Involvement of Lactic Acidosis in Anoxia-Induced Perturbations of Synaptosomal Function

L-Lactate (4-32 mM) added exogenously to resting or depolarised rat forebrain synaptosomes led to a significant decrease in intrasynaptosomal pH. Similarly depolarisation-induced increases in intrasynaptosomal calcium, calcium uptake, and acetylcholine release were all inhibited. These effects mimicked those previously observed in synaptosomes under anoxic conditions and suggest that lactate may be involved in limiting the damage due to calcium accumulation occurring during ischaemia. D-Lactate (added exogenously up to 32 mM) did not produce similar effects on these parameters even though the concentrations of intrasynaptosomal D-lactate reached levels comparable to those obtained with L-lactate (at 8-16 mM exogenous concentration). The results suggest that the mechanism of action of lactate on these parameters is stereospecific for the L-enantiomer. The effect of glucose availability on lactate production was assessed to explore the role of substrate availability on ischaemia/anoxic events. When exogenous glucose was increased (10-60 mM), there was no further increase in lactate production in normoxic synaptosomes, which suggests that glucose is not limiting under these conditions. When glucose was removed, as may occur in complete ischaemia, there was a significant decrease in lactate production after 60 min under anoxic or normoxic conditions. It would seem likely therefore that the mechanism underlying the changes observed in synaptosomes incubated under conditions reflecting complete ischaemia does not involve lactate.     

Effects of Lactic Acidosis on the Function of Cerebral Cortical Synaptosomes

Synaptosomes exposed to anoxic insult produce lactate at a slow rate (measured over 60 min). No measurable damaging effects were produced by prolonged depolarisation, anoxic insult, or exogenous lactate (2-32 mM) either on the synaptic plasma membrane (as judged by release of lactate dehydrogenase and soluble proteins), or on synaptosomal phospholipases (as judged by choline release from membrane phospholipids). Potassium-stimulated acetylcholine release was decreased by incubation in the presence of lactate (2-32 mM), as was potassium- and veratrine-stimulated calcium uptake and the calcium content of depolarised synaptosomes. The intrasynaptosomal pH was also reduced but there was no stimulation of oxygen radical production (as judged by H2O2 generation) by exogenous lactate. The role that lactic acidosis may play in giving rise to the altered calcium homeostasis and decreased acetylcholine release from synaptosomes exposed to anoxic insult is discussed.     

Menadione-treated synaptosomes as a model for post-ischaemic neuronal damage.

Menadione bisulphite increased endogenous oxygen-radical production by rat brain synaptosomes, as indicated by H2O2 generation. Increased oxygen-radical production was also demonstrated in synaptosomes prepared from menadione-treated rats and synaptosomes reoxygenated after an anoxic insult. Acetylcholine synthesis de novo was inhibited in synaptosomes incubated with menadione in vitro, in synaptosomes prepared from menadione-treated animals in vivo, and in depolarized post-anoxic synaptosomes. Intrasynaptosomal free Ca2+ was increased by menadione in vitro (50 microM), but this increase was not due to stimulation of Ca2+ entry into the nerve terminals. Acetylcholine release was stimulated by menadione in vitro, possibly as a consequence of the elevated intrasynaptosomal Ca2+ content. The Ca2+ contents of synaptosomes prepared from menadione (10 mg/kg)-treated animals in vivo and synaptosomes reoxygenated after anoxia were unchanged. In synaptosomes prepared from menadione-treated animals, acetylcholine release was no longer significantly stimulated by K+, whereas it was unchanged from control (normoxic) values in synaptosomes reoxygenated after anoxia. None of these treatments caused any measurable damage to the synaptic plasma membrane (as judged by the release of lactate dehydrogenase), or to synaptosomal phospholipases (as judged by choline release from membrane phospholipids). Synaptosomes prepared from menadione-treated rats were found to be a good model for the study of post-anoxic damage to nerve-terminal function.     

