abolition of mitochondrial Ca2+ transport; however, paradoxically the KCI‐evoked Ca2+ elevation is decreased. It is concluded that the CCCP‐induced increase in cytoplasmic Ca2+ response to KCI is due to inhibition of nonmitochondrial ATP‐dependent transport and that mitochondrial Ca2+ transport enhances entry of Ca2+, perhaps by removing the cation from cytoplasmic sites responsible for feedback inhibition of voltage‐activated Ca2+ channel activity.

Repetitive Action Potentials in Isolated Nerve Terminals in the Presence of 4-Aminopyridine: Effects on Cytosolic Free Ca2+ and Glutamate Release

Abstract: The mechanisms by which an elevated KCl level and the K+‐channel inhibitor 4‐aminopyridine induce release of transmitter glutamate from guinea‐pig cerebral cortical synaptosomes are contrasted. KC1 at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 ± 13 n M above the polarized control. The Ca2+‐dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 ± 0.35 nmol/ mg was released. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl‐induced elevation in [Ca2+]c could be observed in both low‐Na+ medium and in the presence of low concentrations of veratridine. 4‐Aminopyridine at 1 mM increased [Ca2+]c by 143 ± 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4‐aminopyridine administration was slower than that following 30 mM KCl. and a transient spike was less apparent. Consistent with this, the 4‐aminopyridine‐induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4‐Aminopyridine induced the Ca2+‐dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4‐aminopyridine action. In contrast to KCl, glutamate release and the increase in [Ca2+]c with 4‐aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin. Addition of 30 mM KCl to 4‐ami‐nopyridine‐pretreated synaptosomes caused a large transient spike in [Ca2+]c and further release of glutamate. 4‐Aminopyridine failed to increase [Ca2+]c in low‐Na+ media or after addition of low concentrations of veratridine. It is proposed that an unstable membrane potential in the presence of 4‐aminopyridine is amplified by repetitive firing of Na+ channels and that this leads to random opening of transient Ca2+ channels in the synaptosomal population. In contrast, KCl would induce a synchronous activation of transient Ca2+ channels followed by a plateau of low residual channel conductance. By mimicking repetitive stimulation in vivo, 4‐aminopyridine may more closely model physiological excitation than does an elevated KCl level. Our results are difficult to reconcile with a major role for Na+/Ca2+ exchange in the elevation of [Ca2+]c and contradict proposals that 4‐aminopyridine induces release of transmitter from isolated nerve terminals by a mechanism not linked to Na+‐channel firing orCa2+ entry

Ca2+-dependent and Ca2+- independent glutamate release, energy status and cytosolic free Ca2+ concentration in isolated nerve terminals following metabolic inhibition: Possible relevance to hyoglycaemia and anoxia

Hypoglycaemia and anoxia both cause massive release of glutamate from the brain in vivo, and the nature of this release was investigated using guinea-pig cerebral-cortical synaptosomes and iodoacetate and rotenone to simulate the energetic consequences of these conditions. Glutamate release (by continuous fluorimetry), cytoplasmic free Ca2+ (by fura-2), membrane potentials, ATP, ADP and creatine phosphate were determined in parallel, following the addition of iodoacetate or rotenone, alone or in combination. Ca2+-dependent glutamate release had a high energy requirement which could only be satisfied by aerobic glycolysis. Respiration using endogenous substrates, or anaerobic glycolysis following rotenone, caused a progressive inhibition of Ca2+-dependent release, correlating with a decline in the total ATP/ADP ratio and creatine phosphate. With rotenone, an increase in Ca2+-independent glutamate release was observed, correlating with a decline in plasma membrane potential. Only a slight increase in free Ca2+ was seen. Rotenone plus iodoacetate caused an almost immediate collapse of ATP/ADP ratio and a parallel loss of Ca2+-dependent glutamate release before free Ca2+ had risen to a level sufficient for exocytosis. In contrast, Ca2+-independent glutamate release increased. The Ca2+-dependent release of L-glutamate had the characteristics of an exocytotic transmitter release mechanism, being energy-dependent and triggered by elevated cytoplasmic free Ca2+ concentration. A distinct Ca2+-independent release of cytoplasmic glutamate occurred by reversal of the Na+-coupled uptake carrier, which was accelerated by a decline in the Na+ gradient. It is concluded that the Ca2+-independent release of cytoplasmic glutamate may make the major contribution to the excitotoxic release of glutamate in hypoglycaemic and anoxic conditions.