External calcium, intrasynaptosomal free calcium and neurotransmitter release

In a physiological medium the resting membrane potential of synaptosomes from guinea-pig cerebral cortex, estimated from rhodamine 6G fluorescence measurements, was nearly -50mV. This agreed with calculations using the Goldman-Hodgkin-Katz equation. With external [Ca2+] less than or equal to 3 mM veratridine depolarisation (to -30 mV) was accompanied by increases in intrasynaptosomal free calcium concentrations (monitored by entrapped quin2) and parallel increases in total acetylcholine release. With external [Ca2+] greater than 3 mM both intrasynaptosomal free calcium concentrations and transmitter release were paradoxically reduced, providing further evidence for a close correlation between the two events. To support an explanation of these findings based on divalent cation screening of membrane surface charge (increasing the voltage gradient within the membrane and closing voltage-inactivated channels) surface potential measurements were made on synaptic lipid liposomes by using a fluorescent surface-bound pH indicator. These experiments provided evidence for the presence of screenable surface charge on synaptosomes, and it was further shown in depolarised synaptosomes themselves that total external [Ca2+ + Mg2+], and not [Ca2+] alone, set the observed peak in intrasynaptosomal free calcium.     

Effects of In Vitro Anoxia and Low pH on Acetylcholine Release by Rat Brain Synaptosomes

Acetylcholine and choline release from rat brain synaptosomes have been measured using a chemiluminescent technique under a variety of conditions set up to mimic anoxic insult, including conditions of low pH (6.2) and the presence of lactate plus pyruvate as substrate. Lactate plus pyruvate as substrate consistently gave higher respiration rates than glucose alone, but with either substrate (glucose or lactate plus pyruvate) the omission of Ca2+ caused an increase in respiration whereas a low pH caused a decreased respiration. Acetylcholine release under control conditions (glucose, pH 7.4) was Ca2+-dependent, stimulated by high K+ concentrations, and decreased significantly during anoxia but recovered fully after a period of postanoxic oxygenation. Low pH (6.2) suppressed K+ stimulation of acetylcholine release, and after a period of anoxia at low pH the recovery of acetylcholine release was only partial. With lactate plus pyruvate as substrate, the effects of anoxia and/or low pH on acetylcholine release and its subsequent recovery were exacerbated. Choline release from synaptosomes, however, was not affected by anoxic/ionic conditions in the same way as acetylcholine release. At low pH (6.2) there was a marked reduction in choline release both under aerobic and anoxic conditions. These results suggest that acetylcholine release per se from the nerve is very sensitive to anoxic insult and that the low pH occurring during anoxia may be an important contributory factor.     

Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin.

1. Cerebral-cortex synaptosomes were shown to synthesize (14C)acetylcholine after incubation with (14C)choline, and 25mM-KCl released (14C)acetylcholine (but not (14C)choline) into the medium by a Ca2+-dependent and Mg2+-sensitive process. 2. The K+-stimulated release of (14C)acetylcholine was inhibited by more than 80% after preincubation of the synaptosomes with 10(5) mouse lethal doses of botulinum toxin/ml. (14C)choline uptake, (14C)acetylcholine synthesis, intrasynaptosomal K+ and occluded lactate dehydrogenase were unaffected by the toxin. It also failed to prevent the K+-stimulated release of (3H)noradrenaline and (14C)glycine from synaptosomes. 3. Fractionation of hypo-osmotically shocked synaptosomes revealed that more than 75% of the radioactive acetylcholine was in the cytoplasmic compartment, although the vesicle pellet contained more total acetylcholine than the cytoplasmic pool. Consequently the specific radioactivity of acetylcholine in the cytoplasmic pool was almost 5 times that of the vesicles. This distribution was unaffected by preincubation with botulinum toxin. It is concluded that the toxin acts directly on the release of acetylcholine, rather than influencing its storage. 4. After K+-stimulation, toxin-inhibited synaptosomes contained increased amounts of total acetylcholine, which suggests that its rate of synthesis is controlled by depolarization rather than release.     

Dopamine Uptake by Rat Striatal Synaptosomes: A Compartmental Analysis

Abstract: Dopamine (DA) uptake into synaptosomes from rat corpus striatum was studied in the presence of a monoamine oxidase (MAO) inhibitor and dithiothreitol, by means of a filtration technique. Under these conditions a steady state develops rapidly in which the synaptosomal DA content remains constant while the continuing DA uptake is counterbalanced by DA efflux from the synaptosome. Exchange of synaptosomal [³H]DA and [¹⁴C]DA was measured under these conditions. In timecourse experiments it was found that exchange could be described significantly better by a three-compartment model than by a two-compartment model. However, if synaptosomes from reserpine-pretreated animals were used, analysis according to a three-compartment model did not result in a significantly better fit compared with a two-compartment model. Subsequently, kinetic transfer parameters describing DA fluxes between compartments at different DA concentrations were calculated from the fitted exchange curves. A Michaelis-Menten kinetic analysis indicated that only the in-series three-compartment configuration, in which DA is taken up from the medium into one synaptosomal compartment, from which it can subsequently be transferred to a second compartment without direct access to the medium, gave kinetically acceptable results. Transfer parameters in synaptosomes from reserpine-treated rats were comparable to those parameters describing DA transport between the medium and the first intrasynaptosomal compartment as measured under control conditions. Morover, it was found that potassium depolarization of synaptosomes resulted in a release of DA in a quantity similar to that found in the second intrasynaptosomal compartment. It is suggested that the two intrasynaptosomal compartments found correspond to a cytoplasmatic and vesicular DA pool, respectively. The functional significance of these findings is discussed in terms of the regulation of DA levels within the nerve terminal.     

The effect of sodium on depolarization-induced calcium uptake and acetylcholine release by synaptosomes

The influence of external sodium concentration on potassium (depolarizing agent)-stimulated calcium uptake and Ca⁺-dependent acetylcholine release by rat cerebral cortex synaptosomes has been studied. It was found that increased sodium concentration decreases both the Ca²⁺ uptake and the acetylcholine release, whereas a low external sodium concentration is stimulatory.     

Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium

Abstract: The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of d -[³H]aspartate, with a K _i of approximately 0.3 m m . Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of γ-aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmem-brane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of ra-dioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high-affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.     

Conditions Restricting Depolarization-Dependent Calcium Influx in Synaptosomes Reveal a Graded Response of P96 Dephosphorylation and a Transient Dephosphorylation of P65

Temporal changes in the phosphorylation level of synaptosomal phosphoproteins following depolarization of synaptosomes were investigated under conditions restricting calcium influx. High-K+ depolarization in media of low [Na+]o (32 mM during preincubation and depolarization) at pH 6.5 resulted in a pronounced fall in the cytosolic free calcium concentration transient, and in a reduction in the initial K(+)-stimulated 45Ca2+ uptake and endogenous acetylcholine release relative to the values obtained with control synaptosomes (preincubated and depolarized in Na(+)-based media). This reduction was paralleled by a decrease in the rate of dephosphorylation of the synaptosomal protein P96. A slower dephosphorylation of P96 also was observed on exposure to 20 microM veratridine at 0.5 mM external calcium. Our results indicate that, similar to synapsin I phosphorylation, P96 dephosphorylation shows a graded response to the amount of calcium entering the presynaptic terminal. Depolarization of synaptosomes under conditions restricting the influx of calcium revealed a transient dephosphorylation (reversed within 10 s) of the phosphoprotein P65. The possible significance of this finding to the process of neurotransmitter release is discussed.     

Uptake and release of 45Ca by brain microsomes, synaptosomes and synaptic vesicles

Abstract— Microsomes and synaptosomes from rat brain accumulated ^4,5Ca against a concentration gradient by an ATP-dependent process. Calcium accumulation occurred to the same extent in microsomes prepared from white matter and from grey matter, an observation suggesting that calcium uptake may be in part an activity of the axonal membrane. Microsomes and synaptosomes accumulated calcium to a similar extent but less actively than mitochondria. By contrast, synaptic vesicles showed relatively little calcium accumulation. Isotonic concentrations of sucrose, NaCl, KCl and choline chloride inhibited calcium accumulation, with NaCl and KCl the least effective of these inhibitory agents. No consistent effects on calcium uptake were obtained with adenosine 3′,5′-monophosphate, dibutyryl cyclic AMP or the methyl xanthines. Incubation of prelabelled microsomes resulted in a release of ⁴⁵Ca, and ATP inhibited this release process. In the absence of added ATP, isotonic NaCl promoted calcium release to a significantly greater extent than KCl choline chloride or sucrose. In the presence of ATP, these agents all promoted a similar degree of release. Adenosine 3′,5′-monophosphate or agents that affect its metabolism did not significantly affect calcium release. Magnesium ions reduced calcium release under all conditions tested.     

Influence of ionophores which bind calcium on the release of norepinephrine from synaptosomes.

A preparation of synaptosomes isolated from rat brain was used as a model of nerve to study affects of drugs on uptake and release of biogenic amines. The influence of ionophores, which bind calcium, on the release of noripinephrine from synaptosomes was examined to determine their effect on the release of the amine. A23187 induced release of norepinephrine mainly as the amine and this action was enhanced by calcium and depressed by magnetism. X-537A however, released norepinephrine mostly as deaminated metabolites but acted independently of calcium or magnetism. A23187, therefore is thought to be associated at least in part, with exocytotic amine release, possibly by enhancing entry of calcium across the plasma membrane. X-537A on the other hand may act as a carrier of the amine across the vesicular membrane and expose the amine to intrasynaptosomal monoamine oxidase.     

Acetylcholine Incorporation by Cholinergic Synaptic Vesicles from Torpedo marmorata

[3H]Acetylcholine efflux and Na+-K+ ATPase ion pump activity were measured concomitantly in rat cortical synaptosomes. Ouabain (500 microM), strophanthidin (500 microM), and parachloromercuribenzene sulfonate (500 microM) each inhibited ouabain-sensitive 86Rb uptake and elevated [3H]acetylcholine release independently of the external calcium concentration. Veratridine (10 microM), electrical field stimulation (60 V, 60 Hz, 5-ms pulse duration), or the calcium ionophore A23187 (10 micrograms/ml) also inhibited ouabain-sensitive 86Rb uptake and released [3H]acetylcholine, but via a calcium-dependent process. Veratridine-induced [3H]acetylcholine release and ion pump inhibition were correlated over a wide range of drug concentrations and both effects were blocked by pre-treatment with tetrodotoxin (1 microM). The rate of [3H]acetylcholine efflux from superfused synaptosomes was increased within 15 s of exposure to ouabain, strophanthidin, veratridine, A23187, or field stimulation, while ouabain-sensitive 86Rb uptake was significantly decreased within a similar interval. These results suggest that [3H]acetylcholine release is due at least in part to inhibition of Na+-K+ ATPase.     

Synaptosome behaviour is unaffected by weak pulsed electromagnetic fields

The present study examined the effect on rat cortical synaptosomes of a 2 h exposure to 50-Hz electromagnetic fields (EMFs) with a peak magnetic field of 2 mT. We measured modifications of synaptosomal mitochondrial respiration rate, ATP production, membrane potential, intrasynaptosomal Ca(2+) concentration and free iron release. The O(2) consumption remained unvaried in exposed synaptosomes at about 2 nM O(2)/min/mg proteins; ATP production was also unchanged. The intrasynaptosomal Ca(2+) concentration decreased slowly and there was a slight, but non-significant, depolarisation of the synaptosomal membrane. Finally, the free iron release by synaptosomal suspensions, a useful predictor of neuro-developmental outcome, remained unchanged after EMF exposure. On the whole, our results indicate that the physiological behaviour of cortical synaptosomes is not affected by weak pulsed EMFs.     

Occurrence of a Large Ca2+-Independent Release of Glutamate During Anoxia in Isolated Nerve Terminals (Synaptosomes)

Isolated rat cerebral cortical synaptosomes made anoxic by addition of cyanide developed an inhibition of the Ca2+-dependent release of glutamate 2 min after the addition of the metabolic inhibitor when the intrasynaptosomal ATP/ADP ratio decreased below 1.7. In contrast, cyanide induced a continuous efflux of glutamate through a Ca2+-independent pathway that accounted for the release of 25% of total intrasynaptosomal glutamate in 5 min. The results suggest that a Ca2+-independent release of glutamate could be implicated in the neurotoxic action of this amino acid during anoxia.     

Effects of Diltiazem on Bioenergetics, K+ Gradients, and Free Cytosolic Ca2+ Levels in Rat Brain Synaptosomes Submitted to Energy Metabolism Inhibition and Depolarization

Diltiazem was able to decrease the oxygen consumption rate and lactate production in synaptosomes isolated from rat forebrains, both under control and depolarized (40 microM veratridine) conditions, starting from a concentration of 250 microM. This effect was particularly evident when synaptosomes were depolarized by veratridine. This depolarization-counteracting action was evident also when transplasma membrane K+ diffusion potentials were measured after depolarization induced by veratridine and by rotenone with a glucose shortage. The concentrations of ATP, phosphocreatine, and creatine were less sensitive to diltiazem action. The concentration/response relationships were the same as those found for the oxygen consumption were the same as those found for the oxygen consumption rate, lactate production, and K+ diffusion potentials. The effects of 0.5 mM diltiazem in counteracting inhibition of energy metabolism induced by rotenone without glucose were no longer detectable when either Ca2+ or Na+ was absent from the incubation medium of synaptosomes. Diltiazem at the same concentrations (starting from 250 microM) was able to inhibit both the veratridine-induced and the rotenone-without-glucose-induced increase in intrasynaptosomal free Ca2+ levels evaluated with the fluorescent probe quin2. The results are discussed in view of a possible effect of diltiazem on voltage-dependent Na+ channels and the possibility of utilizing this approach for counteracting neuronal failure due to derangement of energy metabolism or hyperexcitation.     

Parameters not influenced by vesamicol: Membrane potential,calcium uptake,and internal calcium concentration of synaptosomes

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca²⁺-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca²⁺-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca²⁺ uptake, and intracellular Ca²⁺ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 M veratridine (from 67±2.3 mV resting membrane potential to 50.7±2.5 mV) was not significantly influenced by vesamicol (1–20 M). Vesamicol (1–20 M) had no effect on either the overall curve of the veratridine-evoked⁴⁵Ca²⁺ uptake or the amount of Ca²⁺ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca²⁺ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 M). The K⁺-evoked (40 mM) increase of Ca²⁺ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 M) vesamicol inhibited both the fall in membrane potential and the elevated Ca²⁺ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na⁺ channels at this concentration. Vesamicol, in lower concentration (20 M) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [¹⁴C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [¹⁴C]choline. The results suggest that vesamicol does not interfere with veratridine-induced changes in isolated nerve terminals other than with the release of acetylcholine, thus further supporting the involvement of a vesamicol-sensitive vesicular transmitter pool in Ca²⁺-dependent veratridine-elicited acetylcholine release.     

NO synthase and xanthine oxidase activities of rabbit brain synaptosomes: Peroxynitrite formation as a causative factor of neurotoxicity

In the present study we demonstrated that synaptosomes isolated from rabbit brain cortex contain NO synthase and xanthine oxidase that can be activated by ultraviolet B radiation and Ca²⁺ accumulation to produce nitric oxide and superoxide which react together to form peroxynitrite. Irradiation of synaptosomes with ultraviolet B (up to 100 mJ/cm²), or increase the intrasynaptosomal calcium concentration using various doses (up to 100 μM) of the calcium ionophore A 23187, a gradual increase in both nitric oxide and peroxynitrite release that was inhibited by N-monomethyl-L-arginine (100 μM) was observed. The rate of nitric oxide release and cyclic GMP production by NO synthase and soluble guanylate cyclase, both located in the soluble fraction of synaptosomes (synaptosol), were increased approximately eight fold after treatment of synaptosomes with Ultraviolet B radiation (100 mJ/cm²). In reconstitution experiments, when purified NO synthase isolated from synaptosol was added to xanthine oxidase, in the presence of the appropriate cofactors and substrates, a ten fold increase in peroxynitrite production at various doses (up to 20 mJ/cm²) of UVB radiation was observed. Ultraviolet B irradiated synaptosomes promptly increased malondialdehyde production with subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1-4-(trimethyl-amino-phenyl)-6-phenyl-hexa-1,3,5-triene. Desferrioxamine (100 μM) tested in Ultraviolet B-irradiated synaptosomes showed a decrease (approximately 80%) in malondialdehyde production with subsequent restoration of the membrane fluidity to that of non-irradiated (control) synaptosomes. Ca²⁺-stimulated ATPase activity was decreased after Ultraviolet B (100 mJ/cm²) radiation of synaptosomes indicating that the subsequent increase of intrasynaptosomal calcium promoted peroxynitrite production by a calmodulin-dependent increase of NO synthase and xanthine oxidase activities. Furthermore, it was shown that UVB-irradiated synaptosomes were subjected to higher oxidative stress by exogenous peroxynitrite (100 μM) compared to non-irradiated (control) synaptosomes. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells lead to the formation of peroxynitrite providing important clues in the role of peroxynitrite as a causative factor in neurotoxicity.     

Calcium-Dependent and -Independent Acetylcholine Release from Electric Organ Synaptosomes by Pardaxin: Evidence of a Biphasic Action of an Excitatory Neurotoxin

Abstract: The effect of pardaxin, a new excitatory neurotoxin, on neurotransmitter release was tested using purely cholinergic synaptosomes of Torpedo marmorata electric organ. Pardaxin elicited the release of acetylcholine with a biphasic dose dependency. At low concentrations (up to 3 × 10⁻⁷ M ), the release was calcium-dependent and synaptosomal structure was well preserved as revealed by electron microscopy and measurements of occluded lactate dehydrogenase activity. At concentrations from 3 × 10⁻⁷ M to 10⁻⁵ M , the pardaxin-induced release of acetylcholine was independent of extracellular calcium, and occluded synaptosomal lactate dehydrogenase activity was lowered, indicating a synaptosomal membrane perturbation. Electron microscopy of 10⁻⁶ M pardaxin-treated synaptosomes revealed nerve terminals depleted of synaptic vesicles and containing cisternae. At higher toxin concentrations ( 10⁻⁵ M ), there were striking effects on synaptosomal morphology and occluded lactate dehydrogenase activity, suggesting a membrane lytic effect. We conclude that, at low concentrations, this neurotoxin is a promising tool to investigate calcium-dependent mechanisms of neurotransmitter release in the nervous system.     

Intrasynaptosomal Free Calcium Concentration During Rat Brain Development: Effects of Hypoxia, Aglycaemia, and Ischaemia

Abstract: The effects of hypoxia, aglycaemia, and hypoxia-aglycaemia on intrasynaptosomal free Ca²⁺ concentration ([Ca²⁺]_i) have been investigated in rat brain synaptosomes prepared from animals aged 5, 10, 15, 20, 25, and 60 days. After 60 min of hypoxia there was no significant difference, when compared with controls, in basal [Ca²⁺]_i or [Ca²⁺]_i following depolarisation in all of the ages studied. Following 60 min of aglycaemia there was no significant difference from controls in [Ca²⁺]_i of synaptosomes prepared from pups of ≤20 days, although a significant rise in [Ca²⁺]_i was seen in preparations from animals >20 days old. Sixty minutes of hypoxia-aglycaemia led to a significant rise in [Ca²⁺]_i only in preparations from animals 15–60 days old. With both aglycaemia and hypoxia-aglycaemia a progressive increase in the magnitude of the rise in [Ca²⁺]_i was seen with development. These data suggest increases in [Ca²⁺]_i in adult nerve terminals following prolonged aglycaemia and hypoxia-aglycaemia but no change following prolonged hypoxia. In contrast, no significant changes in [Ca²⁺]_i values were apparent in neonatal nerve terminals under any of these conditions. In control synaptosomes with glucose and oxygen freely available, a decrease in resting and depolarised [Ca²⁺]_i during development was seen, suggesting a change in calcium homeostasis within the nerve terminal as the brain develops. It is suggested that the mechanism underlying the relative resistance to ischaemic damage of neonatal brain as compared with adult brain may be related to the regulation of calcium at the nerve ending